2,6-dimethylmorpholine

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:

04. Dec 2006 - 05. Mar 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(Aug 1998)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC Directive 2000/32, B. 12 (19 May 2000)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

cis-2,6-dimethylmorpholine

EC Number:

229-353-0

EC Name:

cis-2,6-dimethylmorpholine

Cas Number:

6485-55-8

Molecular formula:

C6 H13 N O

IUPAC Name:

(2S,6R)-2,6-Dimethylmorpholine

Details on test material:

- Name of test material (as cited in study report): Cis-2,6-Dimethylmorpholin
- Physical state: liquid, colorless, clear
- Analytical purity: 96.9% (tolerance +/- 1.0%)
- Impurities (identity and concentrations): no data
- Lot/batch No.: L75-10; date of production: 2006-10-19
- Expiration date of the lot/batch: The stability of the test substance under storage conditions is guaranteed until 01 Dec 2011
- Storage condition of test material: refrigerator

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: ca. 28 g
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program
- Fasting period before study: no
- Housing: individually in Makrolon cages, type MI
- Diet (ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (ad libitum): drinking water from bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2006-11-28 (arrival at testing facility); 2006-12-04 (animals in study) To: 2006-12-06 or 2006-12-07 (sacrifices)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, purified water was used as vehicle.
- Concentration of test material in vehicle: 20, 100, 200 mg/ml bw for the low, mid and high dose group, respectively
- 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 purified 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.
The stability of the test substance at room temperature in the vehicle purified water over a period of 4 hours was verified analytically.

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

sacrifice at 24 and 48 hours post dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 other: mg/kg bw

Remarks:

analytical concentration

Doses / concentrations 2

Dose / conc.:

1 000 other: mg/kg bw

Remarks:

analytical concentration

Doses / concentrations 3

Dose / conc.:

2 000 other: mg/kg bw

Remarks:

analytical concentration

No. of animals per sex per dose:

5 per group (low dose, mid dose, vehicle control, positive control)
8 per group (high dose)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): The stability of cyclophosphamide is well-defined under the selected conditions, since the positive control article is a well-established reference clastogen
- Route of administration: oral
- Doses / concentrations: 20 mg/kg bw

vincristine
- Justification for choice of positive control(s): The stability of vincristine is well-defined under the selected conditions, since the positive control article is a well-established reference aneugen
- Route of administration: i.p.
- Doses / concentrations: 0.15 mg/kg bw

Examinations

Tissues and cell types examined:

femoral bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, at 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline all animals (male and female) survived. The clinical signs observed were piloerection, squatting posture, poor general state, irregular respiration and eyelid closure. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations.
Therefore, 2 000 mg/kg body weight was selected as the highest dose in the main study. 1000 mg/kg bw and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were treated once orally and samples of bone marrow were taken 24 hours and 48 hours after the administration.

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID W (1976 and 1077) and SALAMONE M. et al (1980).
- The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues.
- 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) which was at 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 μL 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.

Staining of the slides
- The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained in Giemsa solution (15 mL Giemsa, 185 mL purified water) for about 15 minutes.
- After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam

METHOD OF ANALYSIS:
Microscopic evaluation
In general, 2000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group were evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occured were also scored. The following parameters were recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei (indicative of clastogenic and/or aneugenic effects)
- 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) (d = diameter of micronucleus, D = cell diameter) (may indicate the possible mode of action, i.e. a clastogenic or a spindle poison effect)
Slides were coded before microscopic analysis.

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 identification and evaluation of a sufficient number of analyzable cells, i.e. ≥ 2000 PCEs and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the untreated animals (vehicle control) has to be within the range of the historical negative control data for the animal strain selected.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical negative control data both for PCEs and for NCEs.
- The positive control substance has to induce a significant increase in the number of PCEs containing 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 negative control value and the range of the historical negative 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 above the concurrent negative control value and is within the historical negative control data range.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (internal system).
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and 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. Significances were identified as follows
* p ≤ 0.05
** p ≤ 0.01

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
In a pretest for the determination of the acute oral toxicity, at 2000 mg/kg bw body weight recommended as the highest dose according to the OECD Guideline all animals (male and female) survived. The clinical signs observed were piloerection, squatting posture, poor general state, irregular respiration and eyelid closure. However, there were no distinct differences in the symptoms between males and females.

RESULTS OF DEFINITIVE STUDY:
1. Microscopic evaluation
The single oral administration of purified water in a volume of 10 mL/kg body weight led to 1.6 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.0 ‰ after the 48-hour sacrifice interval.
After the single administration of the highest dose of 2000 mg/kg bw body weight, 1.1 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.4 ‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of about 1.1 ‰ (1000 mg/kg bw group) and 1.0 ‰ (500 mg/kg bw group) were detected at 24 hour sacrifice interval in each case.
With 17.9 ‰ the positive control substance cyclophosphamide for clastogenicity led to an expected statistically significant increase in the number of polychromatic erythrocytes containing mainly small micronuclei.
Vincristine, a spindle poison substance, produced a statistically significant increase of 49.6 ‰ micronuclei in polychromatic erythrocytes. A significant portion increase, 10.2 ‰ was attributable to large micronuclei.
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.
A distinct inhibition of erythropoiesis induced by the treatment of mice with the test substance was detected at the highest dose of 2000 mg/kg body weight at 48-hour interval.

2. Clinical examinations
The administration of the test substance doses 1000 mg/kg bw and 2000 mg/kg bw led to distinct clinical signs of toxicity (piloerection, hunched posture, reduced general condition, irregular respiration, eyelid closure).
Although, in the pretest all animals survived for at least 2 days after administration of the top dose of 2000 mg/kg bw, in the main experiment animals died at 24-hour sacrifice interval as well as at 48-hour sacrifice interval. Therefore, three additional animals of both dose groups were treated one day later to have the required animal numbers available for scoring. In each of these two high dose groups, totally 3/8 mice died within the first day after dosing.
Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Applicant's summary and conclusion

Conclusions:

According to the results of the present study, the single oral administration of the test substance did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always within the same range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical negative control data. An inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected in the dose of 2000 mg/kg body weight (48-hour sacrifice interval).



As a negative control, male mice were administered merely the vehicle, purified water, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical negative control range.

Both of the positive control substances, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.



Thus, under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.