6-ethyl-5-fluoro-4(3H)-pyrimidone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in vitro test are available.

Ames test:

The mutagenic potential of test item was assessed by exposed to histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100), and diluted in water which was also used as a negative control based on the method as described in OECD 471.

It is concluded that, when tested in water, the test item shows no evidence of mutagenic activity in this bacterial system.

In vitro cychromosome aberration stuty:

A study was performed to assess the ability of the test item to induce chromosomal aberrations in human lymphocytes cultured in vitro as described in OECD 473.

It is concluded that the test item has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

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:

From 1998-10-21 to 1999-01-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch No.: 960509
Purity: 92.3%

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

For lymphocytes:
Human blood was collected aseptically from healthy male donors, pooled and diluted with RPMI 1640 tissue culture medium (Sigma) supplemented with 10% foetal calf serum (Gibco), 20 I.U./mL penicillin/20 μg/mL streptomycin (Imperial) and 2mM glutamine (Imperial). Aliquots (0.4 mL blood : 4.5 mL medium : 0.1 mL phytohaemagglutinin (Life Technologies)) of the cell suspension were placed in sterile universal containers and incubated at 37°C for approximately 48 hours. The cultures were gently shaken once daily to resuspend the cells.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 fraction was prepared from a group of ca. 20 animals (Aroclor 1254-stimulated liver).
- method of preparation of S9 mix: S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter-sterilised before use.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL of S9 mix

Test concentrations with justification for top dose:

- Test concentations: First test: 11.7, 23.4, 46.9, 93.8, 187.5, 375, 750 and 1500 μg/mL with/without S9 mix; Second test: 187.5, 375, 750 and 1500 μg/mL without S9 mix, 375, 750 and 1500 μg/mL with S9 mix
- Justification for top dose: Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system. In this case, the highest final concentration used for subsequent testing was 1500 μg/mL as this gave a final concentration of approximately 10 mM.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle:
The test item was found to be soluble in DMSO at 522.4 mg/mL in the solubility test. On dosing at 1% v/v into aqueous tissue culture medium, to give final concentration of 5224 μg/mL, no precipitate was observed.

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 : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: ca. 48h
- Exposure duration/duration of treatment: First test: 3 h, second test: without S9 mix: 21 h, with S9 mix: 3 h
- Harvest time: 21 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Harvesting and fixation:
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid (Sigma) to each culture at a final concentration of 0.1 μg/mL. After 2 hours incubation, each cell suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with 4 mL of a hypotonic solution (0.075 M KCl prewarmed at 37°C). After a 10 minute period of hypotonic incubation at 37°C, 4 mL of ice-cold fresh fixative (3 parts methanol : 1 part glacial acetic acid) was added with gentle agitation. The cultures were then centrifuged at 500 g for 5 minutes, the supernatant removed, and the cell pellets resuspended in 4 mL fixative. The fixative was replaced further times until it became colourless.
- Slide preparation
The pellets were resuspended, then centrifuged at 500 g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides which were then allowed to air-dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 7.2). After rinsing in buffered water the slides were left to air-dry and then mounted in DPX.

- Microscopic examination:
The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures. From these results the dose level causing a decrease in mitotic index of at least 50% of the solvent control value or, if there was no decrease, the maximum achievable concentration was used as the highest dose level for the metaphase analysis. The intermediate and low dose levels were also selected.
The concentration of each positive control compound selected for analysis was the lowest concentration dosed unless a preliminary scan of metaphase figures indicated an insufficient level of aberrant cells.
The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x1000 using an oil immersion objective. One hundred metaphase figures were examined, where possible, from each culture. Chromosome aberrations were scored according to the classification of the ISCN (1985). Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. The vernier readings of all aberrant metaphase figures were recorded.
The incidence of polyploid metaphase cells, out of 500 metaphase cells, was determined quantitatively for cultures used in the analysis for chromosomal aberrations.
The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973).


