Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
water
Details on exposure:
Single intraperitoneal administration with a volume of 10 ml/kg body weight.
Duration of treatment / exposure:
16, 24 and 48 hours
Frequency of treatment:
single
Post exposure period:
- Sacrifice intervals per dose-group were:
2000 mg/kg: 16, 24 and 48 hours
1000 mg/kg: 24 hours
500 mg/kg: 24 hours
Controls: 24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg
Basis:
nominal conc.
intraperitoneal
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
20 mg cyclophosphamide/kg bw or 0.15 mg vincristine/kg bw, both dissolved in distilled water

Examinations

Tissues and cell types examined:
bone marrow, erythrocytes (see below in "Details of tissue and slide preparation"
Details of tissue and slide preparation:
- Preparation of bone marrow: 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 removed and the cells were resuspended. One drop of this suspension was dropped onto clean microscopic slides.
Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained in eosin and methylene blue solution for 5 minutes, rinsed, placed in fresh distilled water for 2 or 3 minutes and finally stained in Giemsa solution for 12 minutes. After being rinsed twice and clarified with xylene, the preparations were embedded in Corbit-Balsam. Slides were coded before microscopic analysis.

-Evaluations: In general, 1000 polychromatic erythrocytes from each male and female animal of every test group was evaluated and investigated for micronuclei. The normochromatic erythrocytes which occur were also scored. The following parameters were 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,
Number of small micronuclei (d < D/4) and of large micronuclei (d >= D/4)

CRITERIA FOR DOSE SELECTION:
Doses were selected on the basis of a preliminary toxicity study: Here, a dose of 2000 mg/kg bw. led to signs of toxicity including irregular respiration, piloerection abdominal position, apathy and squatting posture; the general state was poor.
Statistics:
No statistical methods were employed in data analysis.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
observed in the highest dose tested

Any other information on results incl. tables

   CLINICAL EXAMINATIONS
The single intraperitoneal administration of the solvent in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms. A dose of 2000 mg/kg bw. of test substance led to irregular respiration, piloerection, abdominal position and apathy about 30 minutes after administration; the general state of some animals was poor. After treatment of the animals with 1000 or 500 mg/kg, only irregular respiration and piloerection were observed after about 30 minutes. After about 1 -2 hours clinical signs were no longer observed. None of the positive control substances caused any evident signs of toxicity.

MICRONUCLEI
Mean polychromatic (normochromatic) erythrocytes containing micronuclei were:
Negative control (24 hrs) 1.5% (1.5%)
2000 mg/kg (16 hrs) 1.2% (1.1%)
2000 mg/kg (24 hrs) 1.7% (0.25%)
2000 mg/kg (48 hrs) 1.6% (1.29%)
1000 mg/kg (24 hrs) 2.4% (1.0%)
 500 mg/kg (16 hrs) 1.2% (0.26%)
Cyclophosphamide (24 hrs) 13.6% (2.33%)
Vincristine (24 hrs) 83.2% (1.72%)



Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Cytogenetic analysis of bone marrow cells of mice in vivo show negative results.
Executive summary:

To assess the cytotoxicity, 2 -pyrrolidone was tested in the Micronucleus Test in Mice in vivo. The study was conducted according to the OECD Guideline 474 and is compliant to GLP requirements.

Administration of test substance did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d D/4) or large micronuclei (d > D/4) did not deviate from the solvent control value at any sacrifice interval. No inhibition of erythropoiesis induced by the treatment of mice with Pyrrolidon-2 was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.