Fluometuron

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June to 16 September 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study The experiments were done according to the EU 2000/32/EC Annex 4E-B17 (2000) = OECD 476 (1997) = US EPA OPPTS 870.5300 (1998).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fluometuron

EC Number:

218-500-4

EC Name:

Fluometuron

Cas Number:

2164-17-2

Molecular formula:

C10H11F3N2O

IUPAC Name:

fluometuron

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: fluometuron, 1,1-dimethyl-3-[3-(trifluoromethyl)phenyl]urea
- Physical state: white powder
- Analytical purity: 98.3% (w/w)
- Batch number: batch no.D-7167
- Date of arrival: 9 November 2001
- Storage conditions: refrigated (ca 4°C) in the dark

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y mouse lymphoma cells were obtained from the MRC Cell Mutation Unit, University of Sussex, Brighton.
These cells are heterozygous at the thymidine kinease locus, TK +/- . Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% dimethyl sulphoxide (DMSO). Cultures were used within seven days of recovery from frozen stock. Cell stocks are periodically checked for freedom from mycoplasma contamination.

Additional strain / cell type characteristics:

other: thymidine kinase deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Final concentrations in µg/ml:
Preliminary toxicity test ( µg/ml): 27.3, 54.7, 109.4, 218.8, 437.5, 875, 1750, 3500

Mutation test, absence -S9 mix:
Test 1: 109.4, 218.8, 437.5, 875, 1750, 3500
Test 2: 25, 50, 100, 125, 150, 175, 200, 250

Mutation test, presence +S9 mix:
Test 1: 109.4, 218.8, 437.5, 875, 1750, 3500
Test 2: 109.4, 218.8, 437.5, 875, 1750, 3500

Vehicle / solvent:

solvent: dimethyl sulphoxide (DMSO) (CAS: 67-68-5).
Fluometuron was soluble at approximately 16.75 mg/ml in DMSO, while 335 mg/ml was on the verge of solubility. The final concentration of DMSO added to the cultures was 1% v/v.

Controls

Controls 1

Positive controls:

yes

Remarks:

in the absence of S9 mix

Positive control substance:

methylmethanesulfonate

Remarks:

significantly increased mutant frequency

Controls 2

Positive controls:

yes

Remarks:

in the presence of S9 mix

Positive control substance:

3-methylcholanthrene

Remarks:

significantly increased mutant frequency

Details on test system and experimental conditions:

Two independent mutation tests were carried out in the presence and absence of an exogenous metabolic activation system (S9 mix) derived from rat livers induced with Aroclor 1254.
Initial toxicity was assessed in a cytotoxicity range finding study prior to the start of the mutation test using a two-fold range of concentrations from 27.3 to 3500 µg/ml of fluometuron in dimethylsulphoxide (DMSO). Cells were exposed to the test substance for 3 hours in the presence and absence of S9 mix and for 24 hours in the absence of S9 mix. For 3 hour exposures, a cell suspension of 2.0 x 1 000 000 cells/ml in R10p was incubated for approximately 30 minutes at 37ºC, then dispensed in 3 ml aliquots before adding either 2 ml of R0 or S9 mix as appropriate. For 24 hour exposures, a cell suspension of 0.3 x 1 000 000 cells/ml in R10p was incubated for approximately 30 minutes at 37ºC, then dispensed in 5 ml aliquots. One culture was prepared for each concentration of the test substance for each test condition. Negative (solvent) controls were tested in duplicate for each test condition. The test substance was formulated and serially diluted in the vehicle. Aliquots of 50 µl of test substance dilution (at 100 times the desired final concentration) or solvent were added to each culture prior to incubation for 3 or 24 hours at 37°C with shaking, as appropriate. At the end of the exposure period, the cells were washed once, resuspended in 20 ml R10p and counted. Cultures were diluted and plated at 16 cells/ml (1.6 cells/well) in R20p (an equal mixture of R10p and R30p). One plate was set up per culture and incubated in a humidified incubator at 37C in an atmosphere of 5% CO2 for at least 7 days. The original cell suspensions were transferred to pre-gassed tissue culture flasks, diluted to 2 x 100 000 cells/ml as required and incubated for 48 hours, with the cell density readjusted to 2 x 100 000 cells/ml with R10p after 24 hours. After 48 hours incubation the number of colonies per plate were counted and the Day0 relative survival calculated. This estimate of toxicity was used to determine the concentrations of test substance to be used in the main tests. Ideally the highest concentration of test substance should reduce Day0 relative survival to 10-20%. There was some evidence of toxicity in the preliminary toxicity test and the maximum concentration tested in the first main test was 3500 µg/ml, the formulations being added at 1% final volume in medium.
Two mutation tests were conducted basis of results in the cytotoxicity range finding test above. Concentrations of 25-3500 µg/ml of fluometuron, dissolved in dimethyl sulphoxide (DMSO), were used in the absence and presence of metabolic activation.
Duplicate 6ml aliquots of a suspension of 2.0 x 1 000 000 cells/ml had 4 ml of R0 media or S9 mix added as appropriate. Aliquots of 100 µl of test substance dilution (at 100 times the desired final concentration), solvent or positive control were added, all cultures were incubated, with shaking, for 3 hours at 37ºC. At least four serial dilutions of the test substance were tested. Toxicity was measured in terms of Day0 relative survival. The concentration range in the first main test was 109.4-3500 µg/ml both in the presence and absence of S9 mix.
Following 3 hour exposure, the cells were washed once, resuspended, cell density measured and cells plated at 16 cells/ml to assess Day0 relative survival with two plates prepared for each solvent control culture. The remaining cell suspensions were transferred to pre-gassed tissue culture flasks, diluted to 2 x 100 000 cells/ml as required and incubated for 48 hours to allow for expression of the mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 100 000 cells/ml with R10p media. After 48 hours the cells were assessed for cloning efficiency (Day2) and mutant frequency by plating in 96 well plates. Cloning efficiency was assessed by plating 1.6 cells per well (16 cells/ml). A single plate was prepared for each treatment and positive control culture and two plates were prepared from each solvent control culture. Mutant frequency was assessed by plating 2 x 1000 cells/well in selective medium. Two plates were prepared for each treatment and positive control culture, four plates for each solvent control culture. The negative (solvent control, i.e. DMSO in the treatment medium) and positive controls were used in each test. 10 and 5 µg/ml of methyl methanesulphonate (MMS) and 2.5 µg/ml of 3-methylchloanthrene (MC) were the positive controls in the absence and presence of metabolic activation, respectively. The plates were placed in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air
After the plates had been incubated for at least 7 days for cloning efficiency, or 10-14 days for mutant frequency, the number of empty wells was assessed for each microtitre plate. This figure was used to calculate the cloning efficiency and mutant frequency. If evidence of an increase in mutant frequency was found, the colony size distribution at each dose was analysed
A second test was carried out, with a 3 hour exposure in the presence and a 24 hour exposure in the absence of S9 mix. In the absence of S9 mix, duplicate 10 ml cultures at 0.3 x 1 000 000 cells/ml were treated for 24 hours with 100 µl of test substance, solvent or positive control. Following this, the procedure was the same as in the 3 hour treatment. The concentration range in the second main test was 109.4-3500 µg/ml in the presence of S9 mix and 25-250 µg/ml in the absence of S9 mix.
The stability of the test substance and the stability and homogeneity of the test substance in the solvent were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.

Evaluation criteria:

see attached background material "Evaluation_of_results__mammalian_cell_mutation_assay_90005273"

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity test on fluometuron, three-hour exposure to 27.3-3500 µg/ml in the absence and presence of S9 mix resulted in Day0 relative survivals of 116-38% and 112-25% respectively. A continuous exposure to 27.3-3500 µg/ml for 24 hours in the absence of S9 mix resulted in a reduction in Day0 relative survival to 1% at a concentration of 437.5 µg/ml and there was a complete loss of cells after exposure to higher concentrations. Concentrations used in the main test were based upon these data.
In Test 1, in the absence of S9 Mix, exposure to 109.4-3500 µg/ml fluometuron for 3 hours resulted in Day0 relative survivals of 106-52%. All cultures were expressed for mutant frequency. The mean relative Day2 cloning efficiencies were 115-65%. No significant increases in mutant frequency were observed after exposure to fluometuron. MMS, the positive control, induced significant increases in mutant frequency.

