3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

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:

30 September 2020 - 11 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Cas Number:

2102522-55-2

Molecular formula:

C20H18N2O4S

IUPAC Name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Details on test material:

Appearance: Off-white powder
Storage conditions: Room temperature (actual range: 21.0 °C to 23.7 °C) in test article storage room.

Specific details on test material used for the study:

Stability: Measured by infrared (IR absorption spectra, confirmed to be stable during the test period.
Solubility: Water, 0.03 mg/L; acetone, 100 mg/mL; DMSO, 50 mg/mL

Treated as 100% purity.

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Mouse is a species commonly used in this type of study and historical control data of this strain are available at the test facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks.
- Weight at study initiation: Preliminary assay: males, 29.7 to 33.2 g; females, 25.7 to 29.0 g. Main assay: males, 30.5 to 34.4 g.
- Assigned to test groups randomly: Yes. Using a computer system (MiTOX, Mitsui E & S Systems Research Inc.), animals were assigned to test groups based on the body weight on the day of group assignment by the stratified random sampling method so that the group mean body weight was comparable among groups. In the main assay, group assignment (including the positive control group) was performed on the day before the start of administration in the negative control and test material groups, and individual body weight of males assigned to test groups ranged from 30.5 to 34.4 g, all of which were within ± 20 % of the mean (32.17 g).
- Housing: Animals were housed in groups of five or three during the quarantine and acclimatisation period, and housed by group after assignment in polycarbonate cages (320 W x 220 D x 135 H, mm), with bedding for experimental animals
- Diet: ad libitum.
- Water: Sapporo City tap water, ad libitum.
- Acclimation period: The period up to the day of group assignment, including the quarantine period (from the day of receipt, quarantine day 1, to quarantine day 6).

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C ± 3 °C (actual range: preliminary assay and main assay; 21 - 23 °C).
- Humidity: 50 % ± 20 % (actual range: preliminary assay; 38 - 53 %, main assay; 44 - 58 %).
- Air changes: 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Artificial lighting for 12 h.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Japanese Pharmacopoeia olive oil
- Justification: Because the test material is hardly soluble in water.
- Concentration of test material in vehicle: Negative control article: 0; test material: 12.5, 25, 50, 100, 200 mg/mL
- Amount of vehicle: 10 mL/kg/time based on the body weight on each day of administration.
- Lot/batch no.: E03300

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Preparation method: The amount of test material required for each dose was accurately weighed out into an agate mortal and pulverized with an agate pestle to mix with a small amount of the vehicle. When the mixture became fluidal, it was transferred to a graduated glass using a syringe. The mortar and pestle were rinsed with the vehicle several times until the rise appeared clear and the rinse was also transferred to the graduated glass. The content of the graduated glass was stirred with a stirrer to make a suspension. To the suspension, the vehicle was added to achieve the prescribed volume and the mixture was stirred to make a homogenous suspension.
- Doses (preliminary study): 125, 250, 500, 1000, 2000 mg/kg/day.
- Doses (main study): 500, 1000, 2000 mg/kg/day.
- Preparation frequency: Dosing suspensions were prepared twice, once for each of the preliminary assay and main assay, and used within two days under refrigerated storage.
- Storage conditions: Airtight and light-resistant glass containers and refrigerated (3.1 °C - 3.6 °C).
- Administration methods and routes: administered by oral gavage using a disposable syringe and a stomach tube into the stomach.

Duration of treatment / exposure:

2 days.

Frequency of treatment:

Twice within approximately 24 h-interval for the negative control article and the test material. Once within 24 h-interval for the positive control article (MMC).

Post exposure period:

On administration days 1 and 2, animals were observed for clinical signs prior to, immediately after and 4 h after administration. On the day of slide preparation animals were observed 21 to 22h (preliminary assay and main assay) after the final administration.
On the day of slide preparation, animals were weighed 21 to 22 h (preliminary assay and main assay) after the final administration with an electronic balance.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Negative control article

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Test material

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Test material

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Test material

No. of animals per sex per dose:

Preliminary assay: 3 males and 3 females per dose
Main assay: 5 male animals per dose (negative control article, test material, positive control article).

Control animals:

yes

Positive control(s):

- Positive control: Mitomycin C (MMC)
- Route of administration: After disinfection of the right lower abdomen with 70 % ethanol swab, the dosing solution was injected into the abdominal cavity using a 25 G x 5/8" disposable injection needle and disposable syringe.
- Doses / concentrations: MMC was dissolved in the vehicle to obtain a concentration of 0.1 mg/mL. The positive control material solution was prepared at the time of use. One dose a day was administered (1 mg/kg/day).

