4,6-Dichloro-5-fluoropyrimidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

studay according to OECD test guideline 471; result: negative (2001)

study according to OECD test guideline 471; result: negative (2003)

study according to OECD test guideline 473; result: positive

Link to relevant study records

Reference

Reference 1

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

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

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.

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%.

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-05-17 to 2001-08-22

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:

21 July 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his locus

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

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: It was made from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice. The animals were prepared unfasted, following the directions of Ames et al. (1975) and Maron and Ames (1983).
- method of preparation of S9 mix: The rats were terminated. Livers were removed under sterile conditions immediately after sacrifice and kept at 4°C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4°C. The livers were washed with cold (4°C), 0.15 M KCl solution (approximately 1 ml KCl per 1 g liver), and then homogenized in fresh, cold (4°C), 0.15 M KCl (approximately 3 ml KCl per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4°C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80°C in small portions. These portions were slowly thawed before use. The S9 mix was freshly prepared (Ames et al., 1973a) and used only on the same day. It was placed in a vessel with a double glass wall until used.
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 mix comprised the amount of S9 fraction (10%) indicated in the tables, 70% cofactor solution and (30-x)% 0.15 M KCl.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF according to information given by the internal sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

other: Nitrofuratoin ; 4-Nitro-1,2-phenylene , 2-Aminoanthracene

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E-06 dilution
- Test substance added in agar (plate incorporation); preincubation
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Rationale for test conditions:

according to OECD test guideline 471

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

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

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

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

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

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

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

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

Summary of the Results with HEC 5725-DCF-Pyrimidine

S9 mix	TA 1535	TA 100	TA 1537	TA 98	TA 102
without	negative	negative	negative	negative	negative
with	negative	negative	negative	negative	negative

Conclusions:

The Ames-test screening, employing doses of up to 5000 µg per plate, showed HEC 5725-DCF-Pyrimidine to produce bacteriotoxic effects, starting at 20 µg per plate. Therefore, 500 µg per plate and above could not be used for assessment. Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility), revealed no biologically relevant variations from the respective negative controls.
In spite of the low doses used, positive controls increased the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity. Despite this sensitivity, no indications of mutagenic effects of HEC 5725-DCF-Pyrimidine could be found at assessable doses of up to 160 µg per plate in any of the Salmonella typhimurium strains used.
Due to these screening-results HEC 5725-DCF-Pyrimidine has to be regarded as non-mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD test guideline 471, strains TA 1535, TA 100, TA 1537, TA 98     TA 102 of S. typhimurium were exposed to 4,6-dichlor-5-fluorpyrimidine (100 % a.i.), in DMSO at concentrations of 10, 16, 50, 160, 500, 1600, 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation and pre-incubation method.

Significant bacteriotoxic effects were seen from 160 µg/plate onwards, thus, mutagenicity was determined between 10 and 160 µg/plate.

4,6-dichlor-5-fluorpyrimidine was tested up to cytotoxic concentrations, there was no increase of the number of revertants as compared to the respective control. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. The substance is not considered to be a mutagen and therefore does not need to be classified as germcell mutagen.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-01-9 to 2003-06-10

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:

21 July 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his locus

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

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: S9 mix was used to simulate the mammalian metabolism of the test substance. It was made from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice. The animals were prepared unfasted, following the directions of Ames et al. (1975) and Maron and Ames (1983).
- concentration or volume of S9 mix and S9 in the final culture medium: S9 mix consists of this cofactor solution, S9 fraction and, if needed, 0.15 M KCI. The
amount of S9 fraction in S9 mix is indicated in the tables in percent. The S9 mix comprised the amount of S9 fraction (10%) indicated in the tables, 70% cofactor solution and (30-x)% 0.15 M KCI. The S9 fraction was derived from the preparation dated May 13, 2002 (protein content 23.8 mg per mL).

Test concentrations with justification for top dose:

5000, 1581, 500, 158, 50, 16, 0 µg/plate first experiment
256, 128, 64, 32, 16, 8, 0 µg/plate second experiment
128, 64, 32, 16, 8, 4, 2, 0 µg/plate second experiment (preincubation)

Vehicle / solvent:

DMSO
The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF according to information given by the internal sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: Nitrofurantoin, 4-Nitro-1,2-phenylene diamine, 2-Aminoanthracene

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1x E-06 dilution
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition, other:
- Any supplementary information relevant to cytotoxicity: Secondly, a toxic effect of the substance
was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

