Benzeneacetyl chloride, 2,5-dimethyl-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-12-13 to 2007-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

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:

-

EC Number:

470-780-9

EC Name:

-

Cas Number:

55312-97-5

Molecular formula:

C10 H11 Cl O

IUPAC Name:

2-(2,5-dimethylphenyl)acetyl chloride

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9-liver mix
- method of preparation of S9 mix: The S9 fraction was isolated in house from the livers of Aroclor 1254 induced male Sprague Dawley rats. The used S9 fraction was derived from the preparation dated August 22, 2006 (color-code black, protein content 37.4 mg per mL).
- concentration or volume of S9 mix and S9 in the final culture medium: For use, frozen aliquots of the S9 fraction were slowly thawed and mixed with a co-factor solution (4:6). The S9 mix contained 40% S9 fraction and was kept in refrigerator and used on the same day.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to first use, each batch was checked for its metabolizing capacity by using 2 µg/mL cyclophosphamide; appropriate clastogenic activity was demonstrated. In addition, each batch was tested in parallel for possible contamination, possible cytotoxic effects and possible clastogenic effects. Only batches without those effects were used.

Test concentrations with justification for top dose:

0, 5, 25, 50, 100, 200, 425, 850, 1700 µg/mL (4h treatment)
0, 50, 100, 200, 425, 850, 1700 µg/mL (18h treatment)

Vehicle / solvent:

DMSO
The test substance was dissolved in a suitable solvent which was selected based on the solubility in the following solvents in the order given. lf possible, deionized water is used as the solvent. Test substances that are not sufficiently soluble in this solvent are dissolved in DMSO, ethanol or acetone and then added to the medium, if this results in a higher final concentration of the test substance in the medium.
For the test item, DMSO was selected as solvent. In this solvent the test item was soluble up to 170 mg/mL.
Precipitation was observed in the culture medium of the pre-test at 1700 µg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1xE+06 cells/culture
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4h and 18 h
- Harvest time after the end of treatment (sampling/recovery times): 4h treatment: 16h and 28h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): 0.2 ml Colcemid-solution (40 µg/mLwater) were added to each flask two hours prior
to the end of the incubation period to arrest the cells in a metaphase-like stage of mitosis (c-metaphase).
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The medium was removed from each flask and cells were removed from the bottom of the flask by trypsinization and suspended in medium. This medium was transferred to a centrifuge tube and spun for approximately 5 minutes at 700 rpm. The supernatant was carefully removed. 1-2 mL of a hypotonic solution (0.4% KCI; 37°C) was added to the tube. Within 4 min, the volume was brought to 6 mL with additional hypotonic solution and cells were resuspended. The cells were sedimented in the centrifuge as before and the supernatant was removed. A few drops of cold (4°C) fixative [ethanolfacetic acid (3:1)] were added and mixed carefully with the cells. The volume was adjusted to 6 ml with the fixative and mixed again with the cells. The mixture was incubated at room temperature for 20 min. Cells were pelleted as before and the supernatant was discarded. Cells were again resuspended in fixative as before and centrifuged. Pelleted cells were resuspended carefully in a small volume of fresh fixative. This suspension wasdropped onto clean slides. The slides were allowed to dry for at least 2 hours. Thereafter, they were submerged in pure methanol for 3 minutes and stained for 15-20 minutes in 3% Giemsa solution. Slides were rinsed twice in water and once in acetone and were then kept in xylene for about 30 minutes. The slides were allowed to dry completely and covered. At least two slides were generated per culture.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): The mitotic index was deterrrined by counting 1000 cells per culture. Duplicate cultures were processed and examined. Coded slides were evaluated using a light microscope at a magnification of about 1000. Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were examined. In most cases at least 100 assessable metaphases were present on one slide prepared from an individual culture. Therefore, the back-up slide which was generated routinely from every culture was normally not utilized for the evaluation. However, in cases when fewer than 100 assessable metaphases were found on the first slide of a culture, the back-up slides were evaluated as well until a total of 100 metaphases was reached. Only metaphases containing the modal chromosome number (22) were analyzed unless exchanges were detected. In this case, metaphases were evaluated even if the chromosome number was not equal to 22.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The classes of structural chromosome damage were defined and recorded by using essentially the terminology of Rieger and Michaelis (1967). Both chromatid and chromosome-type aberrations were assessed. Chromatid-type aberrations are clastogenic effects restricted to one of the two corresponding chromatids. Chromosome-type aberrations are defined as changes expressed in both corresponding sister chromatids at the same locus. The distinction between chromatid and chromosome type aberrations was not made for exchanges.
The different classes of aberrations are characterized as follows:
1. Gap:
A gap is an achromatic lesion within a chromatid arm without obvious dislocation of the chromatid end(s) and smaller than the width of one chromatid. Gaps are found on one chromatid ("gap") or on both chromatids at apparently identical sites ("iso-gap"). The biological relevance of gaps of both types is unclear.
2. Break:
A break is defined as a discontinuity of one chromatid ("break") or both chromatids, at apparently the same locus ("isobreak“), with dislocation of the chromatid ends. The dislocated chromatid end(s) has (have) to be present within the respective meta-phase. In addition, an achromatic lesion within a chromatid arm without obvious dislocation of the chromatid end(s) but larger than the width of one chromatid is also defined as break or as isobreak, if this occurs in parallel on both chromatids of a chromosome.
3. Fragment:
Fragments are parts of chromosomes without centromer. A fragment is the result of a break. The corresponding defective chromosome is not detectable among the chromosomes of the same metaphase. Fragments can be derived from one chromatid ("fragment") or from both corresponding chromatid regions of a chromosome ("isofragment").
4. Deletion:
A deletion occurs as the result of a break. In case of a deletion, one chromatid ("deletion") or both corresponding terminal chromatid parts of a chromosome ("isodeletion") are missing within the metaphase under assessment.
5.Exchange:
This is an exchange of chromatid-parts between different chromosomes (interchange) or within the same chromosome (intrachange).
6. Multiple aberration:
A cell was assessed as to contain "multiple aberrations“ when five or more structural changes (excluding gaps) occur within one metaphase.
In addition to these aberrations, metaphases showing chromosome disintegration as an indication of a cytotoxic effect were also recorded if they were observed. They were counted separately and were not included among the cells that were assessed for aberrations. “Chromosome disintegration" was recorded if fewer than half of the chromosomes reveal characteristic structural features within a given metaphase.
- Determination of polyploidy: Polyploid metaphases observed in a culture in addition to those 100 metaphases analyzed for chromosome aberrations were recorded. The number of polyploid metaphases was not used for assessment.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Evaluation criteria:

