N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

EC Number:

876-151-9

Cas Number:

2292123-68-1

Molecular formula:

C27H24N2O7S2

IUPAC Name:

N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

Test material form:

solid: particulate/powder

Test animals

Species:

mouse

Strain:

ICR

Remarks:

Crl:CD1

Details on species / strain selection:

As required by the guideline. Mice are used widely in micronucleus test because the observation of the micronuclei is easy and the historical data are abundant

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan
- Age at study initiation: 7 weeks
- Weight at study initiation: male (26.5 - 31.3g), female (23.0 - 24.0g)
- Assigned to test groups randomly: yes, under following basis: body weight stratified random sampling method on the day before administration
- Fasting period before study: Not applicable
- Housing: Polycarbonate cages with flat bottom, bedding and enrichment
- Diet (e.g. ad libitum): MF pellets. ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 40-70
- Air changes (per hr): 10-15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item (4.00g) was weighed and suspended in corn oil using a laboratory mixer and ultrasonicator to make a 20 mL of 200mg/mL test item formulation. This formulation was serially diluted with corn oil to prepare 100 and 50.0 mg/mL test item formulations using a magnetic stirrer.

The test item formulations were prepared on the first administration day, stored within a cold place and used within 2 days after preparation.

Duration of treatment / exposure:

Not applicable for dosing by gavage.

Frequency of treatment:

2 doses in a single 24 hour period.

Post exposure period:

1 hour after second dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Mid-dose

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

Low dose - 5 males
Mid-dose - 5 males
High dose - 5 males, 3 females

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

Mitomycin C (MMC)
- Justification for choice of positive control: MMC is lasted as a positive control in the OECD guideline and historical data have been accumulated in the testing facility.
- Route of administration: intraperitoneally
- Doses / concentrations: 2 mg/kg/day

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes (PCE)
Bone marrow erythrocytes (TE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the 28-day repeated dose oral toxicity study of the item. No toxic effect was observed on male or female SD rats administered 1000 mg/kg/day (oral gavage).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Specimens were prepared 24 hours after the second administration in the test item groups. Two specimens per animal were prepared.

DETAILS OF SLIDE PREPARATION:
The animals were euthanised. The femurs were removed and bone marrow cells were collected with approx. 0.8mL of heat-inactive foetal bovine serum into a centrifuge tube, the tube was centrifugated at 1000rpm for 5 minutes. A small amount of the cell suspension was smeared on the glass slide. The smears were completely air-dried and fixed with methanol. Then, the specimen 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.

METHOD OF ANALYSIS:
The specimens of the untreated, the negative and positive controls and all three doses of the test item groups were observed. Slide number were allocated randomly to the specimens. The specimens were microscopically (x1000) in a blinded manner.
1) Frequency of micronuclei
four thousand polychromatic erythrocytes (PCE) per animal (2000 PCE per specimen) were observed and the frequency of micronucleated polychromatic erythroctyes (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 ratio of PCE to TE (PCE/TE) was calculated.

Evaluation criteria:

All the criteria of the MNPCE/PCE in case of 1-3 or 4 were fulfilled, a test item was considered to be positive:
1) outside the distribution of the historical data of the negative control group
2) the does of the test item exhibits a statistically significant increase compared with the concurrent negative control
3) the increase of the MNPCE/PCE is dose-related
4) both micronucleus test and confirmation test are fulfilled in case of 2) and 3).

Statistics:

MNPCE/PCE: The Conditional Binomial (Kastenbaum and Bowman) was conducted in order to compare the MNPCE/PCE in each dose of the test item group and the positive control group with that of the negative control group. The conditional Binomial test was conducted at upper-tailed significance levels of 5% and 1%. The conditional Binomial test was also conducted for the negative control group with the untreated control group.

