4,6-Dichloro-5-fluoropyrimidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-04-05 to 2005-09-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

1

Reference substance name:

-

EC Number:

446-560-3

EC Name:

-

Cas Number:

213265-83-9

Molecular formula:

C4HCl2FN2

IUPAC Name:

4,6-dichloro-5-fluoropyrimidine

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9- liver mix
- source of S9: The S9 fraction was isolated in house from the livers of Aroclor 1254 induced male Sprague Dawley rats. The used S9 fraction was derived from the preparation dated April 6, 2004 (color-code green, protein content 22.9 mg per ml).
- concentration or volume of S9 mix and S9 in the final culture medium: protein content 22.9 mg per ml, The S9 mix contained 40% S9 fraction and was kept in refrigerator and used on the same day.

Test concentrations with justification for top dose:

test concentrations were based on the results from a preliminary cytotoxicity test
0, 0.4, 0.8, 1.2, 1.6, 2, 1.5, 3, 6, 7, 8 µg/mL for the 4h treatment and 18 h harvest time
0, 1.2, 1.6, 2, 6, 7, 8 µg/mL for the 4h treatment and 30 h harvest time

Vehicle / solvent:

DMSO: The test substance was dissolved in a suitable solvent which was selected based on the solubility of HEC 5725-DCF-Pyrimidine in the following solvents in the order given. If possible, deionized water is used as the solvent. Test substances that are not sufficiently soluble in this solvent are dissolved in DMSO, ethanol or acetone and then added to the medium, if this results in a higher final concentration of the test substance in the medium.
For HEC 5725-DCF-Pyrimidine, DMSO was selected as solvent. In this solvent HEC 5725-DCF-Pyrimidine was soluble up to 180 mg/ml.
Precipitation of HEC 5725-DCF-Pyrimidine was observed in the culture medium of the pre-test at 900 ug/ml and above.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 xE+06 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 24 h
- Exposure duration/duration of treatment: 4 h
- Harvest time after the end of treatment (sampling/recovery times): 18 and 30 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. 0.2 mL Colcemid-solution (40 µg/mL water) were added to each flask two hours prior to the end of the incubation period.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The medium was removed from each flask and cells were removed from the bottom of the flask by trypsinization and suspended in medium. This medium was transferred to a centrifuge tube and spun for approximately 5 min at 700 rpm. The supernatant was carefully removed. 1-2 mL of a hypotonic solution (0.4% KCI; 37°C) was added to the tube. Within 4 min, the volume was brought to 6 mL with additional hypotonic solution and cells were resuspended. The cells were sedimented in the centrifuge as before and the supernatant was removed. A few drops of cold (4°C) fixative [ethanol/acetic acid (3:1)] were added and mixed carefully with the cells. The volume was adjusted to 6 mL with the fixative and mixed again with the cells. The mixture was incubated at room temperature for 20 min. Cells were pelleted as before and the supernatant was discarded. Cells were again resuspended in fixative as before and centrifuged. Pelleted cells were resuspended carefully in a small volume of fresh fixative. This suspension was dropped onto clean slides. The slides were allowed to dry for at least 2 h. Thereafter, they were submerged in pure methanol for 3 min and stained for 15-20 min in 3% Giemsa solution. Slides were rinsed twice in water and once in acetone and were then kept in xylene for about 30 min. The slides were allowed to dry completely and covered. At least two slides were generated per culture. All solutions used during this preparation were freshly prepared each time. The Giemsa solution was filtered before usage.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): The mitotic index was determined by counting a total of 1000 cells per culture. Duplicate cultures were processed and examined. Coded slides were evaluated using a light microscope at a magnification of about 1000. Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were examined.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
1. Gap:
A gap is an achromatic lesion within a chromatid arm without obvious dislocation of the chromatid end(s) and smaller than the width of one chromatid. Gaps are found on one chromatid ("gap") or on both chromatids at apparently identical sites ("iso-gap"). The biological relevance of gaps of both types is unclear.
2. Break:
A break is defined as a discontinuity of one chromatid ("break") or both chromatids, at apparently the same locus ("isobreak"), with dislocation of the chromatid ends. The dislocated chromatid end(s) has (have) to be present within the respective metaphase. In addition, an achromatic lesion within a chromatid arm without obvious dislocation of the chromatid end(s) but larger than the width of one chromatid is also defined as break or as isobreak, if this occurs in parallel on both chromatids of a chromosome.
3. Fragment:
Fragments are parts of chromosomes without centromer. A fragment is the result of a break. The corresponding defective chromosome is not detectable among the chromosomes of the same metaphase. Fragments can be derived from one chromatid ("fragment") or from both corresponding chromatid regions of a chromosome ("isofragment").
4. Deletion:
A deletion occurs as the result of a break. In case of a deletion, one chromatid ("deletion") or both corresponding terminal chromatid parts of a chromosome ("isodeletion") are missing within the metaphase under assessment.
5. Exchange:
This is an exchange of chromatid-parts between different chromosomes (interchange) or within the same chromosome (intrachange).
6. Multiple aberration:
A cell was assessed as to contain "multiple aberrations" when five or more structural changes (excluding gaps) occur within one metaphase.