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

Evaluation criteria:

The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (P <0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity.
Evidence of a dose-relationship is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxicity was observed at 1500 μg/mL without S9 mix in the second test. No toxicity were observed in other test conditions.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

FIRST TEST
- Toxicity data:
In both the absence and presence of S9 mix, the test item did not cause any reduction in the mitotic index compared to the solvent control value.
The quantitative analysis for polyploidy showed no statistically significant increase in the number of polyploid metaphase figures when compared to the solvent control.
- Metaphase analysis:
In both the absence and the presence of S9 mix, the test item caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent control.
Both positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

SECOND TEST
- Toxicity data:
In the absence of S9 mix, the test item caused a reduction in the mitotic index to 65% of the solvent control value at the highest dose level, 1500 μg/mL. In the presence of S9 mix, the test item did not cause any reduction in the mitotic index compared to the solvent control value.
The quantitative analysis for polyploidy showed no statistically significant increase in the number of polyploid metaphase cells when compared to the solvent control.
- Metaphase analysis:
In both the absence and the presence of S9 mix, the test item caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent control.
Both positive control compounds, mitomycin C and cyclophospharnide, caused large, statistically significant increases (P<0.001) in the proportion of aberrant cells.

Conclusions:

It is concluded that the test item has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Executive summary:

A study was performed to assess the ability of the test item to induce chromosomal aberrations in human lymphocytes cultured in vitro as described in OECD 473.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

In order to assess the toxicity of the test item to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis:

First test: 375, 750 and 1500 μg/mL with/without S9 mix -3 hours treatment, 18 hours recovery:

Second test: 187.5, 750 and 1500 μg/mL Without S9 mix -21 hours continuous treatment; 375, 750 and 1500 μg/mL With S9 mix -3 hours treatment, 18 hours recovery.

In both the absence and presence of S9 mix, the test item caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control, in either test.

A quantitative analysis for polyploidy was carried out in all tests. No statistically significant increases in the proportion of polyploid cells were seen.

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It is concluded that the test item has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1996-06-07 to 1996-07-08

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:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: Chichibu Lot 950704
Purity: 95.7%

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Liver preparation from Aroclor 1254-induced rats
- method of preparation of S9 mix: S-9 fraction (10% v/v), MgCl2 (8 mM), KC1 (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter-sterilised before use.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S-9 mix

Test concentrations with justification for top dose:

5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

- Justification for choice of solvent/vehicle: At 50 mg/mL, the test item was soluble in water, therefore water was the chosen solvent for use in the subsequent tests.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST
Four concentrations of test substance were assessed for toxicity using the four tester strains. The highest concentration was 50 mg/mL of test substance in the chosen solvent, which provided a final concentration of 5000 μg /plate. Three 10-fold serial dilutions of the highest concentration were also tested. The chosen solvent, water, was used as the negative control.

MUTATION TEST PROCEDURE
The test substance was added to cultures of the four tester strains at five concentrations separated by c. half-log10 intervals. The highest concentration used was 5000 μg/plate. The negative control was the chosen solvent, water. The positive control compounds were also included.
An aliquot of 0.1 mL of a 10 hour bacterial culture and 0.5 mL S-9 mix or 0.5 mL 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 mL of the tea solution was added, followed immediately by 2 mL of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 mL minimal agar. Three petri dishes were used for each dose level. A set of plates were also prepared containing only bacterial culture and S-9 mix or phosphate buffer (0 μg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period revertant colonies were counted using a Seescan Automatic Colony Counter.
At a later date the main test was repeated using the procedures described above with the same concentrations of test substance.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: detected by substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn

Statistics:

The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S9 fraction in the S9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon (1989) and will usually be analysis of variance followed by Dunnett's test.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item was not toxic towards the tester strains. Therefore 5000 pg/plate was chosen as the top dose level in the mutation tests.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations

Conclusions:

When tested in water, the test item shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

The mutagenic potential of test item was assessed by exposed to histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100), and diluted in water which was also used as a negative control based on the method as described in OECD 471.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary toxicity test with dose levels of up to 5000 μg/plate no toxicity was observed. A top dose level of 5000 μg/plate was chosen for the subsequent mutation study, other dose levels used in the mutation assays were: 1500, 500, 150 and 50 μg/plate.

No evidence of mutagenic activity was seen at any dose level of test item in either mutation test.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, when tested in water, the test item shows no evidence of mutagenic activity in this bacterial system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Data:

Ames study: OECD 471, negative

In Vitro Mammalian Chromosome Aberration Test: OECD 473, negative

 

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the test substance should not be classified as germ cell mutagens.