4-Methyl-N-[[[3-(trifluoromethyl)phenyl]amino]carbonyl] benzenesulfonamide

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

Study period:

29 September 2020 to 28 January 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-Methyl-N-[[[3-(trifluoromethyl)phenyl]amino]carbonyl] benzenesulfonamide

EC Number:

850-929-8

Cas Number:

1584-79-8

Molecular formula:

C15H13F3N2O3S

IUPAC Name:

4-Methyl-N-[[[3-(trifluoromethyl)phenyl]amino]carbonyl] benzenesulfonamide

Test material form:

solid

Test animals

Species:

mouse

Strain:

ICR

Remarks:

Crl:CD1

Details on species / strain selection:

Mice, Crl:CD1 (ICR), male and female, from Cahrles River Laboratories Japan (Hino Breeding Center).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Preliminary test (1st): 7 weeks old, male, 14 mice; 6 weeks old, male, 14 mice
Preliminary test (2nd): 7 weeks old, male, 17 mice; 6 weeks old, male, 17 mice
Micronucleus test: 7 weeks old, male, 32 mice


Animals were observed at receipt and healthy mice were quarantined and acclimatized for 6 days. The clinical signs were observed daily during the quarantine and acclimation periods. Body weights were measured at animal receipt, completion of quarantine.

Environmental conditions:
Temperature: 21-25°C
Relative Humidity: 40-70%
Air change rate: 10-15 times/hour
Light cycle: light on 12 hours (07:00-19:00)
Diet: MF pellets ad libitum
Water: ad libitum (3-5 ppm sodium hypochlorite)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

Administration was repeated twice with a 24-hour interval by the oral gavage. The test substance formulations were dispensed using a 1 mL disposable syringe with a disposable stomach sonde for mouse. Administration was carried out at 10mL/kg based on the body weight of the administration dose. The test substance formulation was collected while stirring with a magnetic stirrer.

Duration of treatment / exposure:

For the first preliminary test, animals died at all test substance doses in both sexes within the first day after administration. Therefore, no second treatment was conducted. For the second preliminary test and the micronucleus test the administration was done twice with a 24-hour interval.

Frequency of treatment:

once per day for two days

Post exposure period:

Animals were observed frequently until 1 hour after each administration. In addition, the animals were observed at 1 day after each administration. Body weights were measured from the first administration day to the day after the second administration, i.e. once for 3 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg diet

Doses / concentrations 2

Dose / conc.:

125 mg/kg diet

Doses / concentrations 3

Dose / conc.:

250 mg/kg diet

Doses / concentrations 4

Dose / conc.:

500 mg/kg diet

No. of animals per sex per dose:

5 male animals per dose
5 male animals for the negativea and positive control respectively.

Control animals:

yes

Positive control(s):

Mitomycin C was administered at 10 mL/kg twice at a 24-hour interval by oral gavage.

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

The femur was removed, and the bone marrow cells were collected with approximately 0.8 mL of the heat-inactivated fetal bovine serum into a centrifuge tube, ant the tube was centrifuged at 1000 rpm for 5 minutes. A small amount of the cell suspension was smeared on a glass slide. The smears were completely air-dried and fixed with methanol. then, the specimens were stained with 3 vol% Giemsa solution prepared with 1/15 mol/L phosphate buffer solution (pH 6.8) and treated with 0.004 w/V% citrate aqueous solution.

Evaluation criteria:

The specimens of the negative and the positive control groups were observed, Those of the test substance group of 125, 250 and 500 mg/kg/day were observed. Slide numbers were allocated randomly to specimens and were observed microscopically (*1000) in a coded manner.
(1) Frequency of micronuclei: Four thousand polychromatic erythrocytes (PCE) per animal (2000 PCE per specimen) were observed and the frequency of micro nucleated polychromatic erythrocytes (MNPCE) (MNPCE/PCE) was calculated.
(2= Growth inhibition of bone marrow cells: A total of 500 erythrocytes (TE) per animal (250 TE per specimen) were observed and the ratio of PCE to TE was calculated.

Statistics:

Because the MNPCE/PCE in all doses of test substance was within the range of the historical data of the negative control, the Conditional Binomial test (Kastenbaum and Bowman) was not conducted to compare the MNPCE/PCE in the test substance group with that in the negative control group. The Conditional Binomial test was conducted to compare the MNPCE/PCE in the positive control group with that in the negative control group. The Conditional Binomial test was conducted at upper-tailed significance levels of 5% and 1%.
Judgement of PCE/TE: First, all the data except for the positive control group were tested by Bartlett´s test for homogeneity of variance. Since the variances were homogeneous, Williams´ test was performed. Since there were significant differences with Williams´ test, Dunnett´s test was not performed. The comparisons between the negative and the positive control groups were tested by the F test of homogeneity of variance between the groups. since the results of the F tst showed heterogeneous, Aspin Welch´s t test was performed to compare the mean in the positive control group with that in the negative control group. Significance levels of the tests were 5% for Bartlett´s test and for the F test, both sides of 5% for Williams´ test, and both sides of 5% or 1% for the other tests.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

In the preliminary test at higher concentrations the tested animals died. Concentrations were adjusted for the second preliminary test and the micronucleus test itself.

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It was concluded that the test item did not induce the micronuclei under the present test conditions.

Executive summary:

The means of MNPCE/PCE in the negative and the positive control groups were within the range of the respective historical data in the testing facility. The mean of MNPCE/PCE in the positive control showed a statistically significant increase at both sides of 1 % level compared with the negative control group. In the test substance group, the PCE/TE was 20% or more compared with that of the negative control group at all observation doses. Therefore, it was confirmed that the test was conducted appropriately.
The means of MNPCE/PCE of the test substance group at all observation doses were within the range of the historical data of the negative control. Therefore, the results were judged to be negative.
In the PCE/TE, since no statistically significant decreases were observed in any doses of the test substance group compared to the negative control group, the exposure of the test substance to bone marrow cells was not proved. However, it was considered that the potential to induce micronuclei of the test substance could be evaluated adequately, since the highest observed dose was selected to be MTD in the present test.