- Determination of polyploidy: Additionally observed polyploid metaphases were recorded. The results are shown but were not used for assessment.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Evaluation criteria:

Coded slides were evaluated using a light microscope at a magnification of about 630. The mitotic index was determined by counting 1000 cells per culture. The numbers of mitotic and non-mitotic cells were noted. Duplicate cultures were processed and examined. Coded slides were evaluated using a light microscope at a magnification of about 1000. Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were examined. In most cases at least 100 assessable metaphases were present on one slide prepared from an individual culture. Therefore, the back-up slide which was generated routinely from every culture was normally not utilized for the evaluation. However, in cases when fewer than 100 assessable metaphases were found on the first slide of a culture, the backup slides were evaluated as well until a total of 100 metaphases was reached. Only metaphases containing the modal chromosome number (22) were analyzed unless exchanges were detected. In this case, metaphases were evaluated even if the chromosome number was not equal to 22. The classes of structural chromosome damage were defined and recorded by using essentially the terminology of Rieger and Michaelis (1967). Both chromatid and chromosome-type aberrations were assessed. Chromatid-type aberrations are clastogenic effects restricted to one of the two corresponding chromatids. Chromosometype aberrations are defined as changes expressed in both corresponding sister chromatids at the same locus. The distinction between chromatid and chromosome type aberrations was not made for exchanges.

Statistics:

The statistical analysis was performed by pair-wise comparison of HEC 5725-DCF-Pyrimidine-treated and positive control groups to the respective solvent control group. The mitotic index was statistically analyzed (provided that it was reduced compared to the mean of the respective solvent control) using the one-sided chi²-test. The numbers of metaphases with aberrations (including and excluding gaps) and of metaphases with exchanges were compared (provided that these data superceded the respective solvent control). The statistical analysis followed the recommendations outlined by Richardson et al. (1989). The one-sided chi²-test was used for the statistical evaluation. A difference was considered to be significant if the probability of error was below 5%.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

In the absence of S9 mix Chinese hamster V79 cells were exposed to the test item at concentrations of up to and including 2 µg/mL. Substance precipitation occurred not in the medium. In addition, non-attached cells, apoptotic cells and nucleus disintegration were observed at 1.6 µg/mL and above. Alteration in cell morphology was observed at 1.2 µg/mL and above. With S9 mix cells were exposed to concentrations of up to and including 8 µg/mL. Substance precipitation was not noted. In addition, non-attached cells were observed at 8 µg/mL. Alteration in cell morphology was observed at 6 µg/mL and above, apoptotic cells and nucleus disintegration were observed at 7 µg/mL and above.

Mitotic Index
Mitotic index without S9 mix:
In comparison to the solvent control, the mitotic indices in the treated cultures were relevantly reduced at 1.2 µg/mL and above. The cultures treated with mitomycin C showed no reduction in mitosis rate.
Mitotic index with S9 mix:
In comparison to the solvent control, the treated cultures showed a relevant reduction of the mitosis rate at 7 µg/mL and above. The positive control cyclophosphamide also reduced the mitosis rate.

Survival Index
Survival index without S9 mix:
In comparison to the solvent control, the survival indices in the treated cultures were relevantly reduced at 0.8 µg/mL and above. The cultures treated with mitomycin C showed also a reduction in survival rate.

Survival index with S9 mix:
In comparison to the solvent control, the treated cultures showed a relevant reduction of the survival rate at 6 µg/mL and above. The positive control cyclophosphamide also reduced the survival rate.

Chromosome Aberrations
Numbers of aberrations (listed by class) and numbers of metaphases with aberrations including and excluding gaps and numbers of metaphases with exchanges for individual cultures are listed (in percent).

Test item without S9 mix:
Biologically relevant and statistically significant increases of metaphases with aberrations were detected after total culture times of 18 and 30 h.
The treatment with the positive control mitomycin C resulted in a clear and statistically significant increase of metaphases with aberrations and demonstrated the sensitivity of the test system.

Test item with S9 mix:
Biologically relevant and statistically significant increases of metaphases with aberrations were detected after total culture times of 18 and 30 h.
The positive control cyclophosphamide induced statistically significant and biologically relevant increases of metaphases with aberrations and demonstrated the sensitivity of the test system and the activity of the used S9 mix.

Applicant's summary and conclusion

Conclusions:

After treatment of Chinese hamster V79 cells with HEC 5725-DCF-Pyrimidine concentrations of 0.8, 1.2 and 1.6 µg/mL were used without S9 mix for assessment of the clastogenic potential of 4,6-dichlor-5-fluorpyrimidine. With S9 mix concentrations of 3, 6 and 7 µg/mL were employed. Clear cytotoxic effects were observed. Cultures treated with the test item in the absence and in the presence of S9 mix showed statistically significant and biologically relevant increases of numbers of metaphases with aberrations, starting at 1.2 µg/mL without S9 mix and at 3 µg/mL with S9 mix.
The positive controls mitomycin C and cyclophosphamide induced clear clastogenic effects and demonstrated the sensitivity of the test system and in the case of cyclophosphamide the activity of the used S9 mix. Based on the results of this test, the test item is considered to be clastogenic for mammalian cells in vitro.

Executive summary:

In a mammalian cell cytogenetics assay (Chromosome aberration) according to OECD test guideline 473 (1997), V79 cell cultures were exposed to 4,6-dichlor-5-fluorpyrimidine, (100 % a.i.), in DMSO at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2, 1.5, 3, 6, 7, 8 µg/mL for the 4h treatment and 18 h harvest time and 0, 1.2, 1.6, 2, 6, 7, 8 µg/mL for the 4h treatment and 30 h harvest time with and without metabolic activation [S9-liver mix].

4,6-dichlor-5-fluorpyrimidine was tested up to cytotoxic concentrations. Clear cytotoxic effects were observed. Cultures treated with the test item in the absence and in the presence of S9 mix showed statistically significant and biologically relevant increases of numbers of metaphases with aberrations, starting at 1.2 µg/mL without S9 mix and at 3 µg/mL with S9 mix. Positive controls induced the appropriate response. There was evidence of Chromosome aberrations induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.