2-Thiophenecarboxylic acid, 5-chloro-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Study conducted according to OECD test guideline 471 (screening), result: negative

Study conducted according to OECD test guideline 471, result: negative

Study conducted according to OECD test guideline 473, result: negative

Link to relevant study records

Reference

Reference 1

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

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

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.

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 %.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

report date 2003-02-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His locus

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix; no other information

Test concentrations with justification for top dose:

Between 16 and 5000 µg/Plate or Tube, exact concentrations were not specified

Vehicle / solvent:

Not specified

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

vehicle not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: nitrofurantoin, 4-nitro-1,2-phenylene diamine, 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) not specified
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Not specified
- Test substance added in agar (plate incorporation); preincubation

Rationale for test conditions:

As recommended by the OECD test guideline

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation was seen at 5000 µg/Plate or Tube

Conclusions:

5-Chlorthiophen-2-carbonsäure was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation screening.

Executive summary:

In a reverse gene mutation screening assay in bacteria similar to OECD test guideline 471 (1997), strains TA 98, TA 100, TA 1535, and TA 1537 of S. typhimurium were exposed to 5-Chlorthiophen-2-carbonsäure, (100 % a.i.), at concentrations between 16 and 5000 µg/plate or tube in the presence and absence of mammalian metabolic activation using the plate co-incubation and the pre-incubation method.

5-Chlorthiophen-2-carbonsäure was tested up to limit concentration (5000 µg/plate). There were no increases of revertants in comparison to the control plates. The positive control substance provided the expected increase in revertants numbers. Based on these results 5-Chlorthiophen-2-carbonsäure is not considered to be a mutgen under the conditions of the test.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-07-18 to 2005-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His locus

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9-Mix
- method of preparation of S9 mix: It was made from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice. The animals were prepared unfasted, following the directions of Ames et al. (1975) and Maron and Ames (1983). The rats were terminated. Livers were removed under sterile conditions immediately after sacrifice and kept at 4°C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4°C. The livers were washed with cold (4°C), 0.15 M KCI solution (approximately 1 mL KCI per 1 g liver), and then homogenized in fresh, cold (4°C), 0.15 M KCI (approximately 3 mL KCI per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4°C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80°C in small portions. These portions were slowly thawed before use. The S9 mix was freshly prepared (Ames et al., 1973) and used only on the same day. It was placed in a vessel with a double glass wall until used. The hallow wall was filled with ice to keep the S9 mix cold.
- concentration or volume of S9 mix and S9 in the final culture medium: The amount of S9 fraction in S9 mix is indicated in the tables in percent. The S9 mix comprised the amount of S9 fraction (10 %) indicated in the tables, 70% cofactor solution and (30-10)% 0.15 M KCI. The S9 fraction was derived from the preparation dated September 13, 2005 (protein content 23.4 mg per mL).

Test concentrations with justification for top dose:

0, 50, 158, 500, 1581, 5000 µg/plate
0, 35, 200, 350, 2000, 3500 µg/tube

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: Nitrofurantoin; 4-Nitro-1,2-phenylene diamine; 2-Anthacene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): E+06 dilution
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Summary of Mean Values Without S9 Mix
Table and Group	Strain
	TA 1535	TA 100	TA 1537	TA 98	TA 102
1-5 µg/Plate
0	13	96	6	34	194
50	15	101	6	41	185
158	10	124	5	39	178
500	13	107	5	33	177
1581	9	98	5	27	130
5000	6	51	1	14	25
Na-azide	620
NF		256
4- NPDA			58	147
MMC					553
6-10 µg/tube
0	9	104	7	19	215
35	9	96	6	16	174
200	7	89	7	18	160
350	9	117	6	16	164
2000	6	91	5	17	1041
3500	5	74	3	21	52
Na-azide	627
NF		388
4-NPDA			107	78
Cumene					349
Summary of Mean Values with S9 Mix
Table and Group	Strain
1-5 µg/ Plate	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	9	126	8	32	259
50	7	106	5	32	237
158	6	133	6	33	256
500	6	106	6	35	189
1581	10	89	7	17	130
5000	2	47	-	6	33
2-AA	99	1190	61	693	478
6-10 µg/Tube
0	8	118	7	28	186
35	7	115	8	20	184
200	8	105	6	22	178
350	9	134	6	18	192
2000	6	101	8	20	118
3500	4	84	6	15	61
2-AA	125	1398	199	836	446

Conclusions:

The Salmonella/microsome plate incorporation test, employing doses of up to 5000 pg per plate, showed 5-Chlorthiophen-2-carbonsäure to produce bacteriotoxic effects, starting at 1581 pg per plate. Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility), revealed no biologically relevant variations from the respective negative controls. ln spite of the low doses used, positive controls increased the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity. Despite this sensitivity, no indications of mutagenic effects of 5-Chlorthiophen-2-carbonsäure could be found at assessable doses of up to 5000 pg per plate in any of the Salmonella typhimurium strains used in the assay.
Due to these results 5-Chlorthiophen-2-carbonsäure has to be regarded as non-mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1997), strains TA 98, TA 100, TA 1535, and TA 1537 of S. typhimurium were exposed to 5-Chlorthiophen-2-carbonsäure, (100 % a.i.), in DMSO at concentrations of 0, 16, 50, 158, 500, 1581, 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate co-incubation and the pre-incubation method.

5-Chlorthiophen-2-carbonsäure was tested up to limit concentration (5000 µg/plate). There were no increases of revertants in comparison to the control plates. The positive control substance provided the expected increase in revertants numbers. Based on these results 5-Chlorthiophen-2-carbonsäure is not considered to be a mutgen under the conditions of the test.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reverse gene mutation screening assay in bacteria similar to OECD test guideline 471 (1997), strains TA 98, TA 100, TA 1535, and TA 1537 of S. typhimurium were exposed to 5-Chlorthiophen-2-carbonsäure, (100 % a.i.), at concentrations between 16 and 5000 µg/plate or tube in the presence and absence of mammalian metabolic activation using the plate co-incubation and the pre-incubation method.

5-Chlorthiophen-2-carbonsäure was tested up to limit concentration (5000 µg/plate). There were no increases of revertants in comparison to the control plates. The positive control substance provided the expected increase in revertants numbers. Based on these results 5-Chlorthiophen-2-carbonsäure is not considered to be a mutgen under the conditions of the test.

 

In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1997), strains TA 98, TA 100, TA 1535, and TA 1537 of S. typhimurium were exposed to 5-Chlorthiophen-2-carbonsäure, (100 % a.i.), in DMSO at concentrations of 0, 16, 50, 158, 500, 1581, 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate co-incubation and the pre-incubation method.

5-Chlorthiophen-2-carbonsäure was tested up to limit concentration (5000 µg/plate). There were no increases of revertants in comparison to the control plates. The positive control substance provided the expected increase in revertants numbers. Based on these results 5-Chlorthiophen-2-carbonsäure is not considered to be a mutgen under the conditions of the test.

 

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.

 

Justification for classification or non-classification

Based on the presented information 5-Chlorthiophen-2-carbonsäure does not need to be classified according to Regulation (EC) No. 1272/2008 (CLP) with regard to mutagenicity.