Pyrimidine, 2,4-dichloro-5-fluoro-

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:

4 August 2014 to 5 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

This assay design was based on OECD Guideline 471 (OECD, 1997) and ICH Guidelines S2(R1) (November 2011).

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dichloro-5-fluoropyrimidine

EC Number:

625-810-7

Cas Number:

2927-71-1

Molecular formula:

C4HCl2FN2

IUPAC Name:

2,4-dichloro-5-fluoropyrimidine

Test material form:

solid

Specific details on test material used for the study:

- Source and lot/batch No.of test material: RS7-H81002-035C- Storage condition of test material: Ambient temperature protected from light- Analytical purity: 99.9%

Method

Target gene:

Mutation of the uvrA gene (E. coli) or the uvrB gene (Salmonella)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254-induced rat liver S9 fraction

Test concentrations with justification for top dose:

The test material was evaluated in the mutagenicity assay at doses of 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 micrograms/plate with and without S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dose formulations of the test material and dimethyl sulfoxide were prepared on the day of use- Justification for choice of solvent/vehicle: The volume of Dimetyl Sulfoxide used has been shown not to effect survival or induce mutations in control cultures

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Tester Strain TA98 with S9 activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Tester Strains TA100, TA1535, TA1537 and WP2uvrA with S9 activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Tester Strain TA98 without S9 activitation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Tester Strain TA100 and TA1535 without S9 activitation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Tester Strain TA1537 without S9 activitation

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2uvrA without S9 activitation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation method - the tester strain, test article, and S9 mix (where appropriate) are combined in molten top agar, which is then overlaid onto a minimal bottom agar plate. Following incubation, revertant colonies are counted.DURATION: Plates are incubated for 48 hours at 37oCSELECTION AGENT (mutation assays): Bacterial reverse mutation assayNUMBER OF REPLICATIONS: All test and control articles were evaluated in triplicate platesDETERMINATION OF CYTOTOXICITY: Cytotoxicty is indicated by the presence of "pinpoint" colonies and the absence of background lawn.

Rationale for test conditions:

The bacterial reverse mutation assay has been shown to be a sensitive, rapid, and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes. By using several different tester strains, both base pair substitution and frameshift mutations can be detected. Salmonella and E. coli strains used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his–) or tryptophan (trp–) dependent cells are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan), only those cells which revert to histidine (his+) or tryptophan (trp+) independence are able to form colonies. Trace amounts of histidine or tryptophan added to the media allow all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. his+ or trp+ revertants are readily discernable as colonies against the limited background growth of his– or trp– cells.

Evaluation criteria:

VALID STUDY CRITERIAThe vehicle control (Dimethyl Sulphoxide ) must show a normal range of bacterial colonies for each bacterial strain and should be consistent with historical data.The positive controls Benzo{a}pyrene (BP), 2-aminoanthracene (2AA), 2 -nitrofluorene (2NF), sodium azide (SA), ICR-191 and 4-nitroquinoline-N-oxide must show a mutagenic response and should be consistent with historical data.In the absence of toxicity or precipitation, a maximum treatement concentration of 5000 mcg/plate is sufficiently high to support study validity.POSITIVE RESPONSE CRITERIATest substance is judged to have induced a positive response when a concentration -related increase in revertants is observed in which the number of revertants exceeds the control values by at least 2 -fold (strains TA98, TA100, and WP2uvrA) or at least 3 -fold (strains TA1535 or TA1537) for at least two successive test article concentrations.When the above criteria are met for only one concentration, the determination of a positive response will be made on the basis of scientific judgement relative to the quality of the concentration response finding.NEGATIVE RESPONSE CRITERIAA test article is considered to produce a negative response when the criteria for a positive response is not met and the conditions for a valid assay have been satisfied.

Statistics:

No Statistical analysis was used

Results and discussion

Test results

Test results 1

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:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Test results 3

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:

valid

Positive controls validity:

valid

Test results 4

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:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

These results indicate that LSN2431768 is negative in the Bacterial Reverse Mutation Assay when tested up to toxicity limiting dose levels with and without S9 metabolic activation, and under the conditions of the study protocol used.

Executive summary:

The objective of this study was to evaluate LSN2431768, for its ability to induce reverse mutations at the histidine locus in Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537, and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

LSN2431768 was evaluated in the mutagenicity assay in all five tester strains at dose levels of 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 micrograms/plate with and without S9. LSN2431768 was prepared in dimethylsulfoxide (DMSO) vehicle and formed a transparent, colorless solution.

LSN2431768 treatment-related toxicity was evident at concentrations ≥500 micrograms/plate by reduced numbers of revertant colonies, and by absent or reduced background lawn growth in all strains tested with and without S9. Normal background bacterial lawn growth was observed on all plates at subsequent lower test concentrations indicating there was appropriate bacterial growth during treatment to express a potential mutagenic event. There were no relevant or positive increases in the numbers of revertant colonies observed at any LSN2431768 dose level with any tester strain with or without S9.

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

These results indicate that LSN2431768 is negative in the Bacterial Reverse Mutation Assay when tested up to 5000 micrograms/plate under the conditions of this protocol.