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

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

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

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

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

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

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

Summary of Mean Values Without  S9 Mix
Table and Group	TA 1535	TA 100	TA 1537	TA 98	TA 102
1-5
µg/plate
0	11	165	8	17	175
16	8	141	4	24	138
50	6	136	3	22	66
158	5	105	-	18*	0
500	0	0	0	0	0
1581	0	0	0	0	0
5000	0	0	0	0	0
Na-azide	827
NF		369
4-NPDA			92	204
MMC					522
6-10
µg/plate
0	18	102	6	24	214
8	17	100	6	23	174
16	20	89	4	20	128
32	19	96	4	18	111
64	20	84	2	24	60
128	13	79	-	23	0
256	4	46	-	15	0
Na-azide	765
NF		284
4-NPDA			96	155
Cumene					514
11-15
µg/Tube
0	26	142	6	36	252
2			6		222
4	29	146	6	37	238
8	21	147	5	38	208
16	15	144	7	42	180
32	12	129	5	42	155
64	15	126	5	38	117
128	4	102		25
Na-azide	704
NF		458
4-NPDA			130	181
Cumene					452
Summary of Mean Values With S9 Mix
1-5
µg/plate
0	6	168	9	34	274
16	7	166	6	28	286
50	8	156	3	25	179
158	5	139	-	28	29
500	2	0	-	0	0
1581	0	0	0	0	0
5000	0	0	0	0	0
2-AA	167	1499	269	1401	601
6-10
µg/plate
0	10	109	8	34	275
8	7	109	4	34	281
16	8	98	8	37	241
32	7	87	5	32	202
64	9	81	2	29	140
128	9	71	-	30	9
256	4	58	-	22	0
2-AA	235	1440	296	1334	557
11-15
µg/Tube
0	27	159	10	54	352
2			10		358
4	25	135	9	52	328
8	22	140	11	51	307
16	20	158	7	51	299
32	20	131	10	38	289
64	21	135	5	43	232
128	12	121		36
2-AA	198	1445	423	1120	556

Conclusions:

The Salmonella/microsome test, employing doses of up to 5000 µg per plate, showed 4,6-dichloro-5-fluorpyrimidine to produce bacteriotoxic effects, starting at 32 µg per plate. Therefore, 256 µg per plate and above could not be used for assessment. Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility), revealed no biologically relevant variations from the respective negative controls.
In spite of the low doses used, positive controls increased the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity. Despite this sensitivity, no indications of mutagenic effects of the test item could be found at assessable doses of up to 158 µg per plate in any of the Salmonella typhimurium strains used. Due to these results the test item has to be regarded as non-mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD test guideline 471, strains TA 1535, TA 100, TA 1537, TA 98     TA 102 of S. typhimurium were exposed to 4,6-dichlor-5-fluorpyrimidine (100 % a.i.), in DMSO at concentrations of 5000, 1581, 500, 158, 50, 16, 0 µg/plate first experiment, 256, 128, 64, 32, 16, 8, 0 µg/plate second experiment, 128, 64, 32, 16, 8, 4, 2, 0 µg/plate second experiment (preincubation) in the presence and absence of mammalian metabolic activation using the plate incorporation and pre-incubation method.

Significant bacteriotoxic effects were seen from 32 µg/plate onwards, thus, mutagenicity was determined between 2 and 256 µg/plate.

4,6-dichlor-5-fluorpyrimidine was tested up to cytotoxic concentrations, there was no increase of the number of revertants as compared to the respective control. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. The substance is not considered to be a mutagen and therefore does not need to be classified as germcell mutagen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro:

OECD 471:

In a reverse gene mutation assay in bacteria according to OECD test guideline 471, strains TA 1535, TA 100, TA 1537, TA 98, TA 102 of S. typhimurium were exposed to 4,6-dichlor-5-fluorpyrimidine (100 % a.i.), in DMSO at concentrations of 10, 16, 50, 160, 500, 1600, 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation and pre-incubation method.

Significant bacteriotoxic effects were seen from 160 µg/plate onwards, thus, mutagenicity was determined between 10 and 160 µg/plate.

4,6-dichlor-5-fluorpyrimidine was tested up to cytotoxic concentrations, there was no increase of the number of revertants as compared to the respective control. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. The substance is not considered to be a mutagen and therefore does not need to be classified as germcell mutagen.

 

In a reverse gene mutation assay in bacteria according to OECD test guideline 471, strains TA 1535, TA 100, TA 1537, TA 98, TA 102 of S. typhimurium were exposed to 4,6-dichlor-5-fluorpyrimidine (100 % a.i.), in DMSO at concentrations of 5000, 1581, 500, 158, 50, 16, 0 µg/plate first experiment, 256, 128, 64, 32, 16, 8, 0 µg/plate second experiment, 128, 64, 32, 16, 8, 4, 2, 0 µg/plate second experiment (preincubation) in the presence and absence of mammalian metabolic activation using the plate incorporation and pre-incubation method.

Significant bacteriotoxic effects were seen from 32 µg/plate onwards, thus, mutagenicity was determined between 2 and 256 µg/plate.

4,6-dichlor-5-fluorpyrimidine was tested up to cytotoxic concentrations, there was no increase of the number of revertants as compared to the respective control. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. The substance is not considered to be a mutagen and therefore does not need to be classified as germcell mutagen.

 

OECD 473:

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.

Justification for classification or non-classification

Based on the available information no classification can be made. The genetic toxicity test in bacteria showed no mutagenic effect whereas in the cytogenetic assay performed according to OECD test guideline 473 a positive result was obtained. Further tests are necessary to provide a reliable basis for classification.