6-methyl-2-oxoperhydropyrimidin-4-ylurea

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1998-09-03 to 1999-11-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP. Since this is a read-across from a structural analogue substance (CAS 6104-30-9), the reliability was set from RL1 to RL2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

HESSISCHES MINISTERIUM FÜR UMWELT, ENERGIE, JUGEND, FAMILIE UND GESUNDHEIT, Wiesbaden, Germany

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N''-(isobutylidene)diurea

EC Number:

228-055-8

EC Name:

N,N''-(isobutylidene)diurea

Cas Number:

6104-30-9

IUPAC Name:

N,N''-(2-methylpropane-1,1-diyl)diurea

Details on test material:

- Name of test material (as cited in study report): Isobutylidenediurea (Isobutylidendiharnstoff BG-No. 204)
- Physical state: white powder
- Analytical purity: 90.1 weight %
- Lot/batch No.: 1/1/998
- Expiration date of the lot/batch: the stability of the test item over a period of 10 months was determined analytically
- Stability under test conditions: at least 3 h in 0.5% CMC (vehicle)
- Storage condition of test material: at room temperature
-Supplier: BASF AG, Ludwigshafen, Germany
- Date of synthesis: 1998-04-07

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Füllingsdorf, Switzerland
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 35.0±2.1 g
- Assigned to test groups randomly: yes (not further specified)
- Fasting period before study: 18 h
- Housing: individually in Makrolon Type I cages with wire mesh top (EHRET GmbH, Emmendingen, Germany), bedded with granulated soft wood (ALTROMIN, Lage/Lippe, Germany)
- Diet: pelleted standard diet (ALTROMIN, Lage/Lippe, Germany), ad libitum
- Water: tap water (Gemeindewerke, Roßdorf, Germany), ad libitum
- Acclimation period: at leaast 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 22-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: due to its non-toxicity to the animals
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw (administered in two steps within 24 h)
- Purity: 0.5% aqueous suspension

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test susbtance was formulated in 0.5% CMC.

Duration of treatment / exposure:

24 h

Frequency of treatment:

two applications within 24 h

Post exposure period:

24 h

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

6 males

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA (cyclophosphamide)
- Analytical purity: at least 98%
- Supplier: SIGMA-Aldrich Vertriebs-GmbH, Deisenhofen, Germany
- Route of administration: single oral gavage
- Doses / concentrations: 40 mg/kg bw
- Volume administered: 10 mL/kg bw
- Vehicle: deionised water

Examinations

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The doses applied were selected on the basis of the results of a preliminary acute toxicity study conducted with 3 males and 3 females at doses of 1000, 1500, and 2000 mg/kg bw. The experimental conditions concerning animals strain, vehicle, rout of exposure, frequency of administration, and application volume were the same in the toxicity pre-test as compared to the main genotoxicity study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals received a total of 10 mL/kg bw test material formulation via two oral gavages within a time period of 24 h. The animals were sacrificed 24 h after the last test material administration and submitted to the cell sampling procedure.

DETAILS OF SLIDE PREPARATION:
The slides were air-dried and then stained with May-Grünwald/Giemsa-solution. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
The analysis was performed with coded slides using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed as quotient PCE/NCE.

Evaluation criteria:

The study is considered valid as the following criteria are met:
- the vehcilce controls are in the range of the laboratorie's historical control data 0.04-0.16% PCEs with micronuclei (October 1997 - October 1998, males)
the positive controls show statistically significant increased values (historical range: 1.27-2.82% PCEs with micronuclei (October 1997 - October 1998, males)
- more than 80% of the animals are evaluable

A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this test system.
This can be confirmed by means of the non-parametric Whitney test. However, both biological and statistical significance should be considered together.

Statistics:

The non-parametric Mann-Whitney test was applied in this study.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2000 mg/kg bw (high dose) produced signs of toxicity

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- study type: acute toxicity study
- Dose range: 1000, 1500, and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: reduction of spontaneous activity, eyelid closure, and apathy

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no
- Ratio of PCE/NCE: no dose-related effect on the PCE/NCE ratio observed, which would indicate cytotoxicity
- Appropriateness of dose levels and route: yes (clinical signs of toxicity were observed, indicating systemic availability of the test article)
- Statistical evaluation: no statistically significant increase in micronucleated erythrocytes was observed using the non-parametric Mann-Whitney Test

Any other information on results incl. tables

Tab. 1: Summary of the Micronucleus Test Result (24 h sampling time)

Test group	Dose [mg/kg bw]	PCEs with micronuclei [%]	Range	PCE/NCE
Vehicle control	-	0.130	0-8	2000/1922
Test article	500	0.040	0-3	2000/1922
	1000	0.090	0-3	2000/2217
	2000	0.150	2-5	2000/2017
Positive control	40	1.45	16-46	2000/1996

Vehicle control: CMC (carboxymethyl cellulose), Positive control: CPA (cyclophosphamide)

PCE = polychromatic erythrocytes; NCE = normochromatic erythrocytes

Tab. 2: Results of the pre-experiment for toxicity

Toxic reactions	Hours after first gavage			Hours after second gavage
	1 h	6 h	24 h	1 h	6 h	24 h
2000 mg/kg bw
Reduction of spontaneous activity*	2	3	3	3	3	3
Eyelid closure*	1	1	3	2	2	1
Apathy*	1	2	3	2	2	1
1500 mg/kg bw
Reduction of spontaneous activity (males/females)	2/1	3/3	3/3	3/3	3/3	2/3
Eyelid closure (males/females)	0/0	2/3	2/2	3/3	3/3	1/2
Apathy (males/females)	1/0	2/2	2/2	3/3	3/3	1/1
1000 mg/kg bw
Reduction of spontaneous activity (males/females)	1/2	3/3	3/3	3/3	3/3	3/3
3/3Eyelid closure (males/females)	0/0	2/3	3/3	2/2	3/3	1/2
Apathy (males/females)	0/0	2/2	3/3	2/2	2/3	1/1

* only males tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative