N-phenyl-N-[(trichloromethyl)thio]benzenesulphonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro mutagenicity:

In vitro mutagenic activity was reported in bacterial reverse mutation assays conducted according to OECD TG 471. In mammalian cells, no mutagenic activity was reported in a recent guideline compliant study conducted according to OECD TG 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 80 μg/mL used in the range finding pre-experiment was limited by cytotoxicity of the test item. The concentration range of the main experiments was also limited by cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in any part of the main experiments with and without metabolic activation. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion on in vitro mutagenicity:

In conclusion it can be stated that the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, N-Phenyl-N-[(trichloromethyl)thio]benzene sulphonamide is considered to be non-mutagenic.

In vitro clastogenicity:

The micronucleus test showed a biologically relevant increase in the frequencies of micronucleus containing V79 cells treated with the test item in the absence or in the presence of S9 mix (4 hours treatment). Thus, an independent repeat experiment with extended treatment time in the absence of S9 mix was not performed in accordance with OECD 487. Evaluation of the data indicates that Vulkalent E is a mutagen in the micronucleus test in vitro, when tested up to cytotoxic concentrations in the absence or presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the testing of chemicals 487. In vitro mammalian cell micronucleus test OECD (2010)

Deviations:

no

Principles of method if other than guideline:

The purpose of this study was to investigate whether Vulkalent E (N-phenyl-N-[(trichloromethyl)thio]benzenesulphonamide, CAS no.: 2280-49-1) can induce chromosome breakage (structural chromosomal aberrations) or misdistribution of chromosomes leading to aneuploidy, both of which are measured by an increase of the frequency of micronuclei containing mammalian cells in the absence and presence of an extrinsic metabolizing system.

Vulkalent E, dissolved in tetrahydrofuran, was examined for mutagenic activity in the micronucleus test in vitro. The 4 hours treatment was conducted with concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2.5 and 5 μg/mL without S9 mix and of 0.5, 1, 2.5, 5, 10, 15, 20, 25 and 100 μg/mL with S9 mix.

The in vitro mammalian cell micronucleus test was conducted according to the OECD Guideline for the testing of chemicals 487 (2010).

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

When cytogenetic damages leading to chromosomal breakage (clastogenic effects) or
misdistribution of chromosomes (aneugenic effects) are induced during mitosis, chromosomal
fragments or a whole chromosome may be separated from the main nucleus. In interphase the
separated fragment or chromosome can form a tiny nucleus (micronucleus) that exists
independently of the main nucleus in the cytoplasm.
Micronuclei can be seen in a wide variety of cell types. In the micronucleus test employed in
the present study in vitro cultivated V79 cells were used. V79 cells were derived from fetal
lung tissue of Chinese hamsters and are one of the cell lines most widely used for
mutagenicity studies (3). In common with all cell lines they do not possess the full ability of
mammals to activate promutagenic and procarcinogenic compounds. To overcome this
deficiency the compounds are tested in the presence of an exogenous metabolizing system.
Postmitochondrial supernatant fraction from liver of Aroclor 1254-treated male rats and a
NADPH-generating system have been successfully used in prokaryotic and eukaryotic in vitro
systems for the activation of various compounds.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Postmitochondrial supernatant fraction from liver of Aroclor 1254-treated male rats (S9).

Test concentrations with justification for top dose:

Vulkalent E, dissolved in tetrahydrofuran, was examined for mutagenic activity in the
micronucleus test in vitro. The 4 hours treatment was conducted with concentrations of 0.01,
0.05, 0.1, 0.25, 0.5, 0.75, 1, 2.5 and 5 μg/mL without S9 mix and of 0.5, 1, 2.5, 5, 10, 15, 20,
25 and 100 μg/mL with S9 mix.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Vulkalent E, dissolved in tetrahydrofuran, was examined for mutagenic activity in the

micronucleus test in vitro. The 4 hours treatment was conducted with concentrations of 0.01,

0.05, 0.1, 0.25, 0.5, 0.75, 1, 2.5 and 5 μg/mL without S9 mix and of 0.5, 1, 2.5, 5, 10, 15, 20,

25 and 100 μg/mL with S9 mix.

Without S9 mix cytotoxic effects were observed at 0.25 μg/mL and above after 4 hours

treatment. With S9 mix cytotoxic effects were observed at 15 μg/mL and above. Precipitation

in the medium could be observed starting at 100 μg/mL.

