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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

218-915-0

EC Name:

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

Cas Number:

2280-49-1

Molecular formula:

C13H10Cl3NO2S2

IUPAC Name:

N-phenyl-N-[(trichloromethyl)sulfanyl]benzenesulfonamide

Test material form:

other: solid

Details on test material:

Identification: N-Phenyl-N-[(trichloromethyl)thio]benzenesulphonamide
CAS number: 2280-49-1
Appearance: White solid
Purity: 99.6%
Storage Conditions: At room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Administration / exposure

Route of administration:

oral: gavage

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

orally once by gastric gavage

Doses / concentrations

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

Examinations

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.