2-Thiophenecarboxylic acid, 5-chloro-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Remarks:

expert judgement

Type of information:

other: expert judgement

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

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.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-02-26 to 2007-07-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

27 July 1995

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hsd Cpb:VVU

Details on species / strain selection:

The study was conducted on rats, a species recommended in guidelines for subacute toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmamr GmbH, Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 weeks
- Weight at study initiation: Males: 169 (153 - 184) g
Females: 140 (116 - 158) g
- Housing: At their arrival the animals were placed together, separated by sex, in some Makrolon® cages Type III and transported into the animal room where they were put individually into Makrolon® cages Type Ila up to tattooing. From tattooing to necropsy animals were kept in groups with 2 or 3 animals in Makrolon® cages Type IV. For enviromnental enrichment wooden blocks supplied by Tapvei OY, 73 620, Kortteinen, Finland, were provided to each cage and renewed as necessary.
- Diet (e.g. ad libitum): The diet consisted of a fixed-forrnula standard diet (supplied by Provimi Kliba SA, CH-4303 Kaiseraugst, Switzerland). Diet (Provimi Kliba SA 15 W10 (3883.0. 15 pellet)) was provided in racks integrated in cage-lids for ad libitum consumption except during the 16h urine sampling period.
- Water (e.g. ad libitum): tap water, water was supplied in polycarbonate bottles with a capacity of approximately 300 ml for ad libitum consumption (2 bottles per cage).
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: The nutritive composition and contaminant content ofthe standard diet were routinely checked and analyzed. The tap water complied with the actual German Drinking Water Ordinance ("Trinkwasser-Verordnung” ofDecember 5, 1990; Federal Law Gazette No. 66 edited December 12, 1990, page 2612 and supplemented on May 21, 2001, Federal Law Gazette No. 24, issued on May 28, 2001, page 959 ff). The results of food and water analyses were stored. The available data provided no indication of any effect on the study objective.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-2
- Humidity (%): 55+/-5
- Air changes (per hr):10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Study Initiation Date: February 23, 2007
Experimental Starting Date: February 27, 2007
(=First Day of Treatment)
End of In-Life-Phase: March 29, 2007
Total Duration of Study: 31 days
Duration of Treatment: 29/30 days
Necropsy: March 28, 2007 (males)
March 29, 2007 (females)
0 Experimental Completion Date: May 24, 2007

Route of administration:

oral: gavage

Vehicle:

other: 2% aqueous Cremophor EL®

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated with 2% aqueous Cremophor EL® as suspension at the appropriate concentrations at room temperature and maximally used over the stability period of 8 days. The test substance was administered as a suspension.


VEHICLE
- Justification for use and choice of vehicle (if other than water): 2% aqueous Cremophor EL®
- Concentration in vehicle: 5, 17, 50 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Investigation on homogeneity and stability of the test item was done prior to the study. The correct concentrations and homogeneity in the formulations given to the rats were determined twice by analytical examination during the study.
For analytical investigations, samples from the test item formulations covering the test item concentration range used in the study were taken. These samples were diluted with methanol into the working range and subsequently quantified by a spectrometric method (UV-VIS). The detection wave length is 274 nm. Standard solutions of the authentic test item were used for calibration. Standard solutions of the authentic test item were used for calibration. Linearity, Precision, Specificity, Robustness and Accuracy of the analytical method were evaluated apart from this GLP-study and fulfil the predefined acceptance criteria. Additionally system suitability tests in terms of specificity, precision and linearity indicated, that qualified analytical procedures were followed during the study.
Analysis of blank samples (0 mg/mL) revealed no measurable traces of test item.
The analytical data reveal that the homogeneity of the formulations is given. The content checks assure that during the study appropriate and equal mixture procedures were followed. Formulations are homogenous and stable for at least 8 days at room temperature.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

170 mg/kg bw (total dose)

Dose / conc.:

50 mg/kg bw (total dose)

Dose / conc.:

