Piperidine

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

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)

Version / remarks:

(First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted May 26, 1983, "Micronucleus Test")

Deviations:

yes

Remarks:

Deviation to updated version of OECD TG 474 (1997): 1000 polychromatic erythrocytes analysed per animal (instead of 2000 PCEs)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Piperidine

EC Number:

203-813-0

EC Name:

Piperidine

Cas Number:

110-89-4

Molecular formula:

C5H11N

IUPAC Name:

piperidine

Specific details on test material used for the study:

- Name of test material (as cited in study report): Piperidin
- Physical state: liquid, colourless/yellowish
- Analytical purity: 99.3 %
- Lot/batch No.: 459-60 17 FA. 10000 GR 1 FA. GEZ
- Expiration date of the lot/batch: June 27, 1990
- Stability under test conditions: not indicated
- Storage condition of test material: 4°C, closed container

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Fasting period before study: 18 hours
- Housing: single, Makrolon Type I, with wire mesh top (EBECO, Castrop-Rauxel, Germany)
- Diet (ad libitum): pelleted standard diet (ALTROMIN, Lage/Lippe, Germany)
- Water (ad libitum): tap water
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): not regulated
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

VEHICLE
- Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage): 10 mL/kg body weight orally
- Justification for choice of vehicle: non-toxicity for the animals.

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

Bone marrow preparation intervals: 24, 48, 72 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

24 hours

Doses / concentrations 2

Dose / conc.:

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

Remarks:

24 hours

Doses / concentrations 3

Dose / conc.:

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

Remarks:

24 hours, 48 hours and 72 hours

No. of animals per sex per dose:

6 animals/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamid
- Route of administration: gavage
- Doses / concentrations: 30 mg/kg bw in 10 mL physiological saline /kg bw

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) in the bone marrow of the mouse

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study.
The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours.
Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. The highest dose level used was the maximum tolerated dose or that producing some indication of cytotoxicity. For this dose level additional samples were taken at 48 h and 72 h after treatment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Preparation of the Animals:
The animais were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with 2.0 mL fetal calf serum, using a 5 mL syringe, into 1 mL fetal calf serum. The cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide.

DETAILS OF SLIDE PREPARATION:
The smear was air-dried and then stained with May-Gruenwald/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 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 normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.

Evaluation criteria:

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

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

After treatment with 400 mg/kg bw piperidine 5 out of 36 animals died in the micronucleus assay. One male and one female died at preparation intervals 24 and 48 h, respectively, and one male animal at preparation interval 72 h.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
1. In a first pre-experiment 6 animals (3 males, 3 females) received orally a single dose of 500 mg/kg b.w. piperidine dissolved in aqua dest.. The volume administered was 10 mL/kg b.w..
All treated animals expressed toxic reactions: eyelid closure, apathy, tremor, dyspnoea.
Lethalities: one female within 24 hours, one male and one female within 48 hours.
2. In a second pre-experiment 6 animals (3 males, 3 females) per dose level received orally a single dose of 300 or 400 mg/kg b.w., respectively, piperidine dissolved in aqua dest.. The volume administered was 10 mL/kg b.w..
300 mg/kg b.w.:
All treated animals expressed toxic reactions: eyelid closure, apathy. None of the treated animals died.
400 mg/kg b.w.:
All treated animals expressed toxic reactions: eyelid closure, apathy. None of the treated animals died.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In comparison with the corresponding negative controls there was no substantial enhancement in the freguency of the detected micronuclei at any preparation interval or dose level of the test article. The mean values of micronuclei observed after treatment with piperidine were in the same range as compared to the negative control groups.
- Ratio of PCE/NCE: As compared to the corresponding negative controls the mean number of normochromatic erythrocytes was enhanced after the treatment with highest dose of piperidine, indicating that the test article had cytotoxic properties.
- Positive control: 30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase in induced micronucleus frequency.

Any other information on results incl. tables

Test group	Dose [mg/kg bw]	Sampling time [h]	PCEs with micronuclei [%]	Range	PCE/NCE
solvent	0	24	0.12	0-3	1000/496
test article	40	24	0.12	0-4	1000/441
test article	120	24	0.08	0-3	1000/612
test article	400	24	0.13	0-4	1000/1001
cyclophosphamide	30	24	1.21	4-24	1000/769
solvent	0	48	0.09	0-2	1000/630
test article	400	48	0.14	0-3	1000/1181
solvent	0	72	0.14	0-3	1000/605
test article	400	72	0.14	0-3	1000/435

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay, performed according to GLP and OECD 474, five animals per sex and per dose were treated orally at a single time point with the test item (99.3 % a.i.) at doses of 0, 40, 120, and 400 mg/kg bw (CCR/BG Chemie, 1989). Bone marrow cells were harvested at 24, 48 and 72 h post-treatment. The vehicle was aqua dest..

In the highest dose of 400 mg/kg bw all treated animals expressed toxic reactions such as eyelid closure and apathy. None of the treated animals died. The mean number of normochromatic erythrocytes was enhanced after the treatment with highest dose of the test item as compared to the corresponding negative control, indicating that the test article had cytotoxic properties. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time or dose level. The positive control induced the appropriate response. The study is classified as acceptable (reliability 1) and satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.