2-(4-bromophenyl)-2-methylpropionic acid methyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed in accordance with validated guidelines and in GLP compilance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-bromophenyl)-2-methylpropionic acid methyl ester

EC Number:

450-630-9

EC Name:

2-(4-bromophenyl)-2-methylpropionic acid methyl ester

Cas Number:

154825-97-5

Molecular formula:

C11H13BrO2

IUPAC Name:

methyl 2-(4-bromophenyl)-2-methylpropanoate

Test material form:

other: liquid

Details on test material:

The test material is a liquid with a degree of purity >99%.

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cells in vitro were widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells were chosen because of their relatively small number of chromosomes (diploid number, 2n = 22) and because of the high proliferation rate and a high plating efficiency of untreated cells (normally more than 50%). These facts were necessary for the appropriate perfomance of the study.
The V79 cells are stored over liquid nitrogen (vapour phase) in the cell bank, as large stock cultures allowing the repeated use of the same cell culture batch in experiments.
Routine checking of mycoplasma infections was carried out before freezing. For the experiment thawed cultures were set up in 80 cm2 plastic flasks at 37°C in a 5.0% carbon dioxide atmosphere. 5 x 105 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FCS (foetal calf serum) and subcultures were made every 3-4 days.

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

Duplicate cultures are treated at each concentration. The selection of the concentrations used in experiment I and II based on data from the pre-experiment. The following concentrations were used in the main experiments:

Experiment I:
without metabolic activation: 31.3, 62.5, 125, 150, 175, 200, 250 and 300 µg/mL
with metabolic activation: 62.5, 125, 150, 200, 225, 250, 275 and 300 µg/mL

Experiment II:
without metabolic activation: 6.25, 12.5, 25, 50, 100 and 200 µg/mL
with metabolic activation: 50, 100, 200, 300, 400 and 500 µg/mL

Vehicle / solvent:

The test item FEXO-03 could not be dissolved neither in DMSO nor in ethanol at the concentrations used. The test item was suspended in cell culture medium. Precipitation of the test item was noted at a concentration of 1000 µg/mL and higher. 5000 µg/mL was chosen as highest dose group of the test item for the pre-experiment and tested as suspension.

Controls

Untreated negative controls:

yes

Remarks:

Solvent controls, consisting of solvent or vehicle alone and treated in the same way as the treatment groups were included.

Negative solvent / vehicle controls:

yes

Remarks:

vehicle controls

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Both positive controls are prepared on the day of experiment.

Details on test system and experimental conditions:

The Cells
V79 cells in vitro were widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells were chosen because of their relatively small number of chromosomes (diploid number, 2n = 22) and because of the high proliferation rate and a high plating efficiency of untreated cells (normally more than 50%).
The V79 cells are stored over liquid nitrogen (vapour phase) in the cell bank, as large stock cultures allowing the repeated use of the same cell cultures batch in experiments.
Routine checking of mycoplasma infections was carried out before freezing. For the experiment thawed cultures were set up in 80 cm2 plastic flasks at 37 °C in a 5.0% carbon dioxide atmosphere (95.0% air). 5 x 10exp5 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FCS (foetal calf serum) and subcultures were made every 3-4 days.

Mammalian Microsomal Fraction S9 Mix
Male Wistar rats were induced with Phenobarbital (80 mg/kg bw) and ß-Naphtoflavone (100 mg/kg bw).
The following quality control determinations were performed:
a) Biological activity in the Salmonella typhimurium assay
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in ampoules of 2 and 5 mL and stored at = -75 °C.
The protein concentration in the S9 preparation was 31 mg/mL.

S9 Mix
An appropriate quantity of the S9 supematant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2; 33 mM KCl; 5 mM Glucose-6-phosphate; 4 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.

Experimental Performance
Seeding of the Cultures
Three or four days old stock cultures with higher than 50% confluency were trypsinised at 37 °C for 5 min by adding a trypsine solution in Ca-Mg-free PBS. The enzymatic treatment was stopped with complete culture medium. A single cell suspension was prepared. The trypsine concentration for all subculturing steps was 0.2%. The cells were rinsed with Ca-Mg-free PBS prior to the trypsine treatment. The cells were seeded into Quadriperm dishes which contain microscopic slides (at least 2 chambers per dish and test group). Into each chamber 1 x 10exp04 - 5 x 10exp04 cells were seeded with regard to preparation interval. The medium was minimum essential medium supplemented with 10% FCS.

Treatment
Experiment I: Short time exposure
Two days after seeding of the cells, the culture medium was replaced with serum-free medium containing the test item and 50 µL/mL S9 mix (with metabolic activation). Additional negative and positive controls were performed with and without metabolic activation. 4 h after treatment the cultures were washed twice with PBS and cultured in complete medium for the remaining culture time.
There is no requirement for verification of a clear positive response (OECD guideline 473).

