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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(tetrahydrofuran-3-yl)methanamine
EC Number:
676-405-7
Cas Number:
165253-31-6
Molecular formula:
C5 H11 N O
IUPAC Name:
1-(tetrahydrofuran-3-yl)methanamine
Constituent 2
Reference substance name:
3-Aminomethyl-THF
IUPAC Name:
3-Aminomethyl-THF
Details on test material:
- Name of test material (as cited in study report): 3-Aminomethyl-THF
- Physical state: liquid, colorless, clear
- Analytical purity: 99.9%
- Lot/batch No.: 069/2013 Fr.3 Fl.2

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: 29.4 g (mean)
- Assigned to test groups randomly: yes
- Housing: single housing (polycarbonate cages, type II)
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: al least 5 days

ENVIRONMENTAL CONDITIONS
- 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: water
- Justification for choice of solvent/vehicle: good solubility of the test substance
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test substance was dissolved at dose levels of 300, 600 and 1200 mg/kg bw (prepared immediately before administration)
Duration of treatment / exposure:
24 and 48 Hours
Frequency of treatment:
single application
Doses / concentrations
Remarks:
Doses / Concentrations:
300, 600 and 1200 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 aminals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: orally
- Doses / concentrations: 20 mg/kg bw.

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Pretests for dose selection:
In the pretest to determine the acute oral toxicity in males and females, one male animal died at 2 000 mg/kg body weight. All surviving animals showed severe signs of toxicity. At 1 500 mg/kg, all animals survived with severe signs of toxicity. However, there were no distinct differences in clinical observations between male and female animals. Thus, only male animals will be used in the main experiment.

Test substance concentrations:
The low dose group was given 300 mg/kg body weight or 10 mL/kg body weight of a test substance solution with a concentration of 30 mg/mL.
The intermediate dose group was given 600 mg/kg body weight or 10 mL/kg body weight of a test substance solution with a concentration of 60 mg/mL.
The top dose group was given 1 200 mg/kg body weight or 10 mL/kg body weight of a test substance solution with a concentration of 120 mg/mL.

Preparation of the bone marrow:
The bone marrow was prepared according to the method described by Schmid (7, 8) and Salamone et al. (6).
- The animals were anesthetized with isoflurane and afterwards sacrificed by cervical dislocation. Then the two femora 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 preheated up to 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and 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 with eosin and methylene blue for about 5 minutes. After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2 - 3 minutes. Subsequently, the slides were stained with Giemsa solution for about 15 minutes. After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.


Evaluation criteria:
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 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 the duration 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 (9), i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).
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:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Main Test:
The single oral administration of the vehicle deionized water in a volume of 10 mL/kg body weight led to 1.3‰ polychromatic erythrocytes containing micronuclei after either the 24-hour or 48-hour sacrifice interval.
After the single administration of the highest dose of 1 200 mg/kg body weight, 1.1‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.6‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of 0.8‰ (600 mg/kg group) and 1.4‰ (300 mg/kg group) were detected at a sacrifice interval of 24 hours in each case.
The positive control substance cyclophosphamide led to a statistically significant increase (17.3‰) in the number of polychromatic erythrocytes containing mainly small micronuclei, as expected.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals.
A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected after test substance administration at 48-hour sacrifice interval (1 200 mg/kg body weight; portion of PCEs: 85% of the concurrent vehicle control). Thus, bioavailability of the test substance after oral administration in blood and bone marrow was confirmed.

Clinical examinations:
The single oral administration of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any clinical observations.
The administration of the test substance led to distinct clinical signs of toxicity. At 600 mg/kg body weight hunched posture was observed. At 1 200 mg/kg body weight the animals showed hunched posture and piloerection.
Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight caused any evident signs of toxicity.

Any other information on results incl. tables

Induction of Micronuclei in bone marrow cells

Test group

Sacrifice interval [hrs]

Animal No.

Miconuclei in PCE

PCEs per 2000 erythrocytesc

totala

[‰]

Large MNb

[‰]

Vehicle control

Deionized water

24

5

1.3

0.0

1327

Test substance

300 mg/kg bw.

24

5

1.4

0.3

1282

Test substance

600 mg/kg bw.

24

5

0.8

0.1

1329

Test substance

1 200 mg/kg bw.

24

5

1.1

0.0

1274

Positive control

cyclophosphamide

20 mg/kg bw.

24

5

17.3**

0.1

1391

Vehicle control

Deionized water

48

5

1.3

0.0

1370

Test substance

1 200 mg/kg bw.

48

5

1.6

0.0

1167

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw. = body weight

a = sum of small and large micronuclei

b = large micronuclei (indication for spindle poison effect)

c = calculated number of PCEs per 2 000 erythrocytes (PCE + NCE) when

scoring a sample of up to 10 000 PCE per test group

* = p ≤ 0.05

** = p ≤ 0.01

Applicant's summary and conclusion