Therefore, concentrations of 0.1, 0.25 and 0.5 μg/mL (without S9 mix, 4 hours treatment) 2.5,

10 and 15 μg/mL (with S9 mix, 4 hours treatment) were chosen for reading. Higher

concentrations were excluded from evaluation for micronuclei due to excessive cytotoxicity.

Solvent control (tetrahydrofuran) and appropriate positive controls with known mutagens

(mitomycin C, cyclophosphamide) demonstrated the suitability and sensitivity of the test

system.

The micronucleus test showed a biologically relevant increase in the frequencies of

micronucleus containing V79 cells treated with the test item in the absence or in the presence

of S9 mix (4 hours treatment). Thus, an independent repeat experiment with extended

treatment time in the absence of S9 mix was not performed in accordance with OECD 487.

Evaluation of the data indicates that Vulkalent E is a mutagen in the micronucleus test in

vitro, when tested up to cytotoxic concentrations in the absence or presence of metabolic

activation.

Executive summary:

The micronucleus test showed a biologically relevant increase in the frequencies of

micronucleus containing V79 cells treated with the test item in the absence or in the presence

of S9 mix (4 hours treatment). Thus, an independent repeat experiment with extended

treatment time in the absence of S9 mix was not performed in accordance with OECD 487.

Evaluation of the data indicates that Vulkalent E is a mutagen in the micronucleus test in

vitro, when tested up to cytotoxic concentrations in the absence or presence of metabolic

activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver microsome preparations (S9 mix)

Test concentrations with justification for top dose:

The highest concentration of 80 μg/mL used in the range finding pre-experiment was limited by cytotoxicity of the test item. The concentration range of the main experiments was also limited by cytotoxicity.

0, 1.3, 2.5, 5, 10, 20, 40, 80 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

First experiment: 0.15 μg/mL and above without metabolic activation and at 24.0 μg/mL with metabolic activation. Second experiment: above were noted at 0.45 μg/mL and above without metabolic activation and at 24.0 μg/mL and above with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: N-Phenyl-N-[(trichloromethyl)thio]benzene sulphonamide is considered to be non-mutagenic in this HPRT assay.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest concentration of 80 μg/mL used in the range finding pre-experiment was limited by cytotoxicity of the test item. The concentration range of the main experiments was also limited by cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in any part of the main experiments with and without metabolic activation.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, N-Phenyl-N-[(trichloromethyl)thio]benzene sulphonamide is considered to be non-mutagenic in this HPRT assay.

Executive summary:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, N-Phenyl-N-[(trichloromethyl)thio]benzene sulphonamide is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientifically reliable; non GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

4 bacterial straines tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine synthesis

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 derived from male rat liver

Test concentrations with justification for top dose:

12500, 2500, 500, 100, 20 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Remarks:

Endoxan, Trypaflavin, 2-Amino-anthrazen

Positive control substance:

other: Endoxan, Trypaflavin, 2-Amino-anthrazen

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Cytotoxicity was observed at concentrations above 500 µg/plate

precipitation was observed at or above 2500µg/plate

Biological significant increase in mutationrats were observed at or above 20 µg/plate for TA 1535, TA 100 and TA 98 and at or above 100 µg/plate for TA 1537

Executive summary:

Biologically and statistically increase in mutation rate was observed.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientifically reliable; non GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

4 bacterial straines tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine synthesis

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 derived from male rat liver

Test concentrations with justification for top dose:

12500, 2500, 500, 100, 20 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Remarks:

Endoxan, Trypaflavin, 2-Amino-anthrazen

Positive control substance:

other: Endoxan, Trypaflavin, 2-Amino-anthrazen

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Cytotoxicity was observed at concentrations above 500 µg/plate

precipitation was observed at or above 2500µg/plate

Biological significant increase in mutationrats were observed at or above 20 µg/plate for all strains tested

Executive summary:

Biologically and statistically increase in mutation rate was observed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test item N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD TG 474.

Under the experimental conditions reported, N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat. Therefore, N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide is considered to be nonmutagenic in this in vivo micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The study was performed to investigate the potential of N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD TG 474.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

orally once by gastric gavage

Remarks:

Doses / Concentrations:
24 h preparation interval: 125, 250, and 500 mg/kg b.w.. 48 h preparation interval: 500 mg/kg b.w..
Basis:
nominal conc.

