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Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 May 2006 - 06 September 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, test substance used is methylamine hydrochloride

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methylamine hydrochloride
- Molecular formula (if other than submission substance): CH5N.HCl
- Molecular weight (if other than submission substance): 67.52
- Smiles notation (if other than submission substance): CN.Cl
- InChl (if other than submission substance): 1S/CH5N.ClH/c1-2;/h2H2,1H3;1H
- Structural formula attached as image file (if other than submission substance): see Fig.1 (attached)
- Substance type: alkylamine (salt)
- Physical state: solid, white
- Analytical purity: 99 % (with silver nitrate)
- Stability under test conditions: The stability of the test substance under storage conditions is guaranteed by supplier
- Storage condition of test material: room temperature
- Other: the homogeneity of the test substance is guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance solutions.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: about 30 g
- Assigned to test groups randomly: [yes, under following basis: the animals were assigned to the test groups separately according to a randomization plan prepared with an appropriate computer program.]
- Housing: Makrolon cages, type MI in a fully air-conditioned rooms which central air conditioning
- Diet (e.g. ad libitum): ad libitum (Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

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 selected as the vehicle.
- Concentration of test material in vehicle: 5 g/100 mL, 10 g/100 mL and 20 g/100 mL for 500 mg/kg bw, 1000 mg/kg bw and 2000 mg/kg bw, 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. The pH values of all test substance preparations were between 4.0 - 6.0 prior to dosing without adjustment. 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 water over a period of 4 hours was verified analytically. For the determination of the test substance concentrations in the vehicle, three samples of each dose were taken from the test substance preparations, kept at room temperature until the treatment of the last animal (approx. 1 hour) and then deep-frozen until they were determined analytically. The determination of the concentrations in the vehicle was carried out by means of LC/MS. The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF Aktiengesellschaft, Ludwigshafen, Germany.

DIET PREPARATION: Not applicable. The test material was administered to test animals as dosing solution.
Duration of treatment / exposure:
single administration
Frequency of treatment:
single administration
Post exposure period:
24 h and 48 h
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substances: cyclophosphamide (CPP) and vincristine (VCR), both dissolved in purified water.
- Justification for choice of positive control(s): CPP and VCR are well-established reference clastogens and aneugens respectively.
- Route of administration: CPP was administered once orally and VCR was administered once intraperitoneally.
- Doses / concentrations: 20 mg CPP/kg bw and 0.15 mg VCR/kg bw, each in a volume of 10 mL/kg body weight.

Examinations

Tissues and cell types examined:
bone marrow; erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

In a pretest study to determine the acute oral toxicity in males and females, the recommended highest dose of 2 000 mg/kg body weight was tolerated with no deaths. At 2 000 mg/kg, all animals survived but distinct clinical signs were observed such as piloerection, squatting posture, irregular respiration, eyelid closure and the general state was poor. However, there were no distinct symptomatic differences between male and female animals. Thus, only male animals were used in the main experiment. On account of the test results, 2 000 mg/kg body weight was administered as the highest dose. 1 000 mg/kg and 500 mg/kg body weight were selected as further doses.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Test animals were sacrificed 24 h (vehicle control, all dose groups and positive control dose groups) and 48 h (vehicle control group and the highest dose group) after single administration.

DETAILS OF SLIDE PREPARATION:

• 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.
• 1 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, 2 000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are 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 solvent control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the substance tested.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target.
Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
• 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 or a spindle poison effect.
Slides were coded before microscopic analysis.

OTHER: Since the absolute values shown were rounded but the calculations were made using the unedited values, there may be deviations in the given relative values.
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. ≥ 2 000 PCEs and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs.
• The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.

Assessment criteria:

A finding is considered positive if the following criteria are met:
• 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 and the highest value of the historical control range.

A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
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
Remarks:
The rate of micronuclei was close to 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 control data.
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
other: vehicle control served as negative control
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: single dose of 2000 mg/kg bw
- Solubility: well soluble in water
- Clinical signs of toxicity in test animals: piloerection, squatting posture, irregular respiration, eyelid closure and the general state was poor.
- Rationale for exposure: determination of the acute oral toxicity. The recommended highest dose of 2 000 mg/kg body weight was tolerated with no deaths. On account of the test results, 2 000 mg/kg body weight was administered as the highest dose. 1 000 mg/kg and 500 mg/kg body weight were selected as further doses.