In Test 1, in the presence of S9 mix, exposure to 109.4-3500 µg/ml of fluometuron for 3 hours resulted in Day0 relative survivals of 58-30%. All cultures were expressed for mutant frequency. The mean relative Day2 cloning efficiencies were 140-30%, although for one culture, exposed to 3500 µg/ml, the value relative to the negative control was 21%. A statistically significant increase in mean mutant frequency was observed after exposure to 3500 µg/ml fluometuron where Day0 relative survival was 37% of negative control. MC, the positive control, induced significant increases in mutant frequency.

In Test 2, in the absence of S9 mix, exposure to 25-250 µg/ml of fluometuron for 24 hours resulted in Day0 relative survivals of 114-3%. All cultures exposed were expressed for mutant frequency. The Day2 cloning efficiencies were 99-64% relative to the negative control. No significant increases in mutant frequency were observed after exposure to fluometuron.
MMS, the positive control, induced significant increases in mutant frequency.

In Test 2, in the presence of S9 mix, exposure to 109.4-3500 µg/ml of fluometuron for 3 hours resulted in Day0 relative survivals of 66-37%. All cultures were expressed for mutant frequency.
The Day2 cloning efficiencies were 125-60% relative to the negative control. No significant increases in mutant frequency were observed after exposure to fluometuron. MC, the positive control, induced significant increases in mutant frequency.

The increase in mutant frequency associated with exposure to the highest concentration of fluometuron tested in the presence of S9-mix fell just outside the 95% confidence limit for the historical negative control value, and represented a less than 2-fold increase above concurrent solvent control levels. The effect was confined to one culture, and was not reproduced in main test 2. There were relatively few mutant colonies counted after exposure to this concentration, and a low Day2 cloning efficiency in the duplicate culture with a high mutant frequency. Therefore it is considered to be of no biological significance.

These results did not fulfil the criteria for a positive response.

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 did not demonstrate mutagenic potential

Fluometuron, dissolved in dimethyl sulphoxide, did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

 Fluometuron was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5I78Y cells, from the heterozygous condition at the thymidine kinase locus (TK +/-) to the thymidine kinase deficient genotype (TK -/-). Two independent tests in the absence of exogenous metabolic activation (S9 mix) and two independent tests in the presence of S9 mix were carried out.

 

Toxicity was observed after exposure to fluometuron in all tests in the absence and the presence of S9-mix. In the preliminary toxicity test, 3-hour exposure to 27.3-3500 µg/ml fluometuron in the absence and presence of S9 mix resulted in Day0 relative survival (RS) values of 116-38% and 112-25% respectively. A continuous 24 hour exposure in the absence of S9-mix to 27.3-3500 µg/ml fluometuron reduced Day0 RS to 1% at a concentration of 437.5 µg/ml and there was a complete loss of cells after exposure to higher concentrations. Concentrations used in the main mutagenicity tests were based upon these results. The maximum concentration of fluometuron tested in the main mutagenicity tests was 3500 µg/ml.

In the absence of S9-mix, in main test 1, 3-hour exposure to 109.4-3500 µg/ml fluometuron produced no statistically significant increases in mutant frequency. In main test 2, a 24-hour continuous exposure to 25-250 µg/ml fluometuron in the absence of S9-mix produced no statistically significant increases in mutant frequency. In all tests, methyl methane sulphonate, the positive control substance, significantly increased mutant frequency.

 

In the presence of S9-mix, in main test 1, a statistically significant increase in mutant frequency was associated with a 3-hour exposure to 3500 µg/ml fluometuron, where Day0 relative survival (RS) was 37% of solvent control values. Other concentrations tested (109.4-1750 µg/ml) were not associated with any statistically significant increase in mutant frequency. In main test 2, in the presence of S9-mix, 3-hour exposure to 109.4-3500 µg/ml fluometuron was not associated with statistically significant increases in mutant frequency. In all tests 3-methylcholantherene, the positive control substance, significantly increased mutant frequency.

 

The increase in mutant frequency associated with exposure to the highest concentration of fluometuron tested in the presence of S9-mix fell just outside the 95% confidence limit for the historical negative control value, and represented a less than 2-fold increase above concurrent solvent control levels. The effect was confined to one culture, and was not reproduced in main test 2. There were relatively few mutant colonies counted after exposure to this concentration, and a low Day2 cloning efficiency in the duplicate culture with a high mutant frequency. Therefore it is considered to be of no biological significance.

It was concluded that fluometuron did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.