No. of animals per sex per dose:

Number of animals for the main study: 42 males

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

24 h and 48 h after a single administration of the test item in the bone marrow cells were collected for micronuclei analysis.
Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 4000 polychromatic erythrocytes were scored for micronuclei.
To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Systemic toxicity; ruffled fur, reduced spontaneous activity and eyelid closure in animals treated with the high dose of test item

Vehicle controls validity:

valid

Remarks:

The vehicle of the test item was used as negative control.

Negative controls validity:

valid

Positive controls validity:

valid

Results

Calculation and Results of Individual Data

Pre-Experiment for Toxicity

The animals treated in the pre-experiments received the test item N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide suspended in corn oil once orally. The volume administered was 10 mL/kg b.w.. The following dose level was tested and expressed

clinical symptoms are shown in the table:

	hours post-treatment
Clinical Symptoms	0 - 1	2-4	5-6	24	30	48
1st Pre-experiment: 500 mg/kg b.w.; 2 males / 2 females
Reduction of spontaneous activity	0/0	1/2	1/2	2/1	1/0	0/0
Isolation (Retreat)	0/0	1/0	1/0	0/0	0/0	0/0
Ruffled fur	0/0	2/2	2/2	2/1	2/1	2/1
Hunchback	0/0	1/1	1/0	0/0	0/0	0/0
Eyelid closure (partially)	0/0	1/1	1/1	1/0	0/0	0/0

On the basis of these data 500 mg/kg b.w. were estimated to be suitable.

No substantial differences between sexes in toxicity were observed, so that only male animals were used in the main experiment.

Clinical examinations in the Main Experiment

In the main experiment for each test item dose group 7 males received the test item NPhenyl-N-[(trichloromethyl)thiobenzene-sulphonamide suspended in corn oil once orally.

The volume administered was 10 mL/kg b.w.. The clinical symptoms observed following treatment are shown in the following table for each dose group, which indicates the number of animals with findings.

	hours post-treatment (males)
Clinical symptoms	1	2-4	approx 5	24	48
High dose: 500 mg/kg b.w. (14 males at 1 to 24 h; 7 males at 48 h)
reduction of spontaneous activity	0	7	8	1	0
eyelid closure (partially)	0	4	4	0	0
ruffled fur	0	11	14	14	2
Medium dose: 250 mg/kg b.w. (7 males)
ruffled fur	0	3	4	0	0

The animals treated with the vehicle control (corn oil) and the low dose of the test item did

not express any clinical symptoms.

Summary of Micronucleus Test Results

TestGroup	Dosemg/kgb.w.	samplingtime	meanMN/4000PCE	SDMN/4000PCE	mean %MN/4000	Range MN		RatioPCE/totalEry	PCE% ratioVehicle
						min	max
Corn Oil	0	24	12.0	7.4	0.3	1	22	0.547	100.00
Dose 1	125	24	8.3	4.9	0.2	4	18	0.510	93.24
Dose 2	250	24	10.6	5.3	0.3	5	18	0.474	86.65
Dose 3	500	24	10.3	4.0	0.3	4	17	0.511	93.42
Positive	20	24	68.6	30.7	1.7	47	118	0.432	78.98
Dose 3	500	48	4.7	1.8	0.1	3	8	0.481	87.93

MN = micronuclei

Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw.

Negative control versus test group	Significance	p
Dose 1 - 125 mg N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide/kg b.w.;24 h	-	0.672
Dose 2 - 250 mg N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide/kg b.w.;24 h	-	1.000
Dose 3 - 500 mg N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide/kg b.w.;24 h	-	1.000
Positive Control - 40 mg CPA/kg b.w.; 24 h	+	0.010
Dose 3 - 500 mg N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide/kg b.w.;48 h	-	0.136

- = not significant

+ = significant

Discussuion

The test item N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD TG 474.

The test item was suspended in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w..

24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 4000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 125, 250, and 500 mg/kg b.w..

48 h preparation interval: 500 mg/kg b.w..

As estimated by a pre-experiment 500 mg N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide per kg b.w. was suitable as highest treatment dose.The maximal tolerated dose (MTD) is based on substantial clinical symptoms in the main experiment which included ruffled fur, reduced spontaneous activity and eyelid closure in the animals treated with the high dose of test item. Animals treated with the mid dose level exhibited ruffled fur only. Furthermore, body weight loss was observed in many of the animals treated with the high dose of the test item, and also in some animals treated with the mid dose of the test item. The animals treated with the low dose and the vehicle control did not exhibit

any clinical symptoms or loss in body weight.