Coded slides were evaluated using a light microscope at a magnification of about 630. The mitotic index was deterrrined by counting 1000 cells per culture. The numbers of mitotic and non-mitotic cells were noted. Duplicate cultures were processed and examined. Coded slides were evaluated using a light microscope at a magnification of about 1000. Chromosomes of approximately 200 metaphases per concentration, 100
metaphases from each of two parallel cultures, were examined. In most cases at least 100 assessable metaphases were present on one slide prepared from an individual culture. Therefore, the back-up slide which was generated routinely from every culture was normally not utilized for the evaluation. However, in cases when fewer than 100 assessable metaphases were found on the first slide of a culture, the back-up slides were evaluated as well until a total of 100 metaphases was reached. Only metaphases containing the modal chromosome number (22) were analyzed unless exchanges were detected. In this case, metaphases were evaluated even if the chromosome number was not equal to 22.
The classes of structural chromosome damage were defined and recorded by using essentially the terminology of Rieger and Michaelis (1967). Both chromatid and chromosome-type aberrations were assessed. Chromatid-type aberrations are clastogenic effects restricted to one of the two corresponding chromatids. Chromosome-type aberrations are defined as changes expressed in both corresponding sister chromatids at the same locus. The distinction between chromatid and chromosome type aberrations was not made for exchanges.

Statistics:

The statistical analysis was performed by pair-wise comparison
treated and positive control groups to the respective solvent control group.
The mitotic index was statistically analyzed (provided that it was reduced compared to the mean of the corresponding solvent control) using the one-sided chi²-test.
The numbers of metaphases with aberrations excluding gaps were compared (provided that these data superceded the respective solvent control). The statistical analysis followed the recommendations outlined by Richardson et al. (1989). The one-sided chi²-test was used for the statistical evaluation.
A difference was considered to be significant, if the probability of error was below
5 %.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The survival indices in the treated cultures w/o S9 were relevantly reduced at 1700 µg/mL (4 h treatment) and at 800 µg/mL and above (18 h treatment). Positive control: reductuion in survival after 18h. W S9 reduction from 1700 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: no precipitation observed

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For cell lines: Mitotic index without S9 mix: In comparison to the solvent control, the mitotic indices in the treated cultures were relevantly reduced at 1700 µg/mL (4 hours treatment) and at 800 µg/mL and above (18 hours treatment). The cultures treated with mitomycin C showed no reduction in mitosis rate. Mitotic index with S9 mix:
In comparison to the solvent control, the treated cultures showed no relevant reduction of the mitosis rate. The positive control cyclophosphamide reduced the mitosis rate.
o Definition for chromosome aberrations, including gaps:

Test item without S9 mix: No biologically relevant and statistically significant increases of numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The same was true for a treatment period and total culture time of 18 hours.
The treatment with the positive control mitomycin C resulted in a clear and statistically significant increase of metaphases with aberrations and demonstrated the sensitivity of the test system.

Test item with S9 mix:
No biologically relevant and statistically significant increases of numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excluding gaps: See Details on test system and experimental conditions.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Please refer to any other information on results incl. tables.

Applicant's summary and conclusion

Conclusions:

2,5-dimethyphenylacetic-acid without S9 mix:
No biologically relevant and statistically significant increases of numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The same was true for a treatment period and
total culture time of 18 hours. The treatment with the positive control mitomycin C resulted in a clear and statistically significant increase of metaphases with aberrations and demonstrated the sensitivity of the test system.
2,5-dimethyphenylacetic-acid with S9 mix:
No biologically relevant and statistically significant increases of numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The positive control cyclophosphamide induced statistically significant and biologically relevant increases of metaphases with aberrations and demonstrated the sensitivity of the test system and the activity of the used S9 mix.

Executive summary:

In a mammalian cell cytogenetics assay [Chromosome aberration] according to OECD test guideline 473, V79 cell cultures were exposed to 2,5-dimethylphenylacetic acid (100 % a.i.), in DMSO at concentrations of 0, 5, 25, 50, 100, 200, 425, 850, 1700 µg/mL (4h treatment); 0, 50, 100, 200, 425, 850, 1700 µg/mL (18h treatment) with and without metabolic activation.

2,5-dimethylphenylacetic acid was tested up to precipitating concentrations. Positive controls induced the appropriate response.  There was no evidence of Chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.