0 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels have been selected based on the results obtained in a previous 2-week rat gavage study with 5-Chlorthiophen-2-carbonsäure with 0, 50, 150, 300 and 500 mg/kg b.w. and additionally on the results of single animals treated with 300, 500, 750 and 1000 mg/kg for few days. Results of the 2-week study: Clinical observations, mortality and food intake did not give any evidence for treatment-related effects up to 500 mg/kg. Body weight was inconspicuous in males up to 300 mg/kg and in all female groups. Males at 500 mg/kg showed a slightly delayed body weight gain. Water consumption was increased in males at 150 mg/kg and above and in females at 300 mg/kg and above (up to 46 and 40%). Relative liver weights were higher in both sexes at least at 300 mg/kg and above compared to controls. Relative spleen and kidney weights were slightly higher in females at 500 mg/kg whereas relative prostate weight appeared slightly lower at this dose compared to controls. At necropsy thickened stomach and elongated intestine were observed in both sexes at 300 mg/kg and above. Correspondingly to the increased liver weight, liver appeared enlarged in 300 and/or 500 mg/kg rats; in one high dose rat areas were noted for epididymides. Previous to the 2-week study single animals (1 male and 1 female each) were treated with 300, 500, 750 and 1000 mg/kg for a few days. Rats at 1000 and 750 mg/kg died or had to be killed in moribund condition on day 2 or 3. The 500 mg/kg male had to be killed on day 6 of treatment; no clinical findings were seen in the 500 mg/kg female and the 300 mg/kg rats for a period ofup to 7 days of treatment.
Therefore, the proposed dose levels for the 4-weeks study were 0-50-170-500 mg/kg b.w. of 5-Chlorthiophen-2-carbonsäure.

- Fasting period before blood sampling for clinical biochemistry: non-fasted

- Section schedule rationale (if not random): All animals scheduled for terminal necropsy and all rats to be sacrificed moribund were killed by exsanguination under deep diethyl ether anesthesia and necropsied. Their organs and tissues were subjected to thorough gross pathological examination. The organs and tissues fixed and the methods of fixation are described in the Pathology Report. Animals that died spontaneously were necropsied at the earliest opportunity. From these animals the organs and tissues were handled as described above.
- Dose range finding studies: see above under "rationale for dose-selection"

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily on weekends and public holidays


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 29
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 29
- Animals fasted: No
- How many animals: all

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: on day 28 for 16 h
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Open Field Observation (OFO): once before start and once weekly thereafter; Functional Observational Battery: day 23/24; Motor Activity: day 24/25
- Dose groups that were examined: all
- Battery of functions tested: - home cage pbse_rvation: posture, piloerection, gait abnormalities, involuntary motor
movements, vocalization, others
- observations during handlirgg; ease of removing, reaction to being handled, muscle tone, palpebral closure, lacrimation, nasal discharge, salivation, stains, body temperature, pupil size, pupil response
- open field observations: piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behavior, gait abnormalities, vocalization, arousal, rearing, defecation, urination, others
- the following manipulative tests were additionally performed:
approach response, touch response, auditory response, tail pinch response, righting reflex, grip strength and landing foot splays.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

Optional endpoint(s):

Optional endpoints: No

Statistics:

see under "Any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Daily observations and detailed weekly examinations revealed salivation in one
male at 500 mg/kg (animal no. 19) from day 23 to day 29 and breathing sounds in another male at 500 mg/kg (animal no. 20) on day 16. No clinical signs were seen in treated males up to 170 mg/kg and in all treated females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of treated males and females did not differ toxicologically relevantly from controls up to 170 mg/kg in males and up to 500 mg/kg in females. Males and females at 500 mg/kg showed a slight body weight reduction after the first treatment with the test substance The body weight development was general
slightly reduced in males at this group At the end of treatment, the difference to control was 11%; however, before the start of treatment, the difference to control was already 6%. A transient body weight reduction from day 27 to 28 could be observed in all groups caused by fasting during urine sampling period

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water intake per animal and per kg body weight was markedly increased in both sexes at 500 mg/kg (per animal 27% in males and 35% in females, per kg body weight 41% in both sexes). No toxicologically relevant changes in water intake was deterniind in both sexes up to 170 mg/kg.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological investigation revealed decreased count of erythrocytes (ERY), decreased hemoglobin concentration (HB) as well as decreased hematocrit (HCT) value in both sexes at 500 mg/kg. The count of reticulocytes (RETI) was slightly increased in both sexes at 500 mg/kg (RETI not statistically significantly in females). The mean corpuscular erythrocyte volume (MCV) was statistically significantly increased in males at 500 mg/kg. The MCHC mean in males at 170 mg/kg, which was statistically significantly different from control values, is considered to be of no toxicological relevance, since the difference from control was negligibly low and it did not show a correlation with the dose administered. The count of leucocytes (LEUCO) was increased in females at 500 mg/kg and the counts of neutrophils (NEUTRO) and monocytes (MONO) were increased in both sexes at this dose (mostly p>0.05; increase of LEUCO and MONO was slight). The count of eosinophils (EOS) was slightly decreased in males at 170 mg/kg and above and in females at 500 mg/kg (females: p>0.05).

