Registration Dossier
Registration Dossier
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EC number: 473-810-9 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames Test
Under the conditions of this study, the test material was considered to be non-mutagenic.
Chromosome Aberration
Under the conditions of this study, the test material induced chromosome aberrations in cultured human lymphocytes following a long treatment (over 20 hours) without S9 mix.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 31 May 2005 to 15 July 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- - Histidine requirement in the Salmonella typhimurium strains (Histidine operon)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: The five strains of Salmonella typhimurium were supplied by B.N. Ames' Laboratory (University of California, Berkeley or Oakland Research Institute, USA)
MEDIA USED
- Type and identity of media: The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was placed under agitation in an incubator at 37 °C for about 14 hours, to produce bacterial suspensions
- Properly maintained: Yes. The bacterial strains were stored in liquid nitrogen in cryoprotective medium (1 mL nutrient broth and 0.09 mL dimethylsulfoxide). - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from a liver microsomal fraction of rats induced with Aroclor 1254
- Test concentrations with justification for top dose:
- - Preliminary toxicity test (TA98, TA100 and TA102): 2, 20, 100, 200, 500 and 1000 µg/plate (with and without metabolic activation)
- Main mutagenicity test (all strains): 62.5, 125, 250, 500 and 1000 µg/plate (with and without metabolic activation) - Vehicle / solvent:
- - Solvent: Water for injections
- Justification for choice of solvent/vehicle: The vehicle was selected according to the results of solubility trials performed before the preliminary toxicity test. The test material was insoluble in dimethylsulfoxide, ethanol, acetone and tetrahydrofuran down to 10 mg/mL. It was poorly soluble in water and the limit of solubility was approximately 5 mg/mL. Before use, the vehicle was degassed by sonication for at least 15 minutes and then saturated with nitrogen gas and kept under nitrogen for at least 15 minutes. - 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
- mitomycin C
- other: 2-Anthramine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.
- Plate Incorporation Method: Test material solution (0.2 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45 °C). After rapid homogenisation, the mixture was overlaid onto a Petri plate containing minimum medium.
- Pre-Incubation Method: Test material solution (0.2 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37 °C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
DURATION
- Exposure duration: 48 to 72 hours of incubation at 37 °C
NUMBER OF REPLICATIONS: Testing was performed in triplicate
COLONY COUNTING: Revertants were scored with an automatic counter
DETERMINATION OF CYTOTOXICITY
- Method: 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. - Evaluation criteria:
- ACCEPTANCE CRITERIA
The study was considered valid if the following criteria were fully met:
- The number of revertants in the vehicle controls is consistent with the historical data of the testing facility
- The number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.
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-relationship was considered as a positive result. Reference to historical data or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained. - Key result
- Species / strain:
- other: 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY TOXICITY TEST
No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. No noteworthy toxicity was noted towards the three strains used, with and without S9 mix. The enhanced lawn observed with the TA 102 strain without S9 mix did not allow the scoring of revertants with this strain.
MUTAGENICITY EXPERIMENTS
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test material was poorly soluble in the vehicle and non-toxic in the preliminary toxicity test, the highest dose-level for the main test was the maximum achievable dose-level, taking into account the limit of solubility in the vehicle and the maximum treatment volume used in the testing laboratory for the selected vehicle.
No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. No toxicity was noted towards all the strains used, both with and without S9 mix.
The test material did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. - Conclusions:
- Under the conditions of this study, the test material was considered to be non-mutagenic.
- Executive summary:
The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.
Five strains of Salmonella typhimurium were used, TA 1535, TA 1537, TA 98, TA 100 and TA 102. A preliminary toxicity test was performed to define the dose-levels to be used for the mutagenicity study. The test material was then tested in two independent experiments, with and without a metabolic activation system (S9 mix prepared from a liver microsomal fraction of rats induced with Aroclor 1254). Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method.
The test material was poorly soluble in the vehicle (water) and non-toxic in the preliminary toxicity test, therefore the highest dose-level for the main test was the maximum achievable dose-level. The selected dose-levels were 62.5, 125, 250, 500 and 1000 μg/plate. Each strain was exposed to at least five dose-levels of the test material (three plates/dose-level). After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored.
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.
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. No toxicity was noted towards all the strains used, both with and without S9 mix. The test material did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.
Under the conditions of this study, the test material was considered to be non-mutagenic.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- other information
- Study period:
- 21 July 2005 to 02 November 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro chromosome aberration study in mammalian cells
- Species / strain / cell type:
- lymphocytes: Cultured human lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Human lymphocytes were prepared from whole blood samples obtained from two healthy donors (one male and one female for each experiment) and collected into heparinised sterile tubes.
- Cell cycle length, doubling time or proliferation index: Human lymphocytes have an average cell cycle time of 12 to 14 hours
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium containing 20 % foetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and phytohemagglutinin (PHA). - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from a liver microsomal fraction of rats induced with Aroclor 1254
- Test concentrations with justification for top dose:
- - First experiment: 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5 and 10 mM (with and without S9 mix)
- Second experiment: 0.31, 0.63, 1.25, 2.5, 5 and 10 mM (with and without S9 mix) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Culture medium
- Justification for choice of solvent/vehicle: The test material was freely soluble in culture medium. The culture medium used for dissolution of the test material was previously degassed by sonication for at least 15 minutes and then saturated with nitrogen gas and kept under nitrogen atmosphere for at least 15 minutes. The test material was dissolved in the vehicle at a concentration of 3.96 mg/mL for both mutagenicity experiments.
In the culture medium, the dose-level of 10 mM (corresponding to 1441 μg/mL) showed no precipitate. At this dose-level, the pH was approximately 7.1 (7.4 for the vehicle control) and the osmolality equal to 308 mOsm/kg H2O (305 for the vehicle control). - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
To prepare each culture, 0.5 mL of heparinised whole blood was added to 5 mL of RPMI 1640 medium. The cultures were then placed at 37 °C for 48 hours.
DURATION
In the first experiment, lymphocyte cultures were exposed for 3 hours to the test or control materials, both in the absence and presence of S9 mix, then rinsed. Cells were harvested 20 hours after the beginning of treatment (approximately 1.5 normal cell cycles).
In the second experiment, without S9 mix, cells were exposed continuously to the test or control materials until harvest; with S9 mix, cells were exposed to the test or control materials for 3 hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment (approximately 1.5 normal cell cycles, and 24 hours later).
SPINDLE INHIBITOR (cytogenetic assays): One and a half hours before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis.
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: duplicate cultures per dose/control
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture, whenever possible. Only 50 metaphases/culture were analysed when at least 10 % cells with structural chromosome aberration were observed. All metaphase analyses were performed blind.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (number of cells in mitosis/1000 cells examined), which indicates whether a material induces mitotic inhibition. Mitotic index was determined without blind scoring.
OTHER EXAMINATIONS:
- In addition to structural aberrations, the following numerical aberrations were recorded when encountered: polyploidy and endoreduplication. - Evaluation criteria:
- ACCEPTANCE CRITERIA
This study was considered valid since the following criteria were met:
- The frequency of cells with structural chromosome aberration in the vehicle controls was consistent with the historical data
- The frequency of cells with structural chromosome aberration in the positive controls was significantly higher than that of the controls and consistent with the historical data
EVALUATION CRITERIA
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance was also taken into account in the evaluation of the findings. - Statistics:
- For each test and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the results were compared using the χ2 test, in which p = 0.05 was used as the lowest level of significance.
- Key result
- Species / strain:
- lymphocytes: Cultured human lymphocytes
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: Cultured human lymphocytes
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: Cultured human lymphocytes
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At the final dose-level of 10 mM, the pH values were equivalent to those of the vehicle control culture.
- Effects of osmolality: At the final dose-level of 10 mM, the osmolality values were equivalent to those of the vehicle control culture.
- Precipitation: At the final dose-level of 10 mM, no precipitate was noted.
RESULTS OF THE CHROMOSOME ABERRATION TEST
EXPERIMENTS WITHOUT S9 MIX
- Cytotoxicity: Following the 3-hour treatment, a slight to moderate decrease (29 to 46 %) in mitotic index was noted at dose-levels ≥2.5 mM. Following the 20-hour treatment, a slight to marked decrease (22 to 79 %) in mitotic index was induced at dose-levels ≥1.25 mM. Following the 44-hour treatment, a slight to marked decrease (32 to 69 %) in mitotic index was induced at dose-levels ≥1.25 mM.
- Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows: 2.5, 5 and 10 mM for the 3-hour treatment, the latter being the highest achievable dose-level; 1.25, 2.5 and 5 mM for the 20-hour treatment, the latter inducing 61 % decrease in mitotic index and 1.25, 2.5 and 5 mM for the 44-hour treatment, the latter inducing 65 % decrease in mitotic index.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-hour treatment.
Following the 20-hour treatment, a slight increase in the frequency of cells with structural chromosomal aberrations was noted (4.5 % versus 0.5 % for the vehicle control p<0.05). This frequency was also out of the vehicle control historical range (0.0 to 2.0 %). Following the 44-hour treatment, a significant and dose-related increase in the frequency of cells with structural chromosomal aberrations was noted at dose-levels ≥1.25 mM (2.5 to 16 % versus 1 % for the vehicle control, the vehicle control historical data ranging from 0.0 to 1.0).
EXPERIMENTS WITH S9 MIX
- Cytotoxicity: No noteworthy decrease in mitotic index was noted at either harvest time.
- Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows: 2.5, 5 and 10 mM for the 20-hour harvest time in both experiments and 10 mM for the 44-hour harvest time.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment and at either harvest times.
VALIDITY OF TEST
The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid. - Remarks on result:
- other: 20 and 44 hour treatment
- Conclusions:
- Under the conditions of this study, the test material induced chromosome aberrations in cultured human lymphocytes following a long treatment (over 20 hours) without S9 mix..
- Executive summary:
The potential of the test material to induce chromosomal aberrations in cultured human lymphocytes was investigated in accordance with the standardised guidelines OECD 473 and EU method B10 under GLP conditions.
The test material was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. The test material was dissolved in culture medium. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of mitotic index (MI) in the first experiment was also taken into account. Concurrent positive and negative control cultures were run as applicable.
Dose levels were as follows: First experiment: 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5 and 10 mM (with and without S9 mix); second experiment: 0.31, 0.63, 1.25, 2.5, 5 and 10 mM (with and without S9 mix). For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at 37 °C, for 48 hours.
In the first experiment, lymphocyte cultures were exposed to the test or control materials (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.
For the second experiment, without S9 mix cells were exposed continuously to the test or control materials until harvest; with S9 mix, cells were exposed to the test or control materials for 3 hours and then rinsed. Cells were harvested 20 and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.
One and a half hours before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa.
At the final dose-level of 10 mM, no precipitate was noted and the pH and the osmolality values were equivalent to those of the vehicle control culture.
For the experiments without S9 mix, following the 3-hour treatment, a slight to moderate decrease (29 to 46 %) in mitotic index was noted at dose-levels ≥2.5 mM. Following the 20-hour treatment, a slight to marked decrease (22 to 79 %) in mitotic index was induced at dose-levels ≥1.25 mM. Following the 44-hour treatment, a slight to marked decrease (32 to 69 %) in mitotic index was induced at dose-levels ≥1.25 mM.
For the experiments without S9 mix, the dose-levels selected for metaphase analysis were 2.5, 5 and 10 mM for the 3-hour treatment; 1.25, 2.5 and 5 mM for the 20-hour treatment and 1.25, 2.5 and 5 mM for the 44-hour treatment.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-hour treatment. Following the 20-hour treatment, a slight increase in the frequency of cells with structural chromosomal aberrations was noted (4.5 % versus 0.5 % for the vehicle control p<0.05). This frequency was also out of the vehicle control historical range (0.0 to 2.0 %). Following the 44-hour treatment, a significant and dose-related increase in the frequency of cells with structural chromosomal aberrations was noted at dose-levels ≥1.25 mM (2.5 to 16 % versus 1 % for the vehicle control, the vehicle control historical data ranging from 0.0 to 1.0).
For the experiments with S9 mix, no noteworthy decrease in mitotic index was noted at either harvest time.
For the experiments without S9 mix the dose-levels selected for metaphase analysis were: 2.5, 5 and 10 mM for the 20-hour harvest time in both experiments and 10 mM for the 44-hour harvest time.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment and at either harvest times.
The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid.
Under the conditions of this study, the test material induced chromosome aberrations in cultured human lymphocytes following a long treatment (over 20 hours) without S9 mix.
Referenceopen allclose all
Table 1: Summary of Experiment 1
± S9 Mix |
Concentration (µg/plate) |
Mean number of colonies/plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
||
- |
Solvent 62.5 125 250 500 1000 |
121 113 112 114 123 100 |
19 14 13 19 14 10 |
489 491 493 494 399 228 |
34 20 28 31 28 24 |
9 3 9 5 3 2 |
+ |
Solvent 62.5 125 250 500 1000 |
118 102 115 112 101 97 |
13 7 11 11 14 13 |
512 540 611 512 542 437 |
35 32 27 37 20 24 |
6 11 11 9 4 7 |
Positive Controls |
||||||
- |
Name |
NAN3 |
NAN3 |
MMC |
2NF |
9AA |
Concentration (µg/plate) |
1 |
1 |
0.5 |
0.5 |
50 |
|
Mean no. colonies/plate |
621 |
568 |
1320 |
267 |
1255 |
|
+ |
Name |
2AM |
2AM |
2AM |
2AM |
2AM |
Concentration (µg/plate) |
2 |
2 |
10 |
2 |
2 |
|
Mean no. colonies/plate |
1368 |
322 |
1650 |
1127 |
163 |
|
NAN3 = Sodium Azide
MMC = Mitomycin C
2NF = 2-Nitrofluorene
9AA = 9 -Aminoacridine
2AM = 2 -Anthramine
Table 2: Summary of Experiment 2
± S9 Mix |
Concentration (µg/plate) |
Mean number of colonies/plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
||
- |
Solvent 62.5 125 250 500 1000 |
117 119 123 137 125 107 |
18 15 14 15 14 13 |
301 256 242 228 200 144 |
31 17 18 14 11 15 |
5 6 6 7 6 2 |
+ |
Solvent 62.5 125 250 500 1000 |
135 138 140 110 146 133 |
12 19 13 11 13 14 |
375 324 377 278 296 325 |
16 23 28 21 22 27 |
7 9 7 10 10 4 |
Positive Controls |
||||||
- |
Name |
NAN3 |
NAN3 |
MMC |
2NF |
9AA |
Concentration (µg/plate) |
1 |
1 |
0.5 |
0.5 |
50 |
|
Mean no. colonies/plate |
491 |
497 |
1774 |
179 |
401 |
|
+ |
Name |
2AM |
2AM |
2AM |
2AM |
2AM |
Concentration (µg/plate) |
2 |
2 |
10 |
2 |
2 |
|
Mean no. colonies/plate |
1061 |
153 |
1977 |
1218 |
147 |
|
NAN3 = Sodium Azide
MMC = Mitomycin C
2NF = 2-Nitrofluorene
9AA = 9 -Aminoacridine
2AM = 2 -Anthramine
Table 1: Mitotic Index Results (3-hour treatment)
Dose level (mM) |
Experiment 1 |
Experiment 2 |
||||||
Without S9 mix (20 h harvest) |
With S9 mix (20 h harvest) |
With S9 mix (20 h harvest) |
With S9 mix (44 h harvest) |
|||||
Mean MI (%) |
% of Control |
Mean MI (%) |
% of Control |
Mean MI (%) |
% of Control |
Mean MI (%) |
% of Control |
|
0 |
7.10 |
100 |
5.30 |
100 |
5.55 |
100 |
5.40 |
100 |
0.08 |
7.45 |
105 |
4.20 |
79 |
- |
- |
- |
- |
0.16 |
7.70 |
108 |
5.75 |
108 |
- |
- |
- |
- |
0.31 |
6.30 |
89 |
5.55 |
105 |
3.95 |
71 |
4.50 |
83 |
0.63 |
6.00 |
85 |
5.80 |
109 |
5.35 |
96 |
4.50 |
83 |
1.25 |
6.95 |
98 |
6.90 |
130 |
5.75 |
104 |
4.90 |
91 |
2.5 |
4.60 |
65 |
6.25 |
118 |
4.65 |
84 |
4.40 |
81 |
5 |
5.05 |
71 |
6.05 |
114 |
5.40 |
97 |
5.00 |
93 |
10 |
3.80 |
54 |
5.15 |
97 |
4.10 |
74 |
5.45 |
101 |
MMC 3 µg/mL |
1.60 |
23 |
- |
- |
- |
- |
- |
- |
CPA 12.5 µg/mL |
- |
|
4.45 |
84 |
2.25 |
41 |
- |
- |
CPA 25 µg/mL |
- |
|
1.65 |
31 |
1.60 |
29 |
- |
- |
MI = Mitotic Index
MMC = Mitomycin C
CPA = Cyclophosphamide
Table 2: Mitotic Index Results (longer treatment without S9 mix)
Dose level (mM) |
Experiment 2 |
|||
20 h treatment, 20 h harvest |
44 h treatment, 44 h harvest |
|||
Mean MI (%) |
% of Control |
Mean MI (%) |
% of Control |
|
0 |
3.60 |
100 |
5.70 |
100 |
0.31 |
3.30 |
92 |
4.65 |
82 |
0.63 |
4.00 |
111 |
4.55 |
80 |
1.25 |
2.80 |
78 |
3.45 |
61 |
2.5 |
2.40 |
67 |
3.85 |
68 |
5 |
1.40 |
39 |
2.00 |
35 |
10 |
0.75 |
21 |
1.75 |
31 |
MMC 0.2 µg/mL |
2.30 |
64 |
- |
- |
MI = Mitotic Index
MMC = Mitomycin C
Table 3: Experiment 1 - Results of Chromosome Aberration Test
Treatment and harvest period |
Dose Level (mM) |
Total Number of structural Chromosome Aberrations |
Mean % of Cells with Structural Chromosome Aberrations |
||
(+ Gaps) |
(- Gaps) |
(+ Gaps) |
(- Gaps) |
||
3 h treatment, 20 h harvest (without S9) |
0 |
1 |
1 |
0.5 |
0.5 |
2.5 |
0 |
0 |
0.0 |
0.0 |
|
5 |
1 |
0 |
0.5 |
0.0 |
|
10 |
0 |
0 |
0.0 |
0.0 |
|
MMC 3 µg/mL |
39 |
38 |
28.0 |
27.0*** |
|
3 h treatment, 20 h harvest (with S9) |
0 |
0 |
0 |
0.0 |
0.0 |
2.5 |
0 |
0 |
0.0 |
0.0 |
|
5.0 |
2 |
2 |
1.0 |
1.0 |
|
10 |
0 |
0 |
0.0 |
0.0 |
|
CPA 12.5 µg/mL |
22 |
22 |
18.0 |
18.0*** |
|
MMC = Mitomycin C
CPA = Cyclophosphamide
***p< 0.001(performed only for cells with structural aberrations excluding gaps)
Table 4: Experiment 2 - Results of Chromosome Aberration Test
Treatment and harvest period |
Dose level (mM) |
Total Number of Structural Chromosome Aberrations |
Mean % of Cells with Structural Chromosome Aberrations |
||
(+ Gaps) |
(- Gaps) |
(+ Gaps) |
(- Gaps) |
||
20 h treatment, 20 h harvest (without S9) |
0 |
1 |
1 |
0.5 |
0.5 |
1.25 |
6 |
5 |
3.0 |
2.5 |
|
2.5 |
5 |
5 |
2.5 |
2.5 |
|
5 |
12 |
12 |
4.5 |
4.5* |
|
MMC 0.2 µg/mL |
25 |
25 |
11.3 |
11.3*** |
|
44 h treatment, 44 h harvest (without S9) |
0 |
3 |
2 |
1.5 |
1.0 |
1.25 |
5 |
5 |
2.5 |
2.5 |
|
2.5 |
21 |
20 |
9.5 |
9.5*** |
|
5 |
19 |
18 |
16.0 |
16.0*** |
|
3 h treatment, 44 h harvest (with S9) |
0 |
1 |
1 |
0.5 |
0.5 |
10 |
0 |
0 |
0.0 |
0.0 |
|
3 h treatment, 20 h harvest (with S9) |
0 |
0 |
0 |
0.0 |
0.0 |
2.5 |
2 |
2 |
1.0 |
1.0 |
|
5 |
2 |
1 |
1.0 |
0.5 |
|
10 |
0 |
0 |
0.0 |
0.0 |
|
CPA 25 µg/mL |
36 |
35 |
24.0 |
24.0*** |
|
MMC = Mitomycin C
CPA = Cyclophosphamide
*p< 0.005 (performed only for cells with structural aberrations excluding gaps)
***p< 0.001(performed only for cells with structural aberrations excluding gaps)
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
Mouse Micronucleus
Under the conditions of this study, the test material did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 50, 100 or 200 mg/kg/day.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 June 2005 to 27 December 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- Swiss
- Remarks:
- Swiss lco: OFI (IOPS Caw)
- Details on species / strain selection:
- Rodent species generally accepted by regulatory authorities for this type of study.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: The animals were approximately 6 weeks old on the day of study initiation.
- Weight at study initiation: No data
- Assigned to test groups randomly: Yes. Upon arrival, the animals were randomly allocated to the groups by sex. Subsequently, each group was assigned to a different treatment group.
