Tetrabutylurea

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrabutylurea

EC Number:

224-929-8

EC Name:

Tetrabutylurea

Cas Number:

4559-86-8

Molecular formula:

C17H36N2O

IUPAC Name:

1,1,3,3-tetrabutylurea

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

other: CD / Crl: CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

Supplier: Charles River Laboratories, Germany
Number of animals: 70 (35 males and 35 females), i.e. 5 males and 5 females per group
Age (at start of administration): 31-33 days
Body weight: 86-122 g
Acclimatisation period: At least 5 adaptation days
Diet: Commercial ssniff® R/M-H V1534. Feeding was discontinued approx. 16 hours before administration; only tap water was then available ad libitum.
Housing: The animals were kept in groups of 2 - 3 by sex in MAKROLON cages (type III plus) at a room temperature of 22 +/- 3°C (maximum range) and a relative humidity of 55 +/- 15% (maximum range).
The rooms were lit (about 150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each.
Drinking water: ad libitum

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.8% aqueous hydroxypropylmethylcellulose

Details on exposure:

Administration volume: 20 mL/kg b.w.

Preliminary test
The dose levels had been selected based on a preliminary oral acute toxicity study employing one animal per sex and dose. Three dose levels of 500, 1000 and 2000 mg Tetrabutylurea/kg b.w. were tested. The administration volume was 20 mL/kg b.w. No signs of systemic toxicity were noted up to the highest dose level of 2000 mg/kg b.w.
Hence, three ascending doses of 500, 1000 and 2000 mg Tetrabutylurea/kg b.w. were employed.

Main study
Three dose levels are used for the first sampling time (24 h). The dose levels should cover a range from the maximum toxicity to little or none. Only the highest dose is used at the later sampling time (48 h). The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. The highest dose may also be defined as the dose that produces some indication of toxicity of the bone marrow (e.g. reduction of the proportion of immature erythrocytes among total erythrocytes in the bone marrow). If no toxicity occurs 2000 mg/kg b.w. is the recommended highest reasonable dose level.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

24 or 48 h

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 1000 or 2000 mg/kg bw
Basis:

No. of animals per sex per dose:

5 m / 5 f

Control animals:

yes, concurrent vehicle

Positive control(s):

Negative reference item
0.8% aqueous hydroxypropylmethylcellulose
Route of administration: Oral by gavage
Administration volume: 20 mL/kg b.w.

Positive reference item
Cyclophosphamide
Dose level: 27 mg/kg b.w.
Route of administration: Intraperitoneal
Vehicle: 0.9% NaCl solution
Administration volume: 20 mL/kg b.w.

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

Bone marrow smears were prepared from all treatment groups 24 hours post administration. Additional samples were prepared from the vehicle control and the high dose group 48 hours post administration. The femurs were excised below the knee and at the iliac joint. The bone marrow was flushed out with calf serum and centrifuged at 850 x g for 3 to 5 minutes. The supernatant was removed and the sediment resuspended in a drop of calf serum by using a Pasteur Pipette. Then a smear of 30 to 60 mm length was prepared. After air drying, the preparations were immediately fixed in methanol for 5 minutes. Cells were stained for 6 minutes using filtered Mayers Haemaleum. The slides were rinsed with cold tap water for 5 minutes and then further stained in 0.5% w/v ethanolic eosin solution for 1 minute. The slides were again left to air-dry before being dipped in xylene and mounted.

Analysis
The slides were coded and randomised before microscopic analysis. Two thousand polychromatic erythrocytes per animal were scored for the incidence of micronuclei. The ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was determined for each animal by counting a total of thousand erythrocytes.

Evaluation criteria:

The test chemical was considered as clearly positive in this assay if:
a statistically significant increase in the frequency of micronucleated PCE occurred for at least one dose at one kill time
the frequency of micronucleated PCE at such a point exceeded the historical control range
corroborating evidence was obtained, for example, increased but statistically insignificant frequencies or micronucleated PCE at other doses or kill times, or dose response profiles.

Statistics:

yes

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Tetrabutylurea tested up to the highest reasonable dose level of 2000 mg/kg b.w. by single oral administration showed no mutagenic properties in the rat bone marrow micronucleus test at the two tested sampling times of 24 hours and 48 hours.

Executive summary:

Tetrabutylurea was assayed in an in vivo bone marrow micronucleus test in the rat for the detection of damage to the chromosomes or the mitotic apparatus following single oral administration of the test item. The dose levels had been selected based on a preliminary oral acute toxicity study employing one animal per sex and dose. Three dose levels of 500, 1000 and 2000 mg Tetrabutylurea/kg b.w. were tested. The administration volume was 20 mL/kg b.w. No signs of systemic toxicity were noted up to the highest dose level of 2000 mg/kg b.w. For the main study three ascending doses of 500, 1000 and 2000 mg Tetrabutylurea/kg b.w., p.o. were administered. Further groups received the vehicle (0.8% aqueous hydroxypropylmethylcellulose) and one further group the positive reference item cyclophosphamide (27 mg/kg b.w., i.p.). Each group consisted of 5 male and 5 female rats. Immediately after sacrifice, bone marrow smears were prepared. Two sampling times were employed in this study: 24 hours after administration, samples were prepared from the vehicle, positive reference item and all 3 doses of test item-treated animals; 48 hours after administration, samples were prepared only from the vehicle control and high dose-treated animals. Two thousand (2000) erythrocytes were evaluated per animal.

No signs of systemic toxicity were noted up to the highest dose level of 2000 mg/kg b.w. (sacrifice after 24 or 48 hours). Tetrabutylurea did not increase the incidence of micronucleated polychromatic erythrocytes (PCE) at any of the three tested dose levels of 500, 1000 or 2000 mg/kg b.w. The incidences of PCEs combined for male and female animals were 0.7 or 0.8 micronuclei per 1000 PCEs for the samples collected 24 and 48 hours post administration. The corresponding value for vehicle control (negative reference) was 1.0 or 0.6 micronuclei per 1000 PCEs (sacrifice after 24 or 48 hours, respectively). Administration of cyclophosphamide (positive reference) significantly increased the number of micronuclei to 19.6 per 1000 PCEs.