Description (incidence and severity):

Determination ofplasma enzyme activities revealed increased activities of aspartate aminotransferase (ASAT) in both sexes at 500 mg/kg (p>0 05 % in females) Activity of alanine aminotransferase(ALAT) was slightly but statistically significantly higher in both sexes at 500 mg/kg compared to controls, however, the differences to controls were small and all individual values were within the 2s range of historical data. Determination of substrates in blood samples showed slightly decreased total protein concentration (PROT) in males (p≤ 0.01) as well as increased urea and slightly increased creatinine (CREA) concentrations in females at 500 mg/kg. Glucose concentrations in females at 50 and 170 mg/kg, which were statistically significantly different from control values, are considered to be ofno toxicological relevance since the differences from control were negligibly low and they did not show a correlation with the dose administered.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis showed slightly reduced pH values in 500 mg/kg males (p≤0.05).
The semi-quantitatively determined blood, bilirubin, glucose, urobilinogen were unremarkable in all groups; the concentrations of ketone bodies were slightly increased in both sexes at 170 mg/kg and above. The sediments showed no abnormalities in treated male or female animals compared to respective controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative thymus weights were reduced in both sexes at 500 mg/kg (absolute: 32% in males, 25% in females; relative: 26% in both sexes). Relative liver (15% in males, 38% in females) and kidney (12% in males, 28% in females) weights were increased in both sexes at 500 mg/kg (in females also absolute liver (37%) and kidney (29%) weights were increased; all changes: at least p≤0.05).
Absolute brain and testis weights were slightly reduced in males at 500 mg/kg (brain 11%, testis 14%). Absolute (p>0.05) and relative heart weights were slightly increased in females at 500 mg/kg (14-15%). The determination of the weights ofthe other organs gave no evidence for treatment-related effects.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology revealed minimal to moderate neuronal degeneration with reactive gliosis in the hippocampus (CA1-3), the gyrus dentatus and in the retrosplenial and piriform cortex in 1/5 males and 4/5 females at 500 mg/kg. Slight sciatic nerve fiber degeneration and subsequent moderate thigh muscle atrophy were found in the female no. 39 at 500 mg/kg, which was also affected by thymic atrophy. In the liver, increased glycogen storage ofperiportal hepatocytes was diagnosed in males at 170 mg/kg and above (incidence: 0-0-4-5) and in females at 500 mg/kg (incidence: 0-0-0-5). Decreased hepatocellular fat storage was found in both sexes at 500 mg/kg.
In the spleen, hemopoiesis was slightly increased in females at 500 mg/kg. Corresponding to the observed gross findings, squamous cell hyperplasia and hyper-/dyskeratosis up to a moderate severity level were detected at the limiting ridge of the forestomach to the glandular stomach in both genders at 500 mg/kg and in one male at 170 mg/kg. These findings were accompagnied by basophilia ofthe adjacent glandular epithelium ofthe gastric fundus. Sperm granulomas in 2 males at 500 mg/kg were assessed as occasional findings that are usually found as well in control animals. All other findings not being mentioned in detail are regarded to be individual or strain-, gender-, age-related or handling-derived alterations, which are well documented in publications about laboratory animals and/or are equally distributed among control and treated animals.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (actual dose received)

System:

musculoskeletal system

Organ:

other: sciatic nerve degeneration

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (actual dose received)

System:

nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

Under the conditions described the no-observed-adverse-effect level (NOAEL) for the administration of 5-Chlorthiophen-2-carbonsäure orally by gavage was 50 mg/kg b.w. in male and in female rats.
The NOAEL is based on adverse neurological behaviour observed in the 500 mg/kg group but also in 1 female of the 170 mg/kg group, all other significant findings occurred in the 500 mg/kg dose group only, BW was not affected at all. Based on these results and in absence of any histopathological findings in 50 & 170 mg/kg groups classification for STOT-RE is not warranted.

Executive summary:

In a subacute toxicity study according to OECD guideline 407 (1995) 5-Chlorthiophen-2-carbonsäure (100% a.i.) was administered to Wistar rats 5/sex/dose 2% Chremophor EL® by gavage at dose levels of 0 (control), 50, 170 and 500 mg/kg bw/day for 28 consecutive days.