Experiment II: Short time exposure (with metabolic activation), long time exposure (without metabolic activation)
The treatment with metabolic activation was performed as described above for experiment I. In the experiment without metabolic activation, two days after seeding the cells were incubated with the test item in complete medium (MEM with 10% FCS) for 20 h. The cells were prepared at the end of the incubation. Additional negative and positive controls were tested.
All cultures are incubated at 37 °C in a humidified atmosphere with 5.0% CO2 (95.0% air).

Preparation of the Cultures
17.5 h (4 h and 20 h treatment) and 25.5 h (28 h) after the start of the treatment Colcemid® was added to the cultures (0.2 µg/mL culture medium). 2.5 h later, the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3+l methanol + glacial acetic acid. All the steps were carried out on precision hot plates. After fixation the cells were stained with Giemsa.

Evaluation criteria:

Analysis of Metaphase Cells
All slides, including those of positive and negative controls were independently coded before microscopic analysis. Evaluation of the cultures was performed using microscopes with 100x oil immersion objectives. As structural chromosomal aberrations breaks, fragments, deletions, exchanges and chromosomal disintegration were recorded. Gaps were recorded as well but not included in the calculation of the aberration rates. The definition of a gap is as follows: an achromatic region (occurring in one or both chromatids) independent of its width. The remaining visible chromosome regions should not be dislocated either longitudinally or laterally. At least 200 well spread metaphases per concentration and negative/positive controls were scored for cytogenetic damage.
The cells scored contained 22 ± 1 centromeres. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined by counting the number of mitotic cells in 1000 cells. Additionally the number of polyploid cells was scored. Polyploid means a near tetraploid karyotype in the case of this aneuploid cell line.

Analysis of Relative Cell Density
As an additional parameter for cytotoxic effects of the test item the relative cell density was calculated as the mean of twenty cell counts per test group (cells within the visual field at a 400-fold magnification).

Acceptability of the Assay
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
- the number of aberration found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0%-4.5%,
- the positive control substance should produce biologically relevant increases in the number of cells with structural chromosome aberrations.

Statistics:

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data, both biological and though evaluated statistical significance should be considered together.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

c.a. 250 µg/mL

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

c.a. 250 µg/mL

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Test results 3

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

c.a. 100 µg/mL

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Test results 4

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitate of the test item was noted at 400 µg/mL and higher

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

The following concentrations were evaluated for microscope analysis:
Experiment I:
without metabolic activation: 175, 200 and 250 µg/mL
with metabolic activation: 250, 275 and 300 µg/mL
Experiment II:
without metabolic activation: 25, 50 and 100 µg/mL
with metabolic activation: 300, 400 and 500 µg/mL

Precipitation:
The test item was suspended in culture medium. In experiment I, no precipitate of the test item was noted with and without metabolic activation up to the highest dose group evaluated. In experiment II, precipitate of the test item was noted with metabolic activation at 400 µg/mL and higher but not without metabolic activation up to the highest dose group evaluated.

Toxicity:
In experiment I without metabolic activation, a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at 250 µg/mL (11%). The cell density was also decreased below 70% at 250 µg/mL (49%). Both biologically relevant decreases indicated a cytotoxic effect of the test item at 250 µg/mL. No toxic effect was noted with metabolic activation up to the highest dose evaluated.
In experiment II without metabolic activation, a biologically relevant decrease of the relative mito se index ( decrease below 70% rel. mitose index) was noted at 100 µg/mL (20%). No biologically relevant decrease of the cell density was noted. This result stated a cytostatic effect of the test item at 100 µg/mL. No toxic effect was noted with metabolic activation up to the highest dose evaluated.

Clastogenicity:
In experiment I without metabolic activation the aberration rate of the negative control (2.0%) was within the historical control data of the negative control (0.0% - 4.0%, table 11). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 3.5% (175 µg/mL), 1.5% (200 µg/mL) and 1.3% (250 µg/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.
In experiment I with metabolic activation the munber of aberrant cells noted for the negative control (1.5%) was within the historical control data of the negative control (0.0% - 4.5%). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 2.0% (250 µg/mL), 2.0% (275 µg/mL) and 0.0% (300 µg/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.
In expmiment II without metabolic activation the aberration rate of the negative control (2.5%) was within the historical control data of the negative control (0.0% - 4.0%). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 1.5% (25 µg/mL), 0.0% (50 µg/mL) and 1.5% (100 µg/mL). The number of aberrant cells found in the groups treated with the test item did not show a
biologically relevant increase as compared to the corresponding negative control.
In expe1iment II with metabolic activation the number of aberrant cells noted for the negative control (3.0%) was within the historical control data of the negative control (0.0% - 4.5%). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 3.0% (300 µg/mL), 1.0% (400 µg/mL) and 4.5% (500 µg/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.

Polyploid cells
No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.
EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberration.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

During the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item FEXO-03 did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item FEXO-03 is considered to be non-clastogenic.