Examinations

Tissues and cell types examined:

- 4000 immature erythrocytes for each animal (2000 immature erythrocytes for each slide).
- 500 erythrocytes (250 erythrocytes for each side) for each animal in the main assay.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- In the preliminary assay, no toxic signs (clinical sign and effects on bone marrow) or effects on the body weight were observed in males or females in any dose group. Therefore, males were selected, and 2000 mg/kg/day was set as the highest dose, and two lower doses were selected in a twofold dilution series (3 doses in total) for the main assay.

TREATMENT AND SAMPLING TIMES:
- Slides were prepared 22 to 23 h after the final administration.

DETAILS OF SLIDE PREPARATION:
- Under anaesthesia with isoflurane, animals were euthanised by cervical dislocation and then bone marrow cells in the right and left femurs were flushed with foetal bovine serum.
- The cell suspension obtained was centrifuged at 150 x g (1000 rpm) to remove surplus serum, and a part of the cell suspension obtained was smeared onto a glass slide.
- The slide was air-dried at room temperature and then fixed with methanol.
- 4 slides were prepared for each animal during the main assay.
- After fixation with methanol, 2 slides per animal were coded by personnel other than those engaged in observation of the slides.

METHOD OF ANALYSIS:
- Each slide was stained with 0.005 % acridine orange staining solution, and then washed with 1/15 mol/L phosphate buffer (pH 6.8, instant phosphate buffer).
- A glass cover slip was placed on the slide, and sealed with enamel.
- The slides were observed with a fluorescence microscope (BX50: BX-FLA, Olympus Corporation) at a total magnification of 1000x.
- For the main assay, the incidence of micronucleated immature erythrocytes (incidence of micronuclei) was calculated from 4000 immature erythrocytes for each animal (2000 immature erythrocytes for each slide).
The proporation of immature erythrocytes was calculated from 250 erythrocytes for each animal in the preliminary assay or 500 erythrocytes (250 erythrocytes for each slide) for each animal in the main assay.

Evaluation criteria:

The validity of the test was verified when the mean incidence of micronuclei in the negative and positive control groups were within the range of ± 2 SD of the mean value of respective historical control data.

The result was determined positive when the incidence of micronuclei in any test material group was outside the range of mean ± 2 SD of the mean historical control value in the negative control group, and the incidence of micronuclei in the test material group was significantly higher than in the negative control group by the conditional binominal test in a dose-response manner.

It was also determined to exclude the dose of the test material at which the proportion of immature erythrocytes was less than 20 % of that in the negative control group from the evaluation, however this case did not occur in this study.

Statistics:

Body weight measurements:
- Group means and standard deviations of body weights were calculated and the following statistical analysis was performed using MiTOX.
- The body weights in the negative control and test material groups were analysed for homogeneity of variances by the Bartlett test. When variances were homogenous (p > 0.05), data were analysed by Dunnett's test. When variances were heterogenous (p < 0.05), data were analysed by Steel's test.
- Statistically significance level was less than 5 % for comparison with the control.

Incidence of micronuclei:
- The incidences of micronuclei in all test material groups were within ± 2 SD of the mean historical control value in the negative control group therefore statistical analysis was not performed.
- For the positive control group, these were analysed using the conditional binomial test (Test by the Kastenbaum an Bowman's table) to the negative control group.
- The test was conducted with an upper-tailed significance level of 5 % (represented as p > 0.05 and p > 0.01).

Proportion of immature erythrocytes:
- The following statistical analyses were formed using EXSUS (v. 8.1) and SAS (v. 9.3).
- The proportions were analysed by the Bartlett test. When variances were homogenous (p > 0.05) data were analysed by Dunnett's test, when variances were heterogenous ( p ≤ 0.05) data were analysed by Steel's test.
- The proportions were analysed by the F test (two tailed) for homogeneity. When variances were homogenous (p > 0.05) data were compared by Student's t-test, when variances were heterogenous ( p ≤ 0.05) data were compared by Aspin-Welch's t-test (two tailed).
- Statistically significance level was less than 5 % for comparison with the control.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF PRELIMINARY TEST
- Dose range: 125, 250, 500, 1000 and 2000 mg/kg/day group.
- Clinical signs: No clinical signs were observed in males or females at any dose.
- Body weight: The mean body weights in males or females before the first administration, before the final administration and from 21 to 22 h after the final administration were similar among all doses.
- Proportion of immature erythrocytes: Similar to the negative control groups in all doses and there were no effects on bone marrow.

RESULTS OF DEFINITIVE STUDY
- Clinical sings: No clinical sings were observed in negative control group, at any dose, or the positive control group.
- Body weight: The mean body weights before the first administration, before the final administration and from 21 to 22 h after the final administration were similar to those in the negative control group with no statistically significant differences in all doses.
- The mean body weights from 21 to 22 h after administration in the positive control were similar to those before administration.