No mortality occurred. The Clinical sign observed was salivation in one male for several days and breathing sounds in another male on a single day at 500 mg/kg. No other changes were observed at the weekly detailed clinical observations. Neurotoxicity assessment revealed the following effects: A slightly reduced mean count of rearings in 500 mg/kg males (14.4 -- 10.8 — 11.2 — 6.8); a trend could also be seen in females at this dose (18.6 - 19.0 — 20.2 — 15.2). Correspondingly, MA tests revealed slightly and partly dose-dependently reduced vertical activity in both sexes at 170 and 500 mg/kg. Absolute brain weight was slightly reduced in males at 500 mg/kg (11%). Histopathology revealed minimal to moderate neuronal degeneration with reactive gliosis in the hippocampus, the gyrus dentatus and in the retrosplenial and piriform cortex in 1/5 males and 4/5 females at 500 mg/kg. Slight sciatic nerve fiber degeneration and subsequent moderate thigh muscle atrophy were found in one 500 mg/kg female. In summary, clinical and morphological findings are indicative of a neurotoxicological potential of 5-Chlorthiophen-2-carbonsäure starting at 170 mg/kg.

Body weight was slightly reduced in both sexes at 500 mg/kg after the first treatment with the test substance; the body weight development was generally slightly lower in males at this dose than in controls. At the end of treatment, the difference to control was 11%; however, before the start of treatment, the difference to control was already 6%.

Water intake was markedly increased in both sexes at 500 mg/kg (41% per kg body weight). Serum electrolytes were inconspicuous. Urinalysis showed slightly reduced pH values in 500 mg/kg males and slightly increased ketone body concentrations in both sexes at 170 mg/kg and above (ketone body see also below). Sediments showed no abnormalities in any group. Absolute (females) and relative (both sexes) kidney weights were increased at 500 mg/kg (12% in males, 28-29% in females); histopathology did not reveal any test substance-induced changes. The increase in water intake is interpreted as a consequence of the local irritative effect of 5-Chlorthiophen-carbonsäure on the mucosa of the G1 tract and is not considered an expression of damage to the kidneys as histopathological changes of the kidneys as well as changes in urine and blood parameters indicative of kidney damage are absent.

Hematological investigation revealed decreases in HCT, ERY count and HB concentration in both sexes at 500 mg/kg and the count of RETI was slightly increased in both sexes and the MCV value in malesat this dose. In the spleen, hemopoiesis was slightly increased in females at 500 mg/kg. The count of LEUCO (slight degree) was increased in females and the counts of NEUTRO and MONO (slight degree) were increased in both sexes at 500 mg/kg. The count of EOS was slightly decreased in males at 170 mg/kg and above and in females at 500 mg/kg.

Absolute and relative thymus weights were reduced in both sexes at 500 mg/kg (about 25-32%); in one 500 mg/kg female thymic atrophy was diagnosed.

Clinical laboratory tests revealed increased activity of ASAT and slightly increased activity ofALAT in both sexes at 500 mg/kg (ASAT in females: p>0.05), which is concluded to be adverse. Determination of substrates in blood samples showed slightly decreased total PROT concentration in males and slightly increased CREA and urea concentrations in females at 500 mg/kg. Absolute liver weight in females and relative liver weights in both sexes were increased at 500 mg/kg (15% in males, about 38% in females). Increased glycogen storage of periportal hepatocytes was diagnosed in males at 170 mg/kg and above (incidence: 0-0-4-5) and in females at 500 mg/kg (incidence: 0-0-0-5). Decreased hepatocellular fat storage was found in both sexes at 500 mg/kg. Changes in glycogen and fat storages in the liver as well as in urine ketone body concentrations (see above) may be indicative for metabolic alterations induced by the test substance.

During necropsy dilations of the stomach as well as white areas at the limiting ridge of the forestomach to the glandular stomach were found in both sexes at 500 mg/kg. Correspondingly, squamous cell hyperplasia and hyper-/dyskeratosis up to a moderate severity level were detected in the described area in both sexes at 500 mg/kg and in one male at 170 mg/kg. These findings were accompanied by basophilia of the adjacent glandular epithelium of the gastric fundus and are concluded as adverse.

Absolute, but not relative testes weight was slightly reduced in males at 500 mg/kg (14%). Sperm granulomas in 2 males at 500 mg/kg were assessed as occasional findings that are usually found as well in control animals. Due to the only slight difference to control animals and in the absence of corresponding morphological changes the reduction in testes weight is considered toxicologically not relevant.