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei and ratio of PCE/NCE (for Micronucleus assay):

The single oral administration of purified water in a volume of 10 mL/kg body weight led to 1.4 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 0.8 ‰ after the 48-hour sacrifice interval (Table 1).
After the single administration of the highest dose of 2 000 mg/kg body weight, 1.6 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.2 ‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of about 1.7 ‰ (1 000 mg/kg group) and 2.0 ‰ (500 mg/kg group) were detected after the 24-hour sacrifice interval in each case.
The positive control substance for clastogenicity, cyclophosphamide, led to a clear increase (15%) in the number of polychromatic erythrocytes containing mainly small micronuclei, as expected. Vincristine, a spindle poison agent, produced a 30.6‰ increase in micronuclei in polychromatic erythrocytes. A significant portion of this increase (8.0‰) 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.
No inhibition of erythropoiesis induced by the treatment of mice with Methylamine hydrochloride was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

- Appropriateness of dose levels and route:

The single oral administration of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any signs or symptoms.
The administration of the test substance led to evident clinical signs of toxicity. 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.

Any other information on results incl. tables

Table1: Results of all groups for polychromatic and normochromatic erythrocytes

 

Interval: 24 hours

Interval: 48 hours

 

Total No. of

MN  ( ‰ ) in

Total No. of

MN  (‰ ) in

 

PCE's

NCE's

PCE's

NCE's

PCE's

NCE's

PCE's

NCE's

Vehicle pur. water

10000

3227

1.4

1. 9

10000

3447

0.8

0.9

500 mg/kg

10000

3465

2.0

0.6

 

 

 

 

1000 mg/kg

10000

3770

1. 7

0.5

 

 

 

 

2000 mg/kg

10000

3949

1. 6

0.8

10000

3870

1.2

0.5

CPP 20 mg/kg

10000

4708

15.0**

1. 9

 

 

 

 

VCR 0.15 mg/kg

10000

3813

30.6**

0.3

 

 

 

 

WILCOXON TEST (ONE-SIDED) : *: p <= 0.05, **: p <= 0.01

A pairwise comparison of each dose group with the vehicle control group

PCE's: polychromatic erythrocytes;

NCE's: normochromatic erythrocytes;

MN: micronuclei

Table 2: Differentiation between small and large micronuclei

 

Interval: 24 hours

Interval: 48 hours

 

Total No. of PCE's

Cells  (‰) with

Total No. of PCE's

Cells  (‰ ) with

 

MN.d<D/4

MN.d  ≥   D/4

MN.d<D/4

MN.d  ≥   D/4

Vehicle pur. water

10000

1.4

0.0

10000

0.8

0.0

500 mg/kg

10000

2.0

0.0

 

 

 

1000 mg/kg

10000

1. 7

0.0

 

 

 

2000 mg/kg

10000

1. 6

0.0

10000

1.2

0.0

CPP 20 mg/kg

10000

14.9**

0.1

 

 

 

VCR 0.15 mg/kg

10000

22.6**

8.0**

 

 

 

WILCOXON TEST (ONE-SIDED): *: p 0.05, **: p 0.01

 A pairwise comparison of each dose group with the vehicle control group

PCE's: polychromatic erythrocytes;

MN: micronuclei;

CPP: Cyclophosphamide;

VCR: Vincristine Sulphate;

d = diameter of micronucleus, D = cell diameter.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen of the test, the test substance Methylamine hydrochloride 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 in vivo.
Executive summary:

The substance Methylamine hydrochloride was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method according to the OECD guideline 474. The study was conducted without deviations from the protocol of the guideline and therefore is considered of the highest quality (Klimisch 1). For the purpose of the test, the test substance, dissolved in purified water, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The dose levels were determined in a range-finding study.

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 control range. Both of the positive controls chemicals, 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. Hence, the vehicle and the positive control substances fulfilled validity criteria of the test system.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2 000 mg/kg body weight and in the vehicle controls. In the test groups of 1 000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the single oral administration of Methylamine hydrochloride did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to 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 control data. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen in the test, the test substance Methylamine hydrochloride 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.