INCIDENCE OF MICRONUCLEI
- Negative control group: 0.12 % ± 0.07 % (mean ± SD, n = 5)
- 500 mg/kg/day: 0.10 % ± 0.06 %
- 1000 mg/kg/day: 0.08 % ± 0.04 %
- 2000 mg/kg/day: 0.07 % ± 0.05 %
- The values in the negative control group and test material groups were within the control values (± 2 SD of the mean value) of the historical control data of the test facility.
- Positive control group: 2.79 % ± 0.35 % (mean ± SD, n = 5), statistically significantly higher than in the negative control groups (p ≤ 0.01).
- The value in the positive control group was within the range of control values (± 2 SD of the mean value) of the historical control data of the test facility.

PROPORTION OF IMMATURE ERYTHROCYTES
- Negative control group: 53.2 % ± 2.8 % (mean ± SD, n = 5)
- 500 mg/kg/day: 52.0 % ± 3.9 %
- 1000 mg/kg/day: 55.8 % ± 6.0 %
- 2000 mg/kg/day: 55.7 % ± 8.4 %
- Positive control group: 57.1 % ± 4.2 % (mean ± SD, n = 5). Similar to that in negative control group with no statistically significant differences.

DISCUSSION
- The assay result was negative in the test material groups with no increase in the incidences of micronuclei.
- No effects of the test material on the body weights or clinical signs at any dose of the test material, or no inhibition of the bone marrow which is indicated by a decrease in the proportions of immature erythrocytes was observed.
- Though it is considered appropriate to set the high dose level that causes signs of toxicity, the 2-day successive administration of the doses used in this assay, 2000 mg/kg/day which is the upper limit dose of the single dose toxicity guideline (OECD 474), and 1000 and 500 mg/kg/day set with twofold dilution series, were likely to be sufficiently high doses to assay the potential to induce micronuclei.
- The mean incidence of micronuclei in the negative control group was within the range of the control values of the historical control data of the test facility.
- In the positive control group, the incidences of micronuclei clearly increased and the mean incidence was within the range of the control values of the historical control data of the test facility.
- The results indicated that the test system has sufficient sensitivity and that the potential of the test material to induce micronuclei was appropriately assessed.
- These results confirmed that the test material does not induce micronuclei in mouse bone marrow cells under the conditions of this assay.

Any other information on results incl. tables

Historical control data for micronucleus test (mice)

	Control data (Data: 2015.1 – 2020.9)		Positive control data (Date: 2015.1 – 2020.9)
			Mitomycin C 1 mg/kg
	% MNIE^1)	% IE^2)	% MNIE	% IE
No. of data	41	41	37	37
Mean	0.12	56.9	3.05	54.1
S.D.	0.03	2.7	0.43	4.0
Mean ± 2 S.D.	0.06 – 0.18	51.5 – 62.3	2.19 – 3.91	46.1 – 62.1
Maximum of group mean	0.17	61.8	4.11	61.8
Minimum of group mean	0.06	48.6	2.37	47.6

1) The incidence of micronucleated immature erythrocytes

2) The proportion of immature erythrocytes

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material did not induce micronuclei in mouse bone marrow cells. 

Executive summary:

The ability of the test material to induce micronuclei in mice was investigated in accordance with the standardised guidelines OECD 474, under GLP conditions.

Based on the results of the preliminary assay using male and female mice, the test material was administered by oral gavage to male mice at 500, 1000, and 2000 mg/kg/day twice with approximately 24-h intervals. Japanese Pharmacopoeia olive oil was administered to the negative control group by the same method as in the test material groups, and Mitomycin C was intraperitoneally administered once to the positive control group. Bone marrow slides were prepared 22 to 23 h after the final administration.

No clinical signs were observed in the negative control, test material or positive control.

The mean body weights at each measurement time after administration in the test material groups were similar to those in the negative control group with no statistically significant differences.

The incidence of micronucleated immature erythrocytes (incidence of micronuclei) was 0.12 % ± 0.07 % (mean ± SD, n = 5) in the negative control group and the incidences in the 500, 1000 and 2000 mg/kg/day groups were 0.10 % ± 0.06 %, 0.08 % ± 0.04 % and 0.07 % ± 0.05 %, respectively, which were within the range of the control values of the historical control data of the test facility.

The incidence of micronuclei in the positive control group was 2.79 % ± 0.35 % (mean ± SD, n = 5), which was statistically higher than in the negative control group. This indicated that this assay was performed appropriately.

The proportion of immature erythrocytes in the negative control group was 53.2 % ± 2.8 % (mean ± SD, n = 5), while the proportions in the 500, 1000 and 2000 mg/kg/day groups were 52.0 % ± 3.9 %, 55.8 % ± 6.0 % and 55.7 % ± 8.4 %, respectively, which were similar with no statistically significant differences. Thus, bone-marrow toxicity was not induced by the test material.

Under the conditions of this study, the test material did not induce micronuclei in mouse bone marrow cells.