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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May 2005 to 15 July 2005

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation 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

Method

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The five strains of Salmonella typhimurium were supplied by B.N. Ames' Laboratory (University of California, Berkeley or Oakland Research Institute, USA)

MEDIA USED
- Type and identity of media: The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was placed under agitation in an incubator at 37 °C for about 14 hours, to produce bacterial suspensions
- Properly maintained: Yes. The bacterial strains were stored in liquid nitrogen in cryoprotective medium (1 mL nutrient broth and 0.09 mL dimethylsulfoxide).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from a liver microsomal fraction of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

- Preliminary toxicity test (TA98, TA100 and TA102): 2, 20, 100, 200, 500 and 1000 µg/plate (with and without metabolic activation)
- Main mutagenicity test (all strains): 62.5, 125, 250, 500 and 1000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Solvent: Water for injections
- Justification for choice of solvent/vehicle: The vehicle was selected according to the results of solubility trials performed before the preliminary toxicity test. The test material was insoluble in dimethylsulfoxide, ethanol, acetone and tetrahydrofuran down to 10 mg/mL. It was poorly soluble in water and the limit of solubility was approximately 5 mg/mL. Before use, the vehicle was degassed by sonication for at least 15 minutes and then saturated with nitrogen gas and kept under nitrogen for at least 15 minutes.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.
- Plate Incorporation Method: Test material solution (0.2 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45 °C). After rapid homogenisation, the mixture was overlaid onto a Petri plate containing minimum medium.
- Pre-Incubation Method: Test material solution (0.2 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37 °C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.

DURATION
- Exposure duration: 48 to 72 hours of incubation at 37 °C

NUMBER OF REPLICATIONS: Testing was performed in triplicate

COLONY COUNTING: Revertants were scored with an automatic counter

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Evaluation criteria:

ACCEPTANCE CRITERIA
The study was considered valid if the following criteria were fully met:
- The number of revertants in the vehicle controls is consistent with the historical data of the testing facility
- The number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

EVALUATION CRITERIA
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. No noteworthy toxicity was noted towards the three strains used, with and without S9 mix. The enhanced lawn observed with the TA 102 strain without S9 mix did not allow the scoring of revertants with this strain.

MUTAGENICITY EXPERIMENTS
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test material was poorly soluble in the vehicle and non-toxic in the preliminary toxicity test, the highest dose-level for the main test was the maximum achievable dose-level, taking into account the limit of solubility in the vehicle and the maximum treatment volume used in the testing laboratory for the selected vehicle.
No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. No toxicity was noted towards all the strains used, both with and without S9 mix.
The test material did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Any other information on results incl. tables

Table 1: Summary of Experiment 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	TA102	TA98	TA1537
-	Solvent 62.5 125 250 500 1000	121 113 112 114 123 100	19 14 13 19 14 10	489 491 493 494 399 228	34 20 28 31 28 24	9 3 9 5 3 2
+	Solvent 62.5 125 250 500 1000	118 102 115 112 101 97	13 7 11 11 14 13	512 540 611 512 542 437	35 32 27 37 20 24	6 11 11 9 4 7
Positive Controls
-	Name	NAN3	NAN3	MMC	2NF	9AA
	Concentration (µg/plate)	1	1	0.5	0.5	50
	Mean no. colonies/plate	621	568	1320	267	1255
+	Name	2AM	2AM	2AM	2AM	2AM
	Concentration (µg/plate)	2	2	10	2	2
	Mean no. colonies/plate	1368	322	1650	1127	163

NAN3 = Sodium Azide

MMC = Mitomycin C

2NF = 2-Nitrofluorene

9AA = 9 -Aminoacridine

2AM = 2 -Anthramine

Table 2: Summary of Experiment 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	TA102	TA98	TA1537
-	Solvent 62.5 125 250 500 1000	117 119 123 137 125 107	18 15 14 15 14 13	301 256 242 228 200 144	31 17 18 14 11 15	5 6 6 7 6 2
+	Solvent 62.5 125 250 500 1000	135 138 140 110 146 133	12 19 13 11 13 14	375 324 377 278 296 325	16 23 28 21 22 27	7 9 7 10 10 4
Positive Controls
-	Name	NAN3	NAN3	MMC	2NF	9AA
	Concentration (µg/plate)	1	1	0.5	0.5	50
	Mean no. colonies/plate	491	497	1774	179	401
+	Name	2AM	2AM	2AM	2AM	2AM
	Concentration (µg/plate)	2	2	10	2	2
	Mean no. colonies/plate	1061	153	1977	1218	147

NAN3 = Sodium Azide

MMC = Mitomycin C

2NF = 2-Nitrofluorene

9AA = 9 -Aminoacridine

2AM = 2 -Anthramine

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was considered to be non-mutagenic.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

Five strains of Salmonella typhimurium were used, TA 1535, TA 1537, TA 98, TA 100 and TA 102. A preliminary toxicity test was performed to define the dose-levels to be used for the mutagenicity study. The test material was then tested in two independent experiments, with and without a metabolic activation system (S9 mix prepared from a liver microsomal fraction of rats induced with Aroclor 1254). Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method.

The test material was poorly soluble in the vehicle (water) and non-toxic in the preliminary toxicity test, therefore the highest dose-level for the main test was the maximum achievable dose-level. The selected dose-levels were 62.5, 125, 250, 500 and 1000 μg/plate. Each strain was exposed to at least five dose-levels of the test material (three plates/dose-level). After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. No toxicity was noted towards all the strains used, both with and without S9 mix. The test material did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Under the conditions of this study, the test material was considered to be non-mutagenic.