Registration Dossier

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:
02 Jun 2013 - 23 Sep 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N,N-Bis(3-aminopropyl)methylamine
- Physical state: Liquid, colorless, clear
- Analytical purity: 99.6 area-%
- Lot/batch No.: O 2903
- Stability under test conditions: The stability of the test substance under storage conditions was guaranteed until 16 Apr 2014 as indicated
by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

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: mean: 28.23 g
- Assigned to test groups randomly: yes
- Housing: Makrolon cages, type M II; single housing
- Diet: standardized pelleted feed
- Water: ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): Fully air-conditioned rooms with central air conditioning
- 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 deionized water, deionized water was selected as
vehicle.
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:
single oral administration
Frequency of treatment:
once
Post exposure period:
The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48 hours (highest test substance concentration, vehicle) after the treatment, respectively.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 500, 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPP); vincristine sulfate (VCR)
- Justification for choice of positive control(s): cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison
- Route of administration: gavage (CPP), intraperitoneally (VCR)
- Doses / concentrations: 20 mg (CPP), 0.15 mg (VCR)

Examinations

Tissues and cell types examined:
polychromatic erythrocytes (PCE) and normochromatic erythrocytes (= normocytes / NCE)
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 the highest required dose of 2 000 mg/kg body weight. At 1 00mg/kg, all animals survived showing weak signs of toxicity. The only clinical sign observed was piloerection. However, there were no distinct
differences in the 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 1 000 mg/kg body weight was defined as MTD
(maximum tolerated dose) and was selected as the highest dose in the present cytogenetic study. 500 mg/kg and 250 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES:
At the beginning of the study, the animals were weighed and the substance to be administered or the amount of volume was related to the specific weight of the individual animals.
Male animals per sacrifice interval were given N,N-Bis(3-aminopropyl)methylamine dissolved in deionized water at dose levels of 250 mg/kg, 500 mg/kg and 1 000 mg/kg body weight. Treatment consisted of a single oral administration. The volume administered was 10 mL/kg body weight.
As positive control for clastogenicity, 20 mg/kg body weight cyclophosphamide, dissolved in deionized water, was administered to the animals once orally in a volume of 10 mL/kg body weight. As further positive control for a spindle poison effect, 0.15 mg/kg body weight vincristine sulfate, dissolved in deionized water, was administered to the animals once intraperitoneally in a volume of 10 mL/kg body weight.
Depending on the test group 24 and 48 hours after test substance administration, the animals were sacrificed and both femora each were excised for the preparation of the bone marrow smears.

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by Schmid and Salamone et al.:
- 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 (modified May-Grünwald solution or Wrights solution) 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 (15 mL Giemsa plus 185 mL deionized water) for about 15 minutes.
- After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
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, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).
Slides were coded before microscopic analysis.
Since the absolute values shown were rounded, but further calculation was based on unrounded values, there may be deviations in the relative values given.

OTHER:
Clinical examinations
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. ≥ 2 000 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:
* 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:
yes
Remarks:
piloerection; one animal of the high dose group died before reaching the 48-hour sacrifice intervall
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MICROSCOPIC EVALUATION
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 the 24-hour sacrifice interval or to 0.6‰ after the 48-hour sacrifice interval, respectively.
After the single administration of the highest dose of 1 000 mg/kg body weight, 1,3‰ polychromatic erythrocytes containing micronuclei were
found after 24 hours and 1.8‰ after 48 hours.Besides, the rate of micronucleated cells at delayed 48-hour preparation interval was statistically significant increased compared to the respective vehicle control group.
In the two lower dose groups, rates of micronuclei of 1,2‰ (500 mg/kg group) and 1,8‰ (250 mg/kg group) were detected at a sacrifice interval of 24 hours in each case.
The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (15.1‰) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected.
Vincristine sulfate, a spindle poison, produced a statistically significant increase (23.4‰) in the number of polychromatic erythrocytes containing micronuclei. A significant portion increase, 5.2‰ was attributable to large micronuclei.
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.
No relevant inhibition of erythropoiesis induced by the treatment of NMRI mice with N,N-Bis(3-aminopropyl)methylamine was detected.

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 weak clinical signs of toxicity. One animal of the top dose group died before reaching the 48-hour sacrifice interval. Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine sulfate in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.


TEST SUBSTANCE PREPARATIONS
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.

ANALYSIS OF TEST SUBSTANCE PREPARATIONS
For the determination of the test substance concentrations in the vehicle, 6 samples of each dose (including three retain samples) were taken from the test substance preparations. These were kept at room temperature until the treatment of the last animal (approximately 1 hour) and then were
kept deep-frozen. The determination of the concentrations in the vehicle, three samples per dose, was carried out by means of HPLC. The homogeneity of the samples and the stability of the test substance in the vehicle were confirmed indirectly based on these data.

Any other information on results incl. tables

According to the results of the present study, there are thus no biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (250 mg/kg, 500 mg/kg and 1 000 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) or large micronuclei (d ≥ D/4) did not clearly 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, 1 000 mg/kg body weight was defined as maximum tolerated dose (MTD) due to deaths observed at 2 000 mg/kg body weight. In the main experiment one animal exposed to 1 000 mg/kg body weight died before reaching the intended 48-hour sacrifice interval.

Due to the unexpected lethality of a single animal in the top dose group at 48-hour sacrifice interval only four animals were investigated for cytogenetic damage. However, the 24-hour sacrifice interval is the major interval for the detection of genotoxic effects in case of lacking indication of reduced erythrocyte maturation by test substance treatment, as demonstrated in this study. Therefore, the data of the delayed 48-hour sacrifice interval are of minor relevance. Thus, this deviation has no detrimental impact on the validity and the outcome of this study.

Besides, at delayed 48-hour sacrifice interval at 1 000 mg/kg body weight a statistically significant value (1.8‰ micronucleated PCEs) compared to the respective vehicle control value (0.6‰ micronucleated PCEs) was obtained. However, this observation occurred due to the low rate of micronucleated cells in the concurrent vehicle control group. The micronucleus rate of this dose group was clearly within our historical vehicle control data range and, therefore, this finding has to be regarded as biologically irrelevant.

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.

Applicant's summary and conclusion