1,3-divinylimidazolidin-2-one

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:

experimental study

Adequacy of study:

key study

Study period:

1995-03-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

March 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3-divinylimidazolidin-2-one

EC Number:

237-457-2

EC Name:

1,3-divinylimidazolidin-2-one

Cas Number:

13811-50-2

Molecular formula:

C7H10N2O

IUPAC Name:

1,3-diethenylimidazolidin-2-one

Details on test material:

Name of test substance : N,NI-Divinylimidazolidon
- Analytical purity: 98.2 %
- Batch No.: 229/42
- Manufacture date of the batch: January 19, 1995
- Storage: Refrigerator (protected from light, N2 conditions)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld, Germany
- Assigned to test groups randomly: yes, under following basis: computer program
- Weight at study initiation: 26.6 g (mean)
- Housing: Makrolon cages in groups of 5 separately according to sex
- Diet: Standardized pelleted feed (Kliba Haltungsdiaet, Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: Drinking water from bottles, ad libitum
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70%
- Air changes: Fully air-conditioned rooms
- Photoperiod: Day/night rhythm was 12 hours

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: DMSO

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Male and female animals per sacrifice interval were given test substance dissolved in DMSO.
All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

24 hours (75, 150, 300 mg/kg bw)
48 hours (300 mg/kg bw)

Frequency of treatment:

single intraperitoneal administration

Post exposure period:

None

Doses / concentrations

Remarks:

Doses / Concentrations:
75, 150, 300 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP) and vincristine (VCR)

- Route of administration: Intraperitoneal
- Doses / concentrations: 20 mg/kg bw (CPP) and 0.15 mg/kg bw (VCR) in a volume of 10 mL/kg bw

Examinations

Tissues and cell types examined:

Yes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity deaths were observed down to a dose of 400 mg/kg body weight 300 mg/kg body weight was survived by all animals but led to clinical signs such as piloerection and squatting posture and the general state of the animals was poor.


TREATMENT AND SAMPLING TIMES:
- The two femora were prepared from the animals, and all soft tissues were removed.
After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37'C (about 2 mL/femur).

- The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.

- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.


DETAILS OF SLIDE PREPARATION:
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes.

After being rinsed twice in purified water and clarified in xylene, the preparations were embedded in Corbit-Balsam .


METHOD OF ANALYSIS:

Microscopic

In general, 1000 polychromatic erythrocytes (PCE) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE), which occur, are also scored. The following parameters are recorded:

- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes

This ratio indicates an influence of the test substance specifically on the bone marrow:
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D= cell diameter)


Clinical examinations

After the administration of the test substance the animals were examined for any evident clinical signs of toxicity.

Evaluation criteria:

- The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.

- The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals .

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG ).
The number of micronuclei in polychromatic erythrocytes was analyzed.

A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p <=0 .05, ** for p <= 0 .01) were printed with the group means in the tables. This test was performed one-sided.

This analysis was done separately for each sex and combined for both sexes.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

An inhibition of erythropoiesis induced by the treatment of mice with the test substance was detected at a dose of 300 mg/kg body weight as indicated by the ratio of polychromatic to normochromatic erythrocytes groups.

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 400, 300, 150, 75 mg/kg bw

- Clinical signs of toxicity in test animals: Deaths were observed at a dose of 400 mg/kg bw. 300 mg/kg bw was tolerated by all animals but led to clinical signs such as piloerection and squatting posture and the general state of the animals was poor .
- Evidence of cytotoxicity in tissue analyzed: No


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: None
- Ratio of PCE/NCE: See table below
- Clinical signs of toxicity in test animals: Only squatting posture induced by the vehicle DMSO; piloerection was found after treatment of the animals with 150 mg/kg or 300 mg/kg body weight. One animal of the 300 mg/kg group died 1 hour after test substance administration.

Any other information on results incl. tables

Test substance analysis

The stability of a comperable batch (26300/9) in the vehicle over a period of 4 hours was verified analytically.

 

Test substance preparation analysis

Depending on the dose, about 83 - 104% of the theoretical values could be determined analytically.

 

Feed, water bedding analysis

Were all in the expected range.

 

 

Table 1: Summary table (males+ females): Polychromatic and normochromatic erythrocytes

	Interval 24 hours				Interval 48 hours
	Total No. of PCE´s	NCE´s/PCE´s	MN(0/00) PCE´s	MN (0/00) NCE´s	Total No. of PCE´s	NCE´s/PCE´s	MN(0/00) PCE´s	MN (0/00) NCE´s
Vehicle DMSO	10000	3918	1.2	0.5	10000	4262	1.7	0.5
75 mg/kg bw	10000	4319	1.0	0.7	-	-	-	-
150 mg/kg bw	10000	4712	1.9	0.4	-	-	-	-
300 mg/kg bw	9000	5584	1.7	0.5	10000	7520	2.5	1.2
CCP 20 mg/kg bw	5000	2333	11.0	0.0	-	-	-	-
VCR 0.15 mg/kg bw	5000	3472	59.4	0.9	-	-	-	-

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative