5-fluoro-2-methoxypyrimidin-4(3H)-one

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

473-810-9

EC Name:

-

Cas Number:

1480-96-2

Molecular formula:

Hill formula: C5H5FN2O2 CAS formula: C5H5FN2O2

IUPAC Name:

5-fluoro-2-methoxy-3,4-dihydropyrimidin-4-one

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: white powder
- Storage conditions of test material: at room temperature, protected from humidity and under nitrogen gas

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.