1,1'-(methylenedi-4,1-phenylene)bis(3-butylurea)

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:

2008-09-02 to 2009-05-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

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:

-

EC Number:

416-600-4

EC Name:

-

Cas Number:

77703-56-1

Molecular formula:

C23H32N4O2

IUPAC Name:

3-butyl-1-[4-({4-[(butylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Specific details on test material used for the study:

Analytical purity: >= 99.4 %
Lot/batch No.: 05-12-06

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River; Saint-Germain-sur-l'Arbresle, France;
- Age at study initiation: 5 to 6 weeks;
- Weight at study initiation: males: 150 to 180 g; females: 150 to 187 g;
- Assigned to test groups randomly: yes;
- Fasting period before study: animals were not fasted before study begin;
- Housing: in polypropylene cages, in groups of 2 or 3; by random distribution;
- Diet: No. A04C10 irradiated rat/mouse feed; Supplier: SAFE;
- Water: drinking water softened, treated by osmosis and filtered over 0.2 µm membrane filter; ad libitum;
- Acclimation period: at least 5 days;


ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C;
- Humidity: 55 +/- 15 % R.H.;
- Air changes: 20 air changes per hour;
- Photoperiod: 12 hours light/12 hours dark from 8:00 a.m. to 8:00 p.m.;

Administration / exposure

Route of administration:

subcutaneous

Vehicle:

corn oil

Details on exposure:

Limit test: N

Duration of treatment / exposure:

2 successive administrations at 24-hour intervals;

Frequency of treatment:

2 times;

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

2000 mg/kg bw: 5 males, 5 females;
1000 mg/kg bw: 5 males, 5 females;
500 mg/kg bw: 5 males, 5 females;

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
25 mg/kg bw/day;

Examinations

Tissues and cell types examined:

Bone marrow;
immature (polychromated) erythrocytes;
micronucleated immature erythrocytes;
immature among total erythrocytes;

Details of tissue and slide preparation:

Bone marrow was extracted with foetal calf serum. Cell supensions were centrifugated for 5 minutes at 1000 rpm. Centrifugate was spread on slides. Smears were stained to make possible to distinguish between polychromatic (PCE) and normochromatic (NCE) erythrocytes. PCE are purple whereas NCE are red.

Evaluation criteria:

The mean number of micronuclei observed in the negative control animals must be within the range of the historical values for control animals. The mean number of micronuclei observed in the positive control animals treated with cyclophosphamide must show a statistically significant increase from the negative control animals and higher than the minimal historical value for positive animals. If deaths are observed at the tested dose, the mortality rate must be less than 20 % per group. The dead animals are replaced by those in parallel treatment.

Statistics:

The statistical comparison for the proportion of polychromatic among total erythrocytes and for the weight homogeneity within the sex of each group was performed using the Student's test. Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney U rank test. An analysis of a large number of control results has shown that the distribution of the numbers of micronuclei does not correspond to a Gaussian distribution, but to a Poisson-type distribution.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXICITY ASSAY
Number of sampling times :
one: for negative control and treated groups: 24 hours after the second treatment
one: for positive control group: 24 hours after single treatment

Reference substance :
cyclophosphamide, 25 mg/kg bw, intraperitoneal administration;

Number of polychromatic erythrocytes observed for each animal :
2000 for micronuclei ;
1000 for fraction of PCE among total erythrocytes (PCE+NCE).

No statistically significant decrease in the fraction of PCE among total erythrocytes (PCE+NCE) was observed (this finding provides no evidence that bone marrow cell exposure has occurred). No statistically significant increase in the number of micronuclei was noted at the doses of 2000, 1000 or 500 mg/kg bw/day (x2) in male or in female rats.

Any other information on results incl. tables

SAMPLING TIME (24 H after last treatment)	TEST ITEM   DOSES in mg/kg bw/day (x2)	SEX	Fraction of PCE* (%)		MICRONUCLEI FOR 1000 PCE
			Mean	Student’s t Test (p)	Mean	Mann Whitney (p)
Negative control Group	VEHICLE 10 mL/kg bw	M F M + F	62.9 54.2 58.5		0.70 0.60 0.65
Positive control Group	Cyclosphosphamide 25 mg/kg bw/day (x1)	M F M + F	44.4 37.8 41.1	<0.01 <0.001 <0.001	9.60 8.90 9.25	P<0.01 P<0.01 P<0.001
HAT ISO     TREATED     GROUPS	2000	M F M + F	63.3 48.0 55.7	N.S. N.S. N.S.	0.40 0.80 0.60	N.S. N.S. N.S.
	1000	M F M + F	58.7 53.8 56.3	N.S. N.S. N.S.	0.70 0.60 0.65	N.S. N.S. N.S.
	500	M F M + F	55.1 53.7 54.4	N.S. N.S. N.S.	0.60 0.50 0.55	N.S. N.S. N.S.

N.S.: Non significant at the threshold of p=0.05

PCE: Polychromatic erythocytes

NCE: Normochromatic erythocytes

*Fraction of PCE among total erythrocytes = 100xPCE /(PCE+NCE)

Applicant's summary and conclusion

Conclusions:

HAT ISO was tested for mutagenic activity in the in vivo Micronucleus test in rats. Under the experimental conditions of this test, HAT ISO induced no genotoxic activity. No classification and labelling for mutagenic activity is required according to Regulation 1272/2008/EC (CLP).

Executive summary:

HAT-ISO was investigated by means of the in vivo micronucleus test, in male and female OFA Sprague Dawley rats. Animals were treated by subcutaneous route twice with 2000, 1000 or 500 mg/kg bw/day (x2). Treatment was followed by one sampling time 24 hours after the last treatment. In conclusion, HAT-ISO induced no genotoxic activity under these experimental conditions.