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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-10-06 - 1997-11-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.12: "Other Effects-Mutagenicity: In Vivo Micronucleus Test'. EEC Publication no. L383 (adopted December, 1992).
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing of Chemicals, Proposal for updating guideline 474: Mammalian Erythrocyte Micronucleus Test (Revised draft document, March 1996).
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vivo mammalian erythrocyte (somatic cell) micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
6-amino-1,3-dimethyluracil
EC Number:
229-662-0
EC Name:
6-amino-1,3-dimethyluracil
Cas Number:
6642-31-5
Molecular formula:
C6H9N3O2
IUPAC Name:
6-amino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione
Test material form:
solid: particulate/powder
Remarks:
off-white to yellow
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark, stable under storage conditions

Test animals

Species:
mouse
Strain:
Swiss
Remarks:
CD-1(outbred-SPF-Quality)
Details on species / strain selection:
Recommended test system in international guidelines (e.g. EPA, FDA, OECD, EEC).
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: see attachments, mean weights of the respective treatment groups are given, varying from 33.0±1.6 - 35.8±3.1 g (males) and 25.6±2.2 - 28.8±2.5 g (females)
- Assigned to test groups randomly: Allocated to treatment groups as they came to hand from delivery boxes.
- Fasting period before study: yes, 3-4h
- Housing: The animals were housed in an air-conditioned room. Five animals per sex per group were housed in label led polycarbonate cages containing purified sawdust as bedding material (Woody SPF, supplied by B.M.I., Helmond, The Netherlands). Animals were identified by unique number on the tail.
- Diet (e.g. ad libitum): The animals were provided with standard pelleted laboratory animal diet (Carfil Quality BVBV, Oud-Turnhout, Belgium)
- Water (e.g. ad libitum): tap water
- Acclimation period: The acclimatisation period was at least 5 days before start of treatment under laboratory conditions. On arrival and prior to final assignment to study, all animals were clinically examined to ensure selected animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 15/h
Fluctuations from these optimal conditions were noted, but were considered not to have affected integrity.
- Photoperiod (hrs dark / hrs light): The room is illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 1% Aqueous carboxymethyl cellulose
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 ml/kg body weight.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in 1% (w/v) carboxymethylcellulose (van 8oom B.V., Meppel, The Netherlands). The solution was treated with ultra-sonication to obtain a homogeneous suspension. Test substance concentrations were prepared directly prior to use.
Duration of treatment / exposure:
24h or 48h after single application
Frequency of treatment:
single application
Post exposure period:
24h or 48h
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
maximum tolerated (high) dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
intermediate dose
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
low dose
No. of animals per sex per dose:
5 / sex / dose
exception: females treated with 2000 mg/kg over 24 h (4 animals) and 48 h (6 animals)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, F.R.G.) dissolved in 0.9% (w/v) NaCl (Merck) in Milli-R0 water dosed at a single oral intubation of 50 mg/kg body weight.
- Route of administration: The route of administration and the volume administered of the positive control was the same as those of the test article.

Examinations

Tissues and cell types examined:
femur bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The mice received a single oral intubation of a maximum tolerated (high), an intermediate and a low dose of the test item.
Selection of an adequate dose range for the Micronucleus test was based on a dose range finding study. Two dose groups, each comprising 3 males and 3 females, received a single dose of the test substance. The study duration was three days. During this period mortality and physical condition were recorded daily.
Based on the results of this dose range finding study dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the Micronucleus Test. After two hours, 2000 mg/bw animals were lethargic, then, no abnormalities were noted.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Single treatment, sampling was done after 24 or 48 h.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation at 24 and 48 h according to the above schedule after dosing of the vehicle, the test item and the positive control.
Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a smal1 opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked with the study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were then air-dried and thereafter fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.
Staining of the bone marrow smears: The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

METHOD OF ANALYSIS:
Analysis of the bone marrow smears for micronuclei: All slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
Evaluation criteria:
ACCEPTABILITY OF ASSAY
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (WiIcoxon Rank Sum Test, two-sided test at P (0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

DATA EVALUATION AND STATISTICAL PROCEDURES
A test substance is considered positive in the micronucleus test if: It induced a biologically as well as a statistically significant (WiIcoxon Rank Sum Test; two-sided test at P (0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if: None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups alone.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.
Statistics:
see above

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Animals treated with 2000 and 1000 mg/kg were lethargic on day 1
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: In a dose range finding study 12 animals (3 males and 3 females per group) were dosed orally with 2000 and 1000 mg/kg body weight (groups A and B respectively). 2h after dosinf, animals treated with 2000 mg/kg were lethargic. Based on the results of this dose range finding study dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the Micronucleus Test.
- Clinical signs of toxicity in test animals: Lethargy 2h after dosing, 2 females and 3 males dosed with 2000 mg/kg died.
- Evidence of cytotoxicity in tissue analyzed: Not observed in the range-finding study.
- Rationale for exposure: The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.

RESULTS OF DEFINITIVE STUDY
Mortality and systemic toxic signs: The mortality and systemic toxic signs of the animals treated with 2000 and 1000 mg/kg body weight are presented below. The animals treated with 500 mg/kg body weight showed no abnormalities. Negative and positive control groups showed no abnormalities also. Since two female animals died in group D, 2000 mg/kg body weight with a 48 h sampling time, animal no. 60 of group C, 2000 mg/kg body weight with a 24 h sampling time, was allocated to group D.
The mean number of micronucleated polychromatic erythrocytes scored in the test substance treated groups were compared with the corresponding control groups. Five male and five female animals were used in each treatment group.
- Induction of micronuclei (for Micronucleus assay):
Micronucleated polychromatic erythrocytes: No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of test substance treated animals.
The incidence of micronucleated polychromatic erythrocytes in the control animals were between or equal to the minimum and maximum value of the historical control data range {male: 0 and 3 (mean 0.7)) and {female: 0 and 3 (mean 0.5)}; indicated are means for n=126 and n=105 respectively.
Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.
- Ratio of PCE/NCE (for Micronucleus assay):
Ratio polychromatic to normochromatic erythrocytes: The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes
- Appropriateness of dose levels and route: The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.

Applicant's summary and conclusion

Conclusions:
The study was conducted according to OECD 474 under GLP. Positive and negative controls gave the appropriate results. It is concluded that this test is valid and that the test item is not mutagenic in the micronucleus test under the experimental conditions described in this report.
Executive summary:

6-amino-1,3-dimethyluracil was tested in the Micronucleus Test in mice according to OECD 474 under GLP, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single oral intubation.

Two groups were dosed with 500 mg/kg body weight, two groups were dosed with 1000 mg/kg body weight and two groups were dosed with 2000 mg/kg body weight.

Bone marrow from corresponding vehicle treated groups (A and B) served as negative controls. Bone marrow from a group (I) treated with a single oral intubation of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow was sampled at 24 and 48 hours after dosing. Bone marrow from the positive control group (I), was harvested at 48 hours after dosing only.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with 6-amino-1,3-dimethyluracil.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.

The groups that were treated with 6-amino-1,3-dimethyluracil showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls.

It is concluded that this test is valid and that 6-amino-1,3-dimethyluracil is not mutagenic in the micronucleus test under the experimental conditions described in this report.