Reaction Mass of 6-(1-bromoethyl)-4-chloro-5-fluoropyrimidine and Toluene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June to 9th July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

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:

other: EC Guideline No.92/69 Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”.

Version / remarks:

1992

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:

6-(1-bromoethyl)-4-chloro-5-fluoropyrimidine

Cas Number:

188416-28-6

Molecular formula:

C6H5BrClFN2

IUPAC Name:

6-(1-bromoethyl)-4-chloro-5-fluoropyrimidine

Test material form:

liquid

Details on test material:

Amber Oil

Specific details on test material used for the study:

Identification: UK134,821
Appearance: Clear Yellow Liquid
Batch: 5494/96/1
Purity/Composition: 95.5%
Test item storage: At room temperature in the dark

Method

Target gene:

Histidine and Tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Remarks:

E. coli strain WP 2 uvrA

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

exogenous mammalian metabolic activation system (S9)

Test concentrations with justification for top dose:

Dose-range finding test: 5000 and 1500 µg/plate
First Experiment: Direct Plate Assay with and without: 1500, 500, 150, 50, 15 and 5 µg/plate

Vehicle / solvent:

dimethyl sulfoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

The strains of S. ophimurium were obtained from Profèssor B.N. Ames, University of California, Berkeley, California., USA.
The strain of E. coh was obtained from the National Collections of Industrial and Marine Bacteria. Aberdeen, Scotland.
Batches of the strains were obtained from master stocks held 'in liquid, nitrogen. The test batches were aliquots of nutrient broth cultures and were stored at -80'C. Dimethyl sulphoxide (DMSO) was added to tile cultures at 8% vIv as a cryopreservative. Each batch of frozen strain was tested, where applicable. for cell membrane permeability (rfa mutation), sensitivity to UV light and the pKM101 plasmid which confers resistance to ampicillin. The responses of the strains to a series of diagnostic mutagens were also assessed.
For use in tests an aliquot of frozen culture was added to 25 ml of nutrient broth (Merck No.2) and incubated, with shaking, at 37°C for 10 hours. These cultures provided at least 2 x 10 9 cells per ml which were measured photometrically
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions

Rationale for test conditions:

Rationale: Recommended test system in international guidelines (e.g. OECD, EC).

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for the solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criterie:
(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 tray, it is considered to show evidence of imutagenic activity in this test system. No statistical analysis is performed.
(b) lf treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, in either mutation test, 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), additional testing may be performed in order to resolve the issue of the test substance's mutagenic activity in this test system. Modifications to the experimental method will usually be considered, such as the use of a narrower dose range and different levels of S9 in the mix. Should an increase in revertant colony numbers then be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical anal ysis is performed.
If no clear "positive" response can be obtained, the test dam. may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers.

Statistics:

The statistical procedures used will be those described by Mahon et al (1989) and will usually be analysis of variance followed by Diurnal's test.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only at top dose of 1500ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only at top dose of 1500ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only at top dose of 1500ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only at top dose of 1500ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed in the absence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Selection of an adequate range of doses was based on a dose-range finding test both with and without S9-mix. Eight concentrations,1500 and 5000 µg/plate were tested in triplicate.

HISTORICAL CONTROL DATA
- Positive historical control data: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for WP2uvrA in the absence of S9-mix in the second experiment.
- Negative (solvent/vehicle) historical control data: The negative control values were within the laboratory historical control data ranges.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded tha UK134,821 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in bacterial systems

Executive summary:

In this in vitro assessment of the ininagenie potential of UK-I34.821, histidine dependent auxotrophic mutants of Saimoneila typhimurium (strains TA1535, TA1537, TA98 and TAI 00) and a tryptoplian dependent mutant of Escherichia coh (strain CM891) were exposed to the test substance diluted in dimethyl sulphoxide, which was also used as a negative control. Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). The first was a standard plate incorporation assay.. the second involved a preincubation stage. Dose levels of up to 5000 and 1500ug/plate were tested in the mutation tests. This is the standard limit dose recommended in the regulatory guidelines this assay follows. Other dose levels used were a series of co half-log of dilutions of the highest concentration. Toxicity was observed towards all the tester strains at 5000 and 1500 ug/plate in the first mutation test so 1500 ug/plate was chosen as the top dose level for the second test. In the second test toxicity was observed towards all the strains at 1500 and 5000 ug/plate. No evidence of mutagenic activity was seen at any dose level of UK-434,821 in either mutation WSE. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that, when tested in dimethyl sulphoxide UK-134,821 shows no evidence of mutagenic activity in this bacterial system