2-Thiophenecarboxylic acid, 5-chloro-

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:

2007-02-26 to 2007-06-29

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 Chromosomal Aberration Test)

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-060-6

EC Name:

-

Cas Number:

24065-33-6

Molecular formula:

Hill formula: C5H3ClO2S CAS formula: C5H3ClO2S

IUPAC Name:

5-chlorothiophene-2-carboxylic acid

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 Chinese hamster lung fibroblasts
- Normal cell cycle time (negative control): 12 h

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: Prior to the start of the study Chinese hamster V79 cells from a frozen permanent, which was stored in liquid nitrogen, were normally grown in 20 ml medium and 75 cm² flasks or under comparable conditions. Incubation of the cells was always performed at 37°C in a CO2-incubator (5% CO2). Unless reported othen/vise, cells were grown in medium containing 10% fetal calf serum [FCS = fetal bovine serum (FBS)]. As medium, PAA Ready Mix was used. PAA Ready mix is a commercially available by PAA, Paching, Austria
- Modal number of chromosomes: 22

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9-Mix
- source of S9: 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 green, protein content 36.8 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 cofactor 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, 425, 850 and 1700 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test substance was dissolved in a suitable solventz which was selected based on the solubility of 5-Chlorthiophen-2-carbonsäiure in the following solvents in the order given. If 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 5-Chlorthiophen-2-carbonsäure, DMSO was selected as solvent. In this solvent 5-Chlorthiophen-2-carbonsäure was soluble up to at least 170 mg/mL. Precipitation of 5-Chlorthiophen-2-carbonsäure was not observed in the culture medium of the pre-test.

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: 1

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h and 18 h
- Harvest time after the end of treatment (sampling/recovery times): 18 h and 30 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: 0.2 ml Colcemid-solution (40 µg/mL water) 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 minutes, 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 [ethanol/acetic 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 minutes. 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 was dropped 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 determined by counting 1000 cells per culture. 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.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
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. ln 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 chro-
matid ("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.
- Determination of polyploidy: Polyploid metaphases observed in a culture in addition to those 100 metaphases analyzed for chromosome aberrations were recorded.
- Determination of endoreplication:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI)

Evaluation criteria:

An increased incidence of gaps of both types without concomitant increase of other aberration types was not considered as indication of a clastogenic effect.
A test was considered positive, if there was a relevant and statistically significant increase in the aberration rate.
A test was considered negative, if there was no such increase at any time interval.
A test was also considered negative, if there were statistical significant values, which were, however, within the range of historical negative controls.
A test was considered equivocal, if there was an increase above the range of historical negative controls which was statistically significant but not considered relevant, or if an increase occurred, which was considered relevant, but which was not statistically significant.

Statistics:

The statistical analysis was performed by pair-wise comparison of 5-Chlorthiophen-2-carbonsäure-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 (pro-vided 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

After 4hours treatment of Chinese hamster V79 cells with 5-Chlorthiophen-2-carbonsäure concentrations of 425, 850 and 1700 µg/mL were used without and with S9 mix for assessment of the clastogenic potential of 5-Chlorthiophen-2-carbonsäure. In addition, after 18 hours treatment with 5-Chlorthiophen-2-carbonsäure concentrations of 425, 850 and 1700 µg/mL were read for reading without S9 mix.
None of these cultures treated with 5-Chlorthiophen-2-carbonsäure in the absence or presence of S9 mix showed statistically significant or biologically relevant increases of numbers of metaphases with aberrations.
The positive controls mitomycin C and cyclophosphamide induced clear clastogenic effects and demonstrated the sensitivity of the test system and in the case of cyclophosphamide the activity of the used S9 mix.
Based on the results of this test, 5-Chlorthiophen-2-carbonsäure is considered not to be clastogenic for mammalian cells in vitro.

Executive summary:

In a mammalian cell cytogenetics assay Chromosome aberration according to OECD test guideline 473 (1997)V79 cell cultures were exposed to 5-Chlorthiophen-2-carbonsäure, (% a.i.), in DMSO at concentrations of 0, 425, 850 and 1700 µg/mL with and without metabolic activation [S9 mix].

5-Chlorthiophen-2-carbonsäure was tested up to precipitating concentrations. None of these cultures treated with 5-Chlorthiophen-2-carbonsäure in the absence or presence of S9 mix showed statistically significant or biologically relevant increases of numbers of metaphases with aberrations.

The positive controls mitomycin C and cyclophosphamide induced clear clastogenic effects and demonstrated the sensitivity of the test system and in the case of cyclophosphamide the activity of the used S9 mix. There was no evidence of Chromosome aberrations induced over background.

This study is classified asacceptable. This study satisfies the requirement for Test Guideline [In vitro mammalian cytogenetics OECD 473] for in vitro cytogenetic mutagenicity data.