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EC number: 214-447-6 | CAS number: 1129-42-6
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:
- 1 999
- Report date:
- 1999
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- (1997)
- Deviations:
- no
- 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
- 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
Constituent 1
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
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