1-methyl-2-pyrrolidone

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

1-methyl-2-pyrrolidone

EC Number:

212-828-1

EC Name:

1-methyl-2-pyrrolidone

Cas Number:

872-50-4

Molecular formula:

C5H9NO

IUPAC Name:

1-methylpyrrolidin-2-one

Test material form:

liquid

Details on test material:

- source: BASF AG
- analytical purity: purity > 99.8 %

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG, 88/369
- Analytical purity: 99.8 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: +4°C to +6°C under N2 conditions
- Stability under test conditions: Stability proven by sponsor
- Solubility and stability of the test substance in the solvent/vehicle: Substance was formulated in aqua dest. immediately before administration

FORM AS APPLIED IN THE TEST
- solution

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, D-8741 Sulzfeld, Germany
- Weight at study initiation: not specified
- Assigned to test groups randomly: yes
- Fasting period before study: not indicated
- Housing:
during acclimation period: in Makrolon cages type III, in groups of 5 per sex
during study period: in Makrolon cages type I, individually
- Diet: ad libitum, standardized pellet (Kliba Haltungsdiät; Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: about one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): fully air conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: aqua dest.
- Justification for choice of solvent/vehicle: NMP is soluble and stable in aqua dest. for > 2 hours
- Concentration of test material in vehicle: 9.5 - 38 g/100 mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test groups 2 were given 3800 mg test substance/kg bw or 10 mL/kg bw of a solution with a concentration of 38 g/100 mL
Test groups 3 were given 1900 mg test substance/kg bw or 10 mL/kg bw of a solution with a concentration of 19 g/100 mL
Test groups 4 were given 950 mg test substance/kg bw or 10 mL/kg bw of a solution with a concentration of 9.5 g/100 mL

Duration of treatment / exposure:

24 h (950 and 1900 mg/kg bw); 16, 24 and 48 h (3,800 mg/kg bw)
24 h (solvent control and positive controls)

Frequency of treatment:

one single test substance administration by gavage

Post exposure period:

not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

950 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1 900 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

3 800 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals per sex, dose and sampling interval (for NMP treatment groups and control group)
3 male and 2 female mice for cyclophosphamide treatment group
2 male and 3 female mice for vincristine treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

- Route of administration: gavage
- Doses / concentrations:
cyclophosphamide: 40 mg/kg bw per os (positive agent for clastogenic activity)
vincristine: 0.15 mg/kg intraperitoneal (positive agent for spindle inhibition, aneuploidy)

Examinations

Tissues and cell types examined:

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

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for determination of the acute oral toxicity, deaths were observed down to a dose of 4640 mg/kg bw. The dose level which all animals survived was 3830 mg/kg bw, but signs of toxicity were observed at this dose level: irregular respiration and abdominal position. In addition, the general state of the animal was poor.
Therefore a dose of 3800 mg/kg bw was selected as the highest dose in this micronucleus test. 1900 mg/kg bw and 950 mg/kg bw were selected as additional dose levels.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
All test substance solutions were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animal on the day of the experiment. For control purposes, male and female animals were given the solvent aqua dest. by the same route.

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by Schmid, W. (1976 and 1977).
After cutting the epiphyses, the bone marrow ws flushed out of the diaphysis with ca. 2 mL fetal calf serum. The suspension was reduced by centrifugation and bone marrow smears were prepared onto clean microscopic slides. The slides were stained in eosin and methylene blue and after staining with Giemsa, the preparation were embedded in Entellan.

Evaluation criteria:

1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes are also scored. The following parameters are recorded:
- number of polychromatic erythrocytes
- number of polychromatic erythrocytes containing micronuclei
- number of normochromatic erythrocytes
- ratio of polychromatic to normochromatic erythrocytes
- number of small micronuclei (d < D/4) and large micronuclei (d>= D/4)
d= diameter of micronuclei and D = cell diameter
- number of normochromatic erythrocytes containing micronuclei

Statistics:

Statistical analysis: Fisher's exact test and U-test according to Mann-Whitney

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Apart from irregular respiration and abdominal position that lasted from 1h to 1d, depending on the doses, there was no toxicity observed.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Clinical examinations:
Doses of 3800 mg/kg bw and 1900 mg/kg bw induced irregular respiration and abdominal position immediately after test substance administration. These signs indicate the bioavailability of the test substance. 24 hours after treatment, the animals did not show clinical signs any longer.

- Induction of micronuclei (for Micronucleus assay):
No increased frequency of polychromatic erythrocytes containing either small or large micronuclei (see also table under "Any other information on results incl. tables").
- Ratio of PCE/NCE (for Micronucleus assay): Inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at 3800 mg/kg  bw (approx. 80 % of LD50) after 24 h sacrifice only.

- Statistical evaluation:
There was no statistically significant increase in the rate of polychromatic cells with micronuclei in any animal treated with NMP
The positive controls cyclophosphamide and vincristine induced significant increases in the number of micronucleated polychromatic erythrocytes

Any other information on results incl. tables

Results at 24 h:

 

Dose level (mg/kg bw)	MN/1000 PCE	PCE examined	NCE (found)
0	22 (2.2%)	10000	3423
NMP          950	16 (1.6)	10000	3198
NMP         1900	22 (2.2)	10000	2952
NMP        3800	21 (2.1)	10000	6605
CPP             40	90 (18.0)	5000	2292
VC                 0.15	516 (103)	5000	4132

MN = cells with micronuclei per 100 polychromatic erythrocytes

PCE = poylchromatic erythrocytes

NCE = normochromatic erythrocytes

CP = cyclophosphamide

VC = vincristine

Applicant's summary and conclusion

Conclusions:

The overall result of the in vivo micronucleus assay was a negative outcome.

Executive summary:

NMP was tested for clastogenicity and for the ability to have spindle poison effects in the mouse micronucleus test in NMRI mice. NMP dissolved in aqua dest., was administered as a single dose orally to 5 males and 5 females/test group at dose levels of 3800, 1900 and 950 mg/kg bw in a volume of 10 mL/kg body weight. Concurrent negative control test groups (5 male and 5 female mice) were administered merely the solvent aqua.dest by the same route. As positive control for clastogenicity, 40 mg of cyclophosphamide/kg bw, dissolved in aqua dest., was administered orally to 3 male and 3 female animals. As positive control for spindel poison effects, 0.15 mg of vincristine/kg bw, dissolved in aqua dest., was administered intraperitoneally to 2 male and 2 female animals. The femora of the animals in the respective groups were prepared 16, 24 and 48 hours after test substance administration in the highest dose group of 3800 mg/kg bw. In the test groups of 1900 and 95 mg/kg bw, the negative control group and in the positive control groups, the 24 -hour interval was investigated, only. After staining of the preparations, 1000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normochromatic erythrocytes were also registered. After administration of the test substance, the animals were examined for any evident clinical signs of toxicity.

Doses of 3800 mg/kg bw and 1900 mg/kg bw induced irregular respiration and abdominal position immediately after test substance administration. These signs indicate the bioavailability of the test substance. 24 hours after treatment, the animals did not show clinical signs any longer.

NMP did not increase the frequency of poylchromatic erythrocytes containing either small or large micronuclei.

The positive control substances cyclophosphamide/vincristine sulfate increased clearly the numbers of micronucleated polychromatic erythrocytes indicating the sensitivity of the test system.

These results indicate that NMP has neither a clastogenic effect nor a spindle poison effect in vivo.