CMP

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2B. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

435-030-7

EC Name:

-

Cas Number:

26452-81-3

Molecular formula:

C5H5N2OCl

IUPAC Name:

4-chloro-6-methoxypyrimidine

Test material form:

other: solid

Method

Species / strain

Species / strain / cell type:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 and WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone-induced rat liver S9-mix

Test concentrations with justification for top dose:

5000, 2500, 1000, 500, 200, 100 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

all Salmonella strains and E.Coli WP2 with metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

strain E.Coli WP2 uvrA with metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin HCl

Remarks:

Strain: Salmonella TA98 without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Strain: E.Coli WP2 uvrA, without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Strain: E.Coli WP2P, without metabolic activation

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: acridine mutagen ICR 191

Remarks:

Strain: Salmonella TA1537, without metabolic activation

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains: Salmonella TA1535 and TA100, without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar plate incorporation; preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: tests performed in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: not stated

Evaluation criteria:

- Test data from individual experiments are considered valid if: a) the concurrent solvent control data are acceptable; b) the positive control data show unequivocal positive responses;
- Failure of one or more tester strain/S9 combinations does not invalidate the data for the remainder of a concurrent experiment.
- A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met: a) a statistically significant dose-related increase in the mean number of revertant colonies is obtained; b) a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) which is statistically significant, is observed at one or more concentrations.
- A negative result in a (valid) individual experiment is achieved when: a) there is no statistically significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and b) in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.
- For a positive response in an individual experiment to be considered indicative of an unequivocal positive, i.e. mutagenic, result for that strain/S9 combination, then the observed effect(s) must be consistently reproducible.

Statistics:

An assessment of statistical significance was carried out using a one-tailed Student's t-test (Ehrenberg 1984). The corresponding probability for each dose level was derived by computer using the appropriate degrees of freedom.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strains WP2 and WP2 uvrA in both the presence and absence of S9-mix.

Executive summary:

In a GLP compliant bacterial mutagenicity assay (Ames test), performed according to OECD 471, using four Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and two E. Coli strains (WP2 and WP2 uvrA) the test substance was evaluated at concentrations of 100, 200, 500, 1000, 2500, and 5000 µg per plate in the presence and absence of rat liver derived metabolic activation system (S9-mix). In two separate experiments (one plate incorporation test and one pre-incubation test), the test substance did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any of the strains used, either in the presence or absence of S9-mix.The sensitivity of the test system, and the metabolic activity of the S9-mix, were clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances. It is concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA1535, TA1537, TA98 and TA 100 and E.coli strains WP2 and WP2 uvrA in both the presence and absence of S9-mix.

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