3-morpholinopropylamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 June 1992 to 27 July 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Organisation for Economic Co-operation and Development Guidelines for Testing Chemicals ISBN 92-64-12221-4

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian erythrocyte micronucleus test (migrated information)

Test material

Test material information

Constituent 1

Reference substance name:

3-morpholinopropylamine

EC Number:

204-590-2

EC Name:

3-morpholinopropylamine

Cas Number:

123-00-2

Molecular formula:

C7H16N2O

IUPAC Name:

3-morpholin-4-ylpropan-1-amine

Test material form:

liquid

Specific details on test material used for the study:

- Appearance: clear, colorless liquid
- Storage conditions: at ambient conditions in the container received from the sponsor

Test animals

Species:

mouse

Strain:

CD-1

Remarks:

Hsd:ICR (CD-1)

Details on species / strain selection:

Historically, mice have been used in the MNT and have been shown to exhibit micronuclei indicative of chromosome breakage or lagging chromosomes. Intraperitioneal injection was chosed to maximize bioavailability of the test article to the target cells.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA 01887-0630
- Age at study initiation: 7 weeks.
- Weight at study initiation:
- Fasting period before study: no fasting
- Housing: Mice were housed in stainless steel wire mesh cages with a maximum of five per cage according to sex and dose group. Waste material was removed twice per week
- Diet (e.g. ad libitum): Throughout the study, mice were fed Wayne Rodent Blox and fresh tap water was available ad libitum. No contaminants in the food or water were reasonably expected to interfere with the outcome of this study
- Water (e.g. ad libitum): Water was periodically monitored for contaminants and the records kept on file at Pharmakon Research International Inc.
- Acclimation period: 5 days prior to initiation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20% relative humidity
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: deionized water (diH2O), prepared June 9, 1992 and June 16, 1992 by Pharmakon Research International Inc.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Aliquots of the test article were weighed and diluted with deionized water. Clear solutions were obtained and maintained on a magnetic stirplate at all dose levels evaluated. TEM was dissolved in 0.9% Saline. The test article and TEM solutions were prepared fresh prior to dosing. There were no impurities expected to have been present in the negative control which would have affected the outcome of this assay.
DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: no data

Duration of treatment / exposure:

Each group of mice was comprised of ten animals (five/sex). Each mouse received a single intraperitoneal dose at a volume of 10ml/kg. The test article was administrated at a dose of 80mg/kg to three groups of mice which were sacrificed 24, 48 or 72 hours post-done.

Post exposure period:

The negative control was administrated to one group of mice and sacrificed 48 hours post-dose.
The positive control TEM (0.5mg/kg) was administrated to one group of mice and sacrificed 24 hours post-dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

test substance

Doses / concentrations 2

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Remarks:

TEM (triethylenemelamine)

Doses / concentrations 3

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

deionized water

No. of animals per sex per dose:

Dose range finding study:
- 5/sex/group in the MNT
- 2/sex/group in the DRF

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control TEM (0.5mg/kg) was administrated to one group of mice and sacrificed 24 hours post-dose.

Examinations

Tissues and cell types examined:

All mice were sacrificed by cervical dislocation and their femurs removed. One end of each femur (one proximal, one distal to the ilian end) was opened with scissors until a small opening to the marrow canal was visible.

Details of tissue and slide preparation:

A 1 ml tuberculin syringe, fitted with a 5/8" 25 gauge needle containing approximately 0.2 ml fetal bovine serum, was inserted into the marrow cavity and the marrow gently flushed (to assure maximal dispersion) into a 5 ml round bottom culture tube containing 1.0 ml of fetal bovine serum. Each femur was flushed with fetal bovine serum until the bone appeared almost translucent. The suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatant was removed leaving a small amount of fetal bovine serum with the cell pelet. With a pasteur pipette, the cell pellet was resuspended into a homogeous cell suspension. A small drop of the cell suspension was spread immediatly into an ethanol pre-cleaned glass slide. The smears were quickly dried on a slide warmer (56C), dipped in absolute methanol (2 seconds) and air dried.

Evaluation criteria:

CRITERIA FOR SCORING MICRONUCLEI: Although micronuclei are uniformly round bodies in the cytoplasm of erythrocytes, they may appear almond-, ring-, or teardrop-shaped. Cytoplasmic inclusions which were light reflective, improperly shaped or stained, or which were not in the focal plane of the erythrocyte were considered artifacts and were not scored as micronuclei. Erythrocytes containing one or more than one micronucleus were scored as one micronucleated erythrocyte.

Statistics:

STATISTICAL ANALYSIS:
A test article is considered positive if it produced a statistically significant increase in the number of MPCEs as compared to the negative control. One-tailed t-tests were used to make pairwise comparisons between each treatment group and its concurrent negative control for statistically significant increases in the number of MPCEs. The proportions of PCEs per 1000 erythrocytes per mouse was evaluated by pairwise two-tailed t-tests after an arcsin transformation was performed. Statistical significance was judged at p < or = 0.05 and p < or = 0.01 levels. All comparisons were made for each sacrifice time separately, comparing the treated group versus the concurrent negative control group.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Pharmacotoxic signs in the DRF:
- Mice were observed for death and pharmacotoxic signs immediately, 24, 48 and 72 hours post-dosing. The surviving mice were sacrificed after the last observation.
- Based on the death observed, (one male died in the 100 mg/kg group, both males and one female died in the 250 mg/kg group and all mice died in the 500 and 1000 mg/kg groups), 80 mg/kg was selected (with the sponsor's approval) as the dose for the MNT as an estimate of the maximum tolerated dose.

Pharmacotoxic signs in the MNT:
- no pharmacotoxic signs were observed in mice administered the negative or positive controls.
- analysis of the data for mice treated with the test substance at a dose of 80 mg/kg did not produce a statistically significant increase in the number of MPCEs as compared to the negative control, di-H2O, at the dose and time intervals evaluated. A statistically significant depression in the PCE/NCE ratio was detected in the 72 hour sacrifice group (p<=0.05), indicating that the test substance has reached the target cells in the bone marrow. Mice treated with the positive control produced a statistically significant increase (p<=0.01) in the frequency of MCPE and a statistically significant depression (p<=0.05) in the PCE/NCE ratio.
-In the MNT, both the negative and positive controls are within the range of the test facility historical data.

Applicant's summary and conclusion

Conclusions:

The test substance at 80 mg/kg was considered negative (non-clastogenic) in the mouse MNT at all the time intervals evaluated under the criteria and the experimental conditions of the test protocol.