N-ethylpropylamine

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:

14 April 2013 - 18 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998-08

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N-ethylpropylamine

EC Number:

243-573-4

EC Name:

N-ethylpropylamine

Cas Number:

20193-20-8

Molecular formula:

C5H13N

IUPAC Name:

N-ethylpropan-1-amine

Details on test material:

- Name of the test substance: N-ethylpropylamine
- Test substance No.: 05/0042-3
- Lot/baatch No.: B987 10.05.2011
- Purity: 89.3 corr.area-% Water content: 10.5g/100g
- Physical state: liquid, colorless, clear
- Stability under test conditions: the stability of the test substance under storage conditions throughout the study period was guaranteed until
19 May 2013 as indicated by the sponsor.
- The homogeneity of the test substance was guaranteed by visual inspection.
- Storage condition of test material: room temperature; no direct sunlight

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: mice / Crl:NMRI from Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: 29.08 g
- Assigned to test groups randomly: yes
- Housing: single housing in Makrolon cages, type M II, single housing
- Diet (e.g. ad libitum): standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland);
- Water (e.g. ad libitum): drinking water from bottles; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
Fully air-conditioned rooms with central air conditioning
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: due to the good solubility of the test subsance in water
- Concentration of test material in vehicle: 175, 350 and 700 mg/ml, respectively for the low, mid and high dose groups
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in deionized water.
To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

24 (all groups) or 48 hours (for additional 5 animals in in the vehicle control and in the high dose groups)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

175 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

350 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (10 in the vehicle control and in the high dose groups)

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide (CCP; dissolved in deionized water, 2 mg/ml) and Vincristine sulfate (VCR; dissolved in deionized water, 0.015 mg/ml)
- Route of administration: orally for CCP or intraperitoneally for VCR, each 10 ml/kg bw
- Doses / concentrations: 20 mg/kg bw for CCP and 0.15 mg/kg bw for VCR

Examinations

Tissues and cell types examined:

Bone marrow; polychromatic erythrocytes, normochromatic erythrocytes,

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- In a pretest for the determination of the acute oral toxicity, deaths were observed at 1 000 mg/kg body weight from one hour after test substance administration onward. At 500 mg/kg, all animals survived showing weak signs of toxicity. However, there were no distinct differences in clinical
observations between males and females. Thus, only male animals were used for the cytogenetic investigations as requested by the current OECD Guideline 474.
Based on the data of the pretest a dose of 700 mg/kg body weight was defined as MTD (maximum tolerated dose) and was selected as the highest dose in the present cytogenetic study. 350 mg/kg and 175 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
- The animals were anesthetized and sacrificed by cervical dislocation. Then the two femora were prepared by dissection and removing all soft tissus.
- 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 (FCS; preheated up to 37°C; about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and centrifuged. The supernatant was removed and the precipitate was resuspended in fresh FCS.
- One 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.

METHOD OF ANALYSIS:
- Staining of the slides: the slides were stained with eosin and methylene blue, rinsing in deionized water, and then soaked in deionized water.
The slides were subsequently stained with Giemsa solution, rinsed twice in deionized water, clarifying in xylene and mounted in Corbit-Balsam.
- Microscopic evaluation: in general, 2000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of
every test group, so in total 10000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
Number of polychromatic erythrocytes
Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
Number of normochromatic erythrocytes
Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in theduration of the sacrifice interval.
Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.
Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) [d = diameter of micronucleus, D = cell diameter]
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).

OTHER:
After treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.

Evaluation criteria:

Acceptance criteria: the mouse micronucleus test is considered valid if the following criteria are met:
- the quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2000 PCEs per animal and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data for PCEs.
- The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.
Assessment criteria: a finding is considered positive if the following criteria are met:
- Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows: (1) * p ≤ 0.05; (2) ** p ≤ 0.01. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

distinct clinical signs of toxicity at both top dose groups (700 mg/kg body weight): piloerection, hunched posture, reduced general condition, respiration irregular

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Induction of micronuclei (for Micronucleus assay): according to the results of the present study, there are thus no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (175 mg/kg, 350 mg/kg and 700 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range.
Based on the pretest, 700 mg/kg body weight was defined as maximum tolerated dose (MTD) due to deaths observed at 1 000 mg/kg body weight. The bioavailability of the test substance in bone marrow after oral administration was clearly demonstrated by effects on the ratio of PCEs and NCEs.
In this study, after single oral administration of the vehicle deionized water the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice
intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in these vehicle control
animals was within the range of the historical vehicle control data for PCEs
In addition, both positive control substances, cyclophosphamide and vincristine sulfate, induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Any other information on results incl. tables

Table 1: Summary table – Induction of micronuclei in bone marrow cells

Test group (mg/kg bw)	Sacrifice interval (hours)	Number of animals	Micronuclei in polychromatic erothrocytes		PCEs per 2000 erythrocytes^c
			Total^a(‰)	Large^b(‰)
Vehicle control	24	5	1.2	0.0	1445
Test substance (175)	24	5	1.0	0.0	1551
Test substance (350)	24	5	0.7	0.0	1449
Test substance (700)	24	5	0.8	0.0	1226
Positive control (CCP; 20)	24	5	11.4**	0.0	1616
Positive control (VCR; 0.15)	24	5	29.0**	6.2**	1337
Vehicle control	48	5	1.2	0.1	1415
Test substance (700)	48	5	0.8	0.0	1050
^a: sum of small and large micronuclei;^b: large micronuclei (indication for spindle poison effect); ^c: calculated number of PCEs per 2000 erythrocytes (PCE + NCE) when scoring a sample of up to 10000 PCEs; **: = p ≤ 0.01

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, the test substance N-ethylpropylamine has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.