- Fasting period before study: Animals were not fasted.
- Housing: The animals were housed individually in disposable cages. Each cage contained autoclaved sawdust.
- Diet: ad libitum pelleted maintenance diet
- Water: Drinking water filtered by an FG Millipore membrane (0.22 micron) was provided ad libitum.
- Acclimation period: At least 5 days before the day of treatment
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 30 to 70 % (relative)
- Air changes: At least 12 cycles/hour of filtered non-recycled fresh air
- Photoperiod: - Photoperiod: 12 h/ 12 h light/dark cycle (07:00 - 19:00)
IN-LIFE DATES: Not reported - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: 0.5 % methylcellulose
- Justification for choice of solvent/vehicle: The vehicle was selected according to the results of solubility trials.
- Concentration of test material in vehicle: The test material was suspended in the vehicle in order to achieve the concentrations of 5, 10 and 20 mg/mL.
- Amount of vehicle (if gavage or dermal): Treatment volume of 10 mL/kg. The quantity of material administered to each animal was adjusted according to the most recently recorded body weight. - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: For the main test, the test material was suspended in the vehicle and then homogenised using a magnetic stirrer. The vehicle was degassed by sonication for at least 30 minutes and then kept under nitrogen atmosphere for at least 15 minutes. The preparations were maintained under agitation using a magnetic stirrer during all the treatment period.
- Duration of treatment / exposure:
- The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment.
- Frequency of treatment:
- For the test material solutions and the vehicle control, treatments were administered, with each treatment separated by 24 h hours. For the positive control, one treatment was administered.
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 200 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 5 animals per sex per dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide was used as the positive control
- Route of administration: oral
- Doses / concentrations: 50 mg/kg administered at a dose volume of 10 mL/kg - Tissues and cell types examined:
- The test examined the number of micronucleated polychromatic erythrocytes in the bone marrow of exposed mice.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION
In order to determine the highest dose-level, several preliminary tests were performed on groups of six animals (three males and three females) at dose levels from 100 to 2000 mg/kg/day. Clinical signs and any mortality were recorded for a period of 48 hours. At the end of this period, the animals were killed by CO2 inhalation in excess.
In order to avoid testing of dose-levels inducing too severe toxicity on bone marrow cells, an evaluation of the polychromatic erythrocytes/normochromatic erythrocytes ratio was performed on the animals treated at the maximum tolerated dose (which corresponds to the highest dose-level selected for the main test), by scoring of a total of 1000 erythrocytes per animal.
The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since toxic effects were observed, the choice of the top dose-level was based on the level of toxicity, such that a higher dose-level was expected to induce lethality.
Consequently, 200 mg/kg/day was selected as the top dose-level for the main test. The two other selected dose-levels were 100 and 50 mg/kg/day.
TREATMENT AND SAMPLING
- Plasma level of the test material
Blood samples for the determination of plasma levels of the test material were taken 2 hours following the second treatment. Venous blood (approximately 0.5 mL) was taken into a tube containing lithium heparinate from the orbital sinus of the animals under light isoflurane anaesthesia. The blood was centrifuged (10 min at 4000 rpm, at +4 °C) and the plasma was kept frozen in individual tubes at -20 °C until analysis. The HPLC/UV method was used.
PREPARATION OF THE BONE MARROW SMEARS
At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs of the animals were removed and the bone marrow was flushed out using foetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded for "blind" scoring.
METHOD OF ANALYSIS
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE). - Evaluation criteria:
- For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
- Statistics:
- Normality and homogeneity of variances were tested using a Kolmogorov Smirnov test and a Bartlett test. If normality and homogeneity of variances were demonstrated, the statistical comparison was performed using a Student t-test (2 groups) or a one-way analysis of variance (≥ 3 groups) followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Whitney test (2 groups) or a Kruskall Wallis test (≥ 3 groups) was performed followed by a Dunn test (if necessary).
All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05 for all tests. - Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY TOXICITY TEST
At 2000 mg/kg/day, all animals were found dead 2 hours following the first treatment. At 375 and 500 mg/kg/day, mortality was noted (2/3 males at 500 mg/kg and 1/3 males at 375 mg/kg) in males more than 20 hours following the first administration of the test material. In females, in view of the severe clinical signs noted following the first treatment, the animals were sacrificed prematurely. For the animals treated at 100 and 200 mg/kg/day no mortality was observed in either males or females. Piloerection and sometimes half-closed eyes were noted in males. At 200 mg/kg/day, which was considered to be the maximum tolerated dose, the evaluation of the bone marrow smears performed at this dose-level showed PE/NE ratios consistent with the vehicle control historical data.
