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Toxicological information

Genetic toxicity: in vivo

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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-11-22 to 2011-01-24
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1997-07-21
GLP compliance:
Type of assay:
micronucleus assay

Test material

Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): H-29865
- Physical state: white solid
- Stability under test conditions: the test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Inc. (Raleigh, North Carolina)
- Age at study initiation: approximately 8 weeks old
- Weight at study initiation: males: 28.3 - 35.4 g; females:21.6 - 25.7 g
- Assigned to test groups randomly: yes, they were distributed by computerized, stratified randomization into study groups
- Housing: all animals were housed in solid bottom cages with Shepherd's TM Cob + PLUS TM (i.e., enrichment-containing bedding); At study start, male animals were housed individually and female animals were housed 2 - 3 per cage.
- Diet (ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

- Temperature: 18 - 26°C
- Relative humidity: 30 - 70%
- Photoperiod (hrs dark / hrs light): approximately 12/12

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: aqueous methylcellulose (0.5%) prepared with deionized water
Duration of treatment / exposure:
single dose
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
actual ingested
No. of animals per sex per dose:
Vehicle control group: 10 males / 10 females
500 mg/kg: 10 males / 10 females
1000 mg/kg: 10 males / 10 females
2000 mg/kg: 14 males / 14 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (prepared in deionized water)
- Route of administration: once orally by gavage
- Doses / concentrations: 40 mg/kg (dose volume: 10 mL/kg
- No. of animals per sex per dose: 5 males / 5 females
The positive control group was sampled approximately 24 hours after dosing.


Tissues and cell types examined:
At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for the presence of micronuclei (round, bright yellow-green fluorescing bodies). In the event 2000 PCEs were unattainable for a given animal, the animal was excluded from the statistical evaluation of the scoring results.
Fluorescent microscopy was used to examine the smears. Colour was used to distinguish PCEs (red-orange) from normochromatic erythrocytes (NCEs)(gray-green). Inclusions in PCEs which were improperly shaped or stained or were not in the focal plane of the cell were considered artifacts and not scored as micronuclei. Cells containing more than 1 micronucleus were scored as a single micronucleated PCE (MNPCE). The proportion of PCEs among 1000 total erythrocytes (expressed as the PCE/NCE ratio) was determined for each animal. The proportion of PCEs to total erythrocytes in the test substance-treated animals was not less than 20% of the control value.
Details of tissue and slide preparation:
Dosing concentrations used were determined by the range finding results from an experiment using groups of 3 male animals. The highest dose used for the range finder for the in vivo micronucleus study was the limit dose of 2000 mg/kg bw administered by oral gavage. The animals were observed for clinical signs of toxicity and mortality immediately after dosing and daily thereafter for 2 days (until approximately 48 hours post-dosing). Additional range finding groups of 3 male animals were added if clinical signs of toxicity or mortality were noted at 2000 mg/kg. There were 3 test substance concentrations for the main study: 500, 1000 or 2000 mg/kg bw.

Each group of male and female animals were administered a single dose of the test substance at 500, 1000 or 2000 mg/kg bw, or the vehicle or positive control substances for the main study. The test substance, vehicle, and positve controls were administered by single oral gaveage at a dose volume of 10 mL/kg bw. Approximately 24 and 48 hours after dose administration, treated animals were sacrificed and the bone marrow removed. Additional male and female animals were treated with the highest concentration to allow for unexpected mortality.

Sacrifice and bone marrow collection procedures were conducted according to the laboratory's SOP, with animals placed under general anaesthesia by inhalation of isoflurane prior to sacrifice. Bone marrow was sampled at approximately 24 and 48 hours post-treatment from vehicle control, high-, intermediate-, and low-dose groups. Positive controls were sampled concurrently at the 24-hour interval, only.

Immediately after sacrifice, marrow from both femurs of each animal was aspirated into a syringe containing fetal bovine serum (FBS) and transferred to a centrifuge tube containing FBS. Erythrocytes were collected by centrifugation.
Following centrifugation, most of the supernatant was removed. The pellet was suspended in FBS and a small drop was placed on a pre-cleaned microscope slide. Smears were made using a blood smearing instrument. At least 3 slides per animal were prepared. The slides were air-dried and fixed using absolute methanol. Bone marrow smears were stained in acridine orange, a DNA/RNA specific fluorochrome.

Bone marrow smears from animals in the low and intermediate groups sampled 48 hours after dosing were evaluated only if there was a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) at the highest concentration. Therefore, all groups were evaluated at the 24-hour time point. At the 48-hour time point, only vehicle group and high dose group were evaluated. The low and intermediate groups were not analysed since no positive response was observed for the highest dose group.
Evaluations were conducted on 5 animals/group. At the highest concnetration, 5 males and 5 females were selected for evaluation based on survival until the scheduled sacrifce.

Fluorescent microscopy was used to examine the smears.

A test was considered valid if all of the following conditions were met:
- in the vehicle control group, the mean frequency of MNPCEs did not exceed 10 per 2000 PCEs.
- the range of MNPCE values in the vehicle control animals were within reasonable limits of the laboratory historical control range.
- the positive control induced a statistically significant increase in the frequency of MNPCEs as compared to the vehicle control group at p≤0.05.
Evaluation criteria:
Data were evaluated using scientific judgement taking into account both statistical and biological significance. Results not meeting the indicated criteria for positive or negative findings were evaluated on a case-by-case basis. Further investigation of an equivocal result was not required to obtain a conclusive finding.

The test substance was judged negative if the following conditions were met:
- no statistically significant dose-related increases in the group mean MNPCEs above the concurrent vehicle control value occurred at any concentration of the test substance
- the MNPCE values of the test substance-treated animals were within reasonable limits of the recent (past 3 years) laboratory historical control range.
The test substance was judged positive if the following conditions were met:
- the group mean MNPCEs was statistically significantly increased at one or more concentrations of the test substance compared to the concurrent vehicle control values.
- an accompanying statistically significant dose-response increase in MNPCEs was observed.
Please refer to the field "Any other information on materials and methods incl. tables" below

Results and discussion

Test results
no effects
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
- Dosing solution analysis: data from the analysis of the samples for the study indicated that the test substance was homogeneously mixed, at the targeted concentrations, and stable under the storage conditions for the study. Test substance was not found in the 0 mg/mL control sample.
- Body weights and body weight gains: no significant changes in body weight or body weight gain in either male or female animals administered the test substance.
- no clinical signs of toxicity were observed at any timepoint at any dose level in male and female animals exposed to the test substance. No abnormalities were detected in the vehicle or positive control groups. No mortality occurred during the study.

- no statistically significant increases in micronucleated PCE frequency were observed in any evaluated test substance-treated group of male or female animals at any timepoint.
- no depressions in the frequency of PCE/total erythrocytes were detected at any timepoint in either male or female animals exposed to the test substance.
- statstically significant increases in MNPCE frequency were found in cyclophosphamide-treated animals of both sexes. A statistically significant depressions in PCE frequency were observed in female animals administered cyclophosphamide.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
The test item did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in the bone marrow of mice when administered up to the limit dose of 2000mg/kg bw via oral route.