Relative heart weight was slightly increased in females at 500 mg/kg (14%); without histopathological correlate, no toxicological relevance is assumed.

 

 

Under the conditions described the no-observed-adverse-effect level (NOAEL) for the administration of 5-Chlorthiophen-2-carbonsäure orally by gavage was 50 mg/kg b.w. in male and in female rats.

Cross-reference 4

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

adopted 24 February, 1987

GLP compliance:

yes

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan/Winkelmann GmbH, 33178 Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Rationale for use of males (if applicable)
- Age at study initiation: 9-13 weeks
- Weight at study initiation: males: 237-253 g; females: 202-222 g
- Housing: The animals were caged individually in polycarbonate cages on low dust wood
granulate bedding (J. Rettenmaier & Söhne, 73494 Rosenberg, Germany)
- Historical data:
- Diet (e.g. ad libitum): The animals received the standard diet “Provimi Kliba 3883.0. 1 5 Maus/Ratte Haltung, Kaiseraugst Switzerland” ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 d
- Method of randomisation in assigning animals to test and control groups: The animals were assigned to their groups by randomization. The random list was based on evenly distributed chance numbers especially generated for the study by a software application.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±5
- Air changes (per hr):10
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: 30.0 cm²
- % coverage: 10%
- Type of wrap if used: wet gauze-layer (6.0 cm x 5.0 cm = 30.0 cm²) of a
,,Cutiplast® steril“ coated with air-tight ,,Leukoflex®“. The gauze strip was placed on the rat’s back and secured in place using ,,Peha®-Haft“ cohesive stretch tape and additionally covered with a "Lomir biomedical Inc rat jacket", which was connected with a safety pin to the stretch tape to ensure that the animals could not ingest the test substance.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the area was rinsed with tepid water using soap and gently patting the area dry.
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 15.8 - 16.9 mg/cm² (male) 13.5 - 14.8 mg/cm² (female)

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

1

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations: at least once daily, weighing weekly
- Necropsy of survivors performed: yes
- Clinical signs including body weight
- Other examinations performed: clinical signs, body weight

Statistics:

An assessment ofthe LD50 was made based on the applied dose and dose-response curve, respectively.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the present investigations, 5-Chlorthiophen-2-carbonsäure is to be regarded to have the following LD50 values:
LD50 rat, male : > 2000 mg/kg body weight
rat, female : > 2000 mg/kg body weight
So it is regarded as non-toxic after dermal application.

Executive summary:

In an acute dermal toxicity study according to OECD test guideline 402 (1987), one young adult male and female Wistar rats (1/sex) were dermally exposed to 5-Chlorthiophen-2-carbonsäure (100 % a.i) for 24 hours to 10 % of body surface area at a doses of 2000 mg/kg bw.  Animals then were observed for 14 days.

Dermal LD50 Males/Females => 2000 mg/kg bw

5-Chlorthiophen-2-carbonsäure is of low Toxicity based on the LD50 value for male and female Wistar rats. Each animal of the high dose groups (2000 mg/kg bw) survived 14 days.

 Based on the results the substance does not need to be classified according to Regulation (EU) 1272/2008 (CLP) and the Globally Harmonized System for Calssification and Labelling of Chemicals (GHS) as acute toxic via the dermal route.

 

Cross-reference 5

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

17 Decmber 2001

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan/Winkelmann G1nbH, 5960 AD Horst, Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Fasting period before study: 16-24 h
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 161 g - 177 g
- Housing:The animals were group caged conventionally in polycarbonate cages on low dust wood granulate bedding (J. Rettenmaier & Söhne, 73494 Rosenberg, Germany).
- Diet (e.g. ad libitum): The animals received the standard diet “Provimi Kliba 3883.0.15 Maus/Ratte Haltung, Kaiseraugst Switzerland” ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days
- Method of randomisation in assigning animals to test and control groups: The random list was based on evenly distributed chance numbers by a software application.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22±2
- Humidity (%): 55±5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

with the aid of 2% Cremophor EL

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200, 30 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw


MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw


CLASS METHOD (if applicable) acute toxic class
- Rationale for the selection of the starting dose: The starting dose level should be that which is most likely to produce mortality in some of the dosed animals. Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step, i.e.:
- no further testing is needed,
- dosing ofthree additional animals, with the same dose,
- dosing of three additional animals at the next higher or the next lower dose level
The substance is tested using a stepwise procedure, each step using three animals of a single sex (normally females). The procedure is described in the flow charts of Annex 2, OECD guideline 423.