PCE/TE: All data except for the positive control were tested by Bartlett's test for homogeneity of variance. Since the variance were homogeneous, Williams' test was performed. Since there were no significant differences, Dunnett's test was performed to compare the mean in the negative control group with each dose of the test item group.
The comparisons between the negative and the positive control group, and the untreated and negative control groups were tested by the F test for homogeneity of variance between the groups. Since the results of the F test showed homogeneity, Student's t-test was performed to compare the mean in the positive control group with that in the negative control group and the mean in the negative control group with that the untreated group. Significance levels of the tests were 5% for the Bartlett's test and the F test, both sides of 5% for Williams' test and both sides of 5% and 1% for the other tests.

Results and discussion

Test results

Test results 1

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean of the MNPCE/PCE in the untreated control group was within the range of the negative historical data in the testing facility. The means of MNPCE/PCE in the negative and 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 was showed a statistically significant increase at both sides of 1% level compared with the negative control group. In the test item group, the PCE/TE was 20% or more compared with that of the negative control group at all observation doses. Therefore, the results were judged to be negative.
In the PCE/TE, since no statistically significant differences were observed in any doses of the test item group compared to the negative control group, the exposure of the test item to bone marrow was not proved. However, it was considered that the potential to induce micronuclei of the test item could be evaluated adequately, since 2000 mg/kg/day that was the maximum dose in the test method was set in the present test.

Any other information on results incl. tables

Clinical signs were observed at the following time points:

until 1 hour after administration; before 2nd administration; until 1 hour after administration; one day after administration

No abnormalities were observed in male or female mice.

Table 1: Body weights of male mice in micronucleus test

 

Animal: 7 weeks old Crl:CD1 (ICR) male mice

Number of administrations: twice in a 24-hour period (positive control: single administration)

Administration route: oral administration by gavage (positive control: intraperitoneal administration)

Volume of administration: 10mL/kg

 

Administration group	Dose (mg/kg/day)	Animal no.	Body weight (g)
			The 1^st administration day	The 2^nd administration day	One day after 2^nd administration
			Before administration	Before administration
Untreated control	0	101	30.5	31.3	31.5
		102	30.0	30.4	30.5
		103	31.3	31.9	32.2
		104	26.5	26.9	27.2
		105	30.1	30.2	30.7
	Mean±S.D.		29.7 ± 1.85	30.1 ± 1.94	30.4 ± 1.92
Negative control (corn oil)	0	106	29.5	30.1	30.4
		107	29.0	29.5	30.4
		108	31.1	31.3	31.7
		109	28.5	28.7	29.3
		110	29.1	29.5	29.9
	Mean±S.D.		29.4 ± 0.99	29.8 ± 0.97	30.3 ± 0.88
Test item	500	111	29.2	29.2	29.5
		112	29.5	29.3	30.1
		113	31.5	31.6	31.8
		114	28.9	29.8	30.7
		115	30.7	31.1	31.7
	Mean±S.D.		29.9 ± 1.00	30.2 ± 1.09	30.7 ± 1.03
	1000	116	29.7	30.0	30.5
		117	29.2	29.3	29.2
		118	31.2	31.9	32.8
		119	28.3	29.0	29.5
		120	26.9	29.4	29.8
	Mean±S.D.		29.1 ± 1.60	29.9 ± 1.16	30.4 ± 1.45
	2000	121	29.7	30.1	30.1
		122	28.3	28.4	28.5
		123	30.8	30.9	31.6
		124	28.8	28.9	29.4
		125	28.0	27.2	27.8
	Mean±S.D.		29.1 ± 1.14	29.1 ± 1.45	29.5 ± 1.47
Positive control	2	126	30.3	30.9	30.9
		127	27.5	29.1	29.1
		128	29.8	30.6	30.6
		129	28.3	28.6	28.6
		130	30.2	30.7	30.7
	Mean±S.D.		29.2 ± 1.25	29.3 ± 1.23	30.0 ± 1.05

S.D.: Standard Deviation

MMC: Mitomycin C

 

Table 2: Body weights of female mice in micronucleus test

 

Animal: 7 weeks old Crl:CD1 (ICR) male mice

Number of administrations: twice in a 24-hour period (positive control: single administration)

Administration route: oral administration by gavage (positive control: intraperitoneal administration)