The mean number of polychromatic erythrocytes (PCE) was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide did not have any cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item.

The mean values of micronuclei observed after treatment with N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide were below to the value of the vehicle control group.

20 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial and statistically significant increase of induced micronucleus

frequency (p>0.01).

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, it can be stated that under the experimental conditions reported, the test item N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat.
Therefore, N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide is considered to be nonmutagenic in this in vivo micronucleus assay.

Executive summary:

This study was performed to investigate the potential of N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD TG 474.

The test item was suspended in corn oil, which was also used as vehicle control. The dose volume administered orally was 10 mL/kg b.w..

24 h and 48 h after a single administration of the test item in the bone marrow cells were collected for micronuclei analysis.

Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 4000 polychromatic erythrocytes were scored for micronuclei.

To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 125, 250, and 500 mg/kg b.w..

48 h preparation interval: 500 mg/kg b.w..

The highest dose (500 mg/kg b.w.) was estimated by a pre-experiment. This maximal tolerated dose (MTD) is based on substantial clinical symptoms in the main experiment which included ruffled fur, reduced spontaneous activity and eyelid closure in animals treated with the high dose of test item. Animals treated with the mid dose level exhibited ruffled fur only. Furthermore, body weight loss was observed in many of the animals treated with the high dose of the test item, and also in some animals treated with the mid dose of the test item. The animals treated with the low dose and the vehicle control did not exhibit any clinical symptoms or loss in body weight.

The observed systemic toxicity at the tested doses is indicative for a systemic distribution of the test item. Thus, bioavailability of the test item under the tested conditions is assumed.

After treatment with the test item the number of polychromatic erythrocytes (PCE) was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronulclei at any preparation interval after administration of the test item and with any dose level used. 20

mg/kg b.w. cyclosphosphamide administered orally was used as positive control which induced a substantial and statistically significant increase in cells with micronuclei (p<0.01).

In conclusion, it can be stated that under the experimental conditions reported, the test item N-Phenyl-N-[(trichloromethyl)thio] benzenesulphonamide did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat.

Therefore, N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide is considered to be nonmutagenic in this in vivo micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide is positive in the bacterial reverse mutation assay and an in-vitro MNT.

The compound was negative in a cell mutation assay in V79 cells (OECD TG 476).

N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD TG 474.

Under the experimental conditions reported, N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat. Therefore, N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide is considered to be nonmutagenic in this in vivo micronucleus assay.

Overall, the compound is not considered to be mutagenic.

Additional information

In vitro mutagenicity:

In vitro mutagenic activity was reported in bacterial reverse mutation assays conducted according to OECD TG 471. In mammalian cells, no mutagenic activity was reported in a recent guideline compliant study conducted according to OECD TG 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 80 μg/mL used in the range finding pre-experiment was limited by cytotoxicity of the test item. The concentration range of the main experiments was also limited by cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in any part of the main experiments with and without metabolic activation. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion on in vitro mutagenicity:

In conclusion it can be stated that the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, N-Phenyl-N-[(trichloromethyl)thio]benzene sulphonamide is considered to be non-mutagenic.

In vitro clastogenicity:

The micronucleus test showed a biologically relevant increase in the frequencies of micronucleus containing V79 cells treated with the test item in the absence or in the presence of S9 mix (4 hours treatment). Thus, an independent repeat experiment with extended treatment time in the absence of S9 mix was not performed in accordance with OECD 487. Evaluation of the data indicates that Vulkalent E is a mutagen in the micronucleus test in vitro, when tested up to cytotoxic concentrations in the absence or presence of metabolic activation.

In-vivo mutagenicity:

The test item N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD TG 474.

Under the experimental conditions reported, N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat. Therefore, N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide is considered to be nonmutagenic in this in vivo micronucleus assay.

Overall, the compound is considered to be not mutagenic

Justification for classification or non-classification

N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide is positive in the bacterial reverse mutation assay and an in-vitro MNT.

The compound was negative in a cell mutation assay in V79 cells (OECD TG 476).

N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD TG 474.

Under the experimental conditions reported, N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat. Therefore, N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide is considered to be nonmutagenic in this in vivo micronucleus assay.

Overall, the compound is considered to be not mutagenic.