Doses:

2000 and 300 mg/kg bw

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: weighing: weekly; Observations: at least once per day
- Necropsy of survivors performed: yes
- Clinical signs including body weight
- Other examinations performed: clinical signs, body weight

Statistics:

The LD50 value was estimated according to OECD - Guideline for Testing of Chemicals No. 423 -"Acute Oral Toxicity - Acute Toxic Class Method"; adopted:
December 17, 2001.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

>= 300 - <= 2 000 mg/kg bw

Based on:

test mat.

Mortality:

All animals of the high dose group died within 5h to 2d.

Clinical signs:

other:

Gross pathology:

All animals treated with 2000 mg/kg bw. died during the observation period. The following changes were observed:
liver, discoloration, spotted, spleen, discoloration, pale; diminished in size, kidneys, discoloration, pale.
The necropsies performed at the end ofthe study revealed no particular findings in animals treated with 300 mg/kg bw.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

According to OECD guideline 423 the LD50 of 5-Chlorthiophen-2-carbonsäure is ≥ 300 mg/kg bw ≤ 2000 mg/kg bw (Category 4 ofthe Globally Harmonized Classification System).

Executive summary:

In an acute oral toxicity study according to OECD guideline 423, adopted 17 December 2001, 9 female, fasted, 10-12 weeks old Wistar strain rats were given a single oral dose of 5-Chlorthiophen-2-carbonsäure in demineralized water containing 2% Cremophor by gavage at a dose of 2000 and 300 mg/kg bw and observed for 14 days.

3 animals of the 2000 mg/kg bw group died between 5h and 2d after dosing. Clinical signs shown by the animals that suvived, i.e. the 300 mg/kg bw dose group, was only increased water intake.

No other signs were recorded.

The body weight and the body weight development of the animals were not affected by the treatment.

In animals that died during the observation period the following changes were detected:

liver, discoloration, spotted, spleen, discoloration, pale; diminished in size, kidneys, discoloration, pale.

 

No gross pathologic changes were observed in animals sacrificed at the end of the study period.

Oral LD50 ≥ 300 mg/kg bw ≤ 2000 mg/kg bw

Data source

Reference

Reference Type:

other: statement regarding Toxicokinetics

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Objective of study:

absorption

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Toxicokinetic statement based on available physico-chemical and toxicological data according to REACH Guidance R.7

GLP compliance:

no

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

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The toxicokinetics of 5-Chlorthiophen-2-carbonsäure are based on the studies performed within the process of registration of a new chemical under the Chemicals Act, Base Set. Experimental toxicokinetic studies have not been performed. The physico-chemical characteristics of 5-Chlorthiophen-2-carbonsäure (readily soluble in water (1.01 g/L at 20 °C), log POW of 1.95) and the molecular mass of 162.6 g/mol are in a range suggestive of absorption from the gastro-intestinal tract subsequent to oral ingestion. This assumption is confirmed by the data on acute and subacute oral toxicity. The findings of the subacute oral toxicity study indicate systemic availability of the test substance. The NOAEL of this study was established at 50 mg/kg body weight/day in male and female animals. The onset of growth retardation early during the study (after the first treatment with the test substance) suggests rapid absorption from the GI tract.
Water solubility, n-octanol/water partition coefficient and molecular weight of 5-Chlorthiophen-2-carbonsäure are in ranges which favor dermal absorption. Therefore, absorption through the skin may be assumed a route of exposure. However, in a study assessing the acute dermal toxicity of 5-Chlorthiophen-2-carbonsäure to rats a dose of 2000 mg/kg body weight was tolerated by male and female animals without mortalities, clinical signs, effects on body weight development and gross pathological findings. In acute skin and eye irritation studies in rabbits no systemic intolerance reactions have been observed and no sensitizing effect has been identified in the Local Lymph Node Assay. Based on the results of the in vitro genotoxicity tests it is concluded that DNA-reactive metabolites of 5-Chlorthiophen-2-carbonsäure will most probably not be generated in mammals in the course of hepatic biotransformation.

Applicant's summary and conclusion

Conclusions:

The test item is considered to be readily intestinally absorbed. Dermal absorption seems to be reduced in comparison to oral absorption based on the results from the acute dermal toxicity and skin and eye irritation studies.