Volume of administration: 10mL/kg

 

Administration group	Dose (mg/kg/day)	Animal no.	Body weight (g)
			The 1^st administration day	The 2^nd administration day	One day after 2^nd administration
			Before administration	Before administration
Test Item	2000	131	24.0	23.3	23.6
		132	23.0	23.4	23.2
		133	23.5	23.3	23.7
	Mean±S.D.		23.5 ± 0.5	23.3 ± 0.06	23.5 ± 0.36

S.D.: Standard Deviation

 

Table 3: Results of observation of specimens in male mice in micronucleus test

 

Animal: 7 weeks old Crl:CD1 (ICR) male mice

Number of administrations: twice in a 24-hour period (positive control: single administration)

Administration route: oral administration by gavage (positive control: intraperitoneal administration)

Volume of administration: 10mL/kg

 

Administration group	Dose (mg/kg/day)	Sampling time after the 2^nd Administration (hours	Animal no.	PCE/TE^a)	MNPCE/PCE^b)
Untreated control	0		101	45.6	0.150
			102	44.0	0.050
			103	40.0	0.125
			104	30.8	0.100
			105	52.8	0.150
		Mean±S.D.		43.6 ± 8.08	0.115 ± 0.0418
		Max/Min.		52.8 / 30.8	0.150 / 0.050
Negative control (corn oil)	0		106	50.2	0.100
			107	54.6	0.125
			108	48.0	0.150
			109	52.0	0.075
			110	49.6	0.200
		Mean±S.D.		50.9 ± 2.52	0.130 ± 0.0481
		Max/Min.		54.6 / 48.0	0.200 / 0.075
Test item	500		111	47.4	0.125
			112	53.4	0.150
			113	44.8	0.075
			114	38.4	0.025
			115	49.2	0.050
		Mean±S.D.		46.6 ± 5.57	0.085 ± 0.0518
		Max/Min.		53.4 / 38.4	0.150 / 0.025
	1000		116	54.8	0.050
			117	43.2	0.000
			118	45.2	0.075
			119	54.0	0.175
			120	59.2	0.125
		Mean±S.D.		51.3 ± 6.80	0.085 ± 0.0675
		Max/Min.		59.2 / 43.2	0.175 / 0.000
	2000		121	66.4	0.125
			122	52.4	0.000
			123	62.4	0.225
			124	44.0	0.050
			125	52.8	0.050
		Mean±S.D.		55.6 ±8.88	0.090 ± 0.0877
		Max/Min.		66.4 / 44.0	0.225 / 0.000
Positive control	2		126	55.0	7.650
			127	53.6	8.325
			128	56.6	6.000
			129	49.0	4.650
			130	47.4	5.700
		Mean±S.D.		52.3 ± 3.95	6.465 ± 1.4966**
		Max/Min.		56.6 / 47.4	8.325 / 4.650

PCE: polychromatic erythrocytes; TE: total erythrocytes; MNPCE: Micronucleated polychromatic erythrocytes

S.D.: Standard Deviation

MMC: Mitomycin C

1. a) Five hundred erythrocytes per animal were observed


2. b) Four thousand polychromatic erythrocytes per animal were observed

**: Significant increase compared with negative control at p<0.01 [Conditional Binomial Test (Kastenbaum and Bowman)]

 

Table 4: Historical data of micronucleus test with mice in testing facility

 

Control	MNPCE/PCE (%, Values in parentheses indicate 95% control limit)
	Mean	Lower control limit	Upper control limit
Negative control	0.08	0.02 (0.02)	0.15 (0.14)
Positive control	5.38	3.38 (3.48)	7.38 (7.28)

MNPCE: Micronucleated polychromatic erythrocytes

The latest 20 test data completed by January 2020 were used

In the positive control, MMC was administered intraperitoneally with a single dose at 2 mg/kg.

Upper and lower control limits were calculated from the number MNPCE using X chart

The value was converted to the frequency from the number of MNPCE

    

Applicant's summary and conclusion

Conclusions:

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