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:
2013-04-19 - 2013-11-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant 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)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

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 (information from the breeder), male
- Weight at study initiation (mean): 28.4 g
- Assigned to test groups randomly: yes
- Housing: individual
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Photoperiod: 12 hours light from 6.00 - 18.00 hours; 12 hours darkness from 18.00 - 6.00 hours

IN-LIFE DATES: From: 2013-04-19 To: 2013-11-04

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: deionized water
- Justification for choice of vehicle: good solubility of the test substance
- Concentration of test material in vehicle: 15, 30 or 60 mg/mL
- Amount of vehicle: 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:
up to 48 hrs after single oral administration
Frequency of treatment:
single oral dose
Post exposure period:
Depending on the test group 24 (all dose groups) and 48 hours (0 and 600 mg/kg bw) after test substance administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 300 or 600 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10 animals (0 and 600 mg/ kg bw)
5 animals (150, 300 mg/kg bw and positive control groups)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPP)
- Justification for choice of positive control: standard for clastogenic effects
- Route of administration: oral
- Doses / concentrations: 2 mg/mL

Vincristine sulfate (VCR)
- Justification for choice of positive control: standard for spindle poisoning
- Route of administration: intraperitoneally
- Doses / concentrations: 0.015 mg/mL

Examinations

Tissues and cell types examined:
Bone marrow was collected and bone marrow smears (slides) were prepared. Bone marrow cells (polychromatic and normochromatic erythrocytes) were examined microscopically for the presence of micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a pretest for the determination of the acute oral toxicity, deaths or moribund animals were observed at 1000 mg/kg bw . At 500 mg/kg bw, all animals survived showing weak signs of toxicity. The clinical signs observed were piloerection and hunched posture. 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 600 mg/kg bw was defined as MTD (maximum tolerated dose) and was selected as the highest dose in the present cytogenetic study. 300 mg/kg and 150 mg/kg bw were administered as further doses.

DETAILS OF SLIDE PREPARATION:
Preparation of the bone marrow :
- The two femora were prepared from the animals sacrificed by