PLASMA LEVEL OF THE TEST MATERIAL
For animals given 200 mg/kg/day, two hours following the second treatment, mean ± SD plasma levels achieved were 344 ± 32 μg/mL and 358 ± 22 μg/mL in males and females, respectively. These results demonstrated that bone marrow cells were exposed to the test material.
MICRONUCLEUS ASSAY (CYTOGENETIC TEST)
No clinical signs and no mortality were observed in the animals of both sexes given 50, 100 or 200 mg/kg/day. For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test material were equivalent to those of the vehicle control group. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the testing facility historical data.
Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under the experimental conditions. The study was therefore considered valid. - Conclusions:
- Under the conditions of this study, the test material did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 50, 100 or 200 mg/kg/day.
- Executive summary:
A study was performed in vivo to investigate the potential of the test material to induce structural or numerical damage in bone marrow cells of mice in accordance with the standardised guidelines OECD 474 and EU method B.12 under GLP conditions.
A preliminary toxicity test was performed to define the dose-levels to be used for the main cytogenetic study. In the main study, three groups of five male and five female Swiss lco: OFI (IOPS Caw) mice were given oral administrations of the test material at dose-levels of 50, 100 or 200 mg/kg/day, over a 2-day period. One group of five males and five females received the vehicle (0.5 % methylcellulose) under the same experimental conditions and acted as control group. One group of five males and five females received the positive control test material (Cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.
The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic erythrocyte (NE) ratio was established by scoring a total of 1000 erythrocytes (PE+ NE).
No clinical signs and no mortality were observed in the animals of both sexes given 50, 100 or 200 mg/kg/day. Results of analysis for the plasma levels of the test material, in animals given 200 mg/kg/day, demonstrated that bone marrow cells were exposed to the test material.
For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test material were equivalent to those of the vehicle control group. The mean values of MPE, as well as the PE/NE ratio, for the vehicle and positive controls were consistent with the historical control data. Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under the experimental conditions. The study was therefore considered valid.
Under the conditions of this study, the test material did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 50, 100 or 200 mg/kg/day.
Reference
Table 1: Summary of Results of the Cytogenetic Test
Group |
Doses (mg/kg/day) |
MPE/1000PE |
PE/NE Ratio |
Time of sacrifice after the last administration (h) |
||
Mean |
SD |
Mean |
SD |
|||
Males |
||||||
Vehicle |
- |
0.1 |
0.2 |
0.3 |
0.1 |
24 |
Test material |
50 |
0.6 |
0.4 |
0.4 |
0.1 |
|
100 |
0.6 |
0.4 |
0.4 |
0.1 |
||
200 |
0.7 |
0.6 |
0.5 |
0.1 |
||
CP |
50 |
17.6* |
4.6 |
0.5 |
0.1 |
|
Females |
||||||
Vehicle |
- |
0.6 |
0.5 |
0.5 |
0.1 |
24 |
Test material |
50 |
0.4 |
0.4 |
0.5 |
0.1 |
|
100 |
0.6 |
0.4 |
0.5 |
0.2 |
||
200 |
0.5 |
0.6 |
0.5 |
0.2 |
||
CP |
50 |
16.8* |
7.7 |
0.6 |
0.2 |
|
MPE: Micronucleated Polychromatic Erythrocytes
PE: Polychromatic Erythrocytes
NE: Nonnochromatic Erythrocytes
SD: standard deviation
*p <0.05
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
There are three studies available to address this endpoint; all were awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
Ames Test
The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.
Five strains of Salmonella typhimurium were used, TA 1535, TA 1537, TA 98, TA 100 and TA 102. A preliminary toxicity test was performed to define the dose-levels to be used for the mutagenicity study. The test material was then tested in two independent experiments, with and without a metabolic activation system (S9 mix prepared from a liver microsomal fraction of rats induced with Aroclor 1254). Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method.
The test material was poorly soluble in the vehicle (water) and non-toxic in the preliminary toxicity test, therefore the highest dose-level for the main test was the maximum achievable dose-level. The selected dose-levels were 62.5, 125, 250, 500 and 1000 μg/plate. Each strain was exposed to at least five dose-levels of the test material (three plates/dose-level). After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored.
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.
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. No toxicity was noted towards all the strains used, both with and without S9 mix. The test material did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.
Under the conditions of this study, the test material was considered to be non-mutagenic.
Chromosome Aberration
The potential of the test material to induce chromosomal aberrations in cultured human lymphocytes was investigated in accordance with the standardised guidelines OECD 473 and EU method B10 under GLP conditions.
The test material was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. The test material was dissolved in culture medium. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of mitotic index (MI) in the first experiment was also taken into account. Concurrent positive and negative control cultures were run as applicable.
Dose levels were as follows: First experiment: 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5 and 10 mM (with and without S9 mix); second experiment: 0.31, 0.63, 1.25, 2.5, 5 and 10 mM (with and without S9 mix). For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at 37 °C, for 48 hours.
In the first experiment, lymphocyte cultures were exposed to the test or control materials (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.
For the second experiment, without S9 mix cells were exposed continuously to the test or control materials until harvest; with S9 mix, cells were exposed to the test or control materials for 3 hours and then rinsed. Cells were harvested 20 and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.
One and a half hours before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa.
At the final dose-level of 10 mM, no precipitate was noted and the pH and the osmolality values were equivalent to those of the vehicle control culture.
For the experiments without S9 mix, following the 3-hour treatment, a slight to moderate decrease (29 to 46 %) in mitotic index was noted at dose-levels ≥2.5 mM. Following the 20-hour treatment, a slight to marked decrease (22 to 79 %) in mitotic index was induced at dose-levels ≥1.25 mM. Following the 44-hour treatment, a slight to marked decrease (32 to 69 %) in mitotic index was induced at dose-levels ≥1.25 mM.
For the experiments without S9 mix, the dose-levels selected for metaphase analysis were 2.5, 5 and 10 mM for the 3-hour treatment; 1.25, 2.5 and 5 mM for the 20-hour treatment and 1.25, 2.5 and 5 mM for the 44-hour treatment.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-hour treatment. Following the 20-hour treatment, a slight increase in the frequency of cells with structural chromosomal aberrations was noted (4.5 % versus 0.5 % for the vehicle control p<0.05). This frequency was also out of the vehicle control historical range (0.0 to 2.0 %). Following the 44-hour treatment, a significant and dose-related increase in the frequency of cells with structural chromosomal aberrations was noted at dose-levels ≥1.25 mM (2.5 to 16 % versus 1 % for the vehicle control, the vehicle control historical data ranging from 0.0 to 1.0).
For the experiments with S9 mix, no noteworthy decrease in mitotic index was noted at either harvest time.
For the experiments without S9 mix the dose-levels selected for metaphase analysis were: 2.5, 5 and 10 mM for the 20-hour harvest time in both experiments and 10 mM for the 44-hour harvest time.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment and at either harvest times.
The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid.
Under the conditions of this study, the test material induced chromosome aberrations in cultured human lymphocytes following a long treatment (over 20 hours) without S9 mix.
Mouse Micronucleus Test
A study was performed in vivo to investigate the potential of the test material to induce structural or numerical damage in bone marrow cells of mice in accordance with the standardised guidelines OECD 474 and EU method B.12 under GLP conditions.
A preliminary toxicity test was performed to define the dose-levels to be used for the main cytogenetic study. In the main study, three groups of five male and five female Swiss lco: OFI (IOPS Caw) mice were given oral administrations of the test material at dose-levels of 50, 100 or 200 mg/kg/day, over a 2-day period. One group of five males and five females received the vehicle (0.5 % methylcellulose) under the same experimental conditions and acted as control group. One group of five males and five females received the positive control test material (Cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.
The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic erythrocyte (NE) ratio was established by scoring a total of 1000 erythrocytes (PE+ NE).
No clinical signs and no mortality were observed in the animals of both sexes given 50, 100 or 200 mg/kg/day. Results of analysis for the plasma levels of the test material, in animals given 200 mg/kg/day, demonstrated that bone marrow cells were exposed to the test material.
For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test material were equivalent to those of the vehicle control group. The mean values of MPE, as well as the PE/NE ratio, for the vehicle and positive controls were consistent with the historical control data. Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under the experimental conditions. The study was therefore considered valid.
Under the conditions of this study, the test material did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 50, 100 or 200 mg/kg/day.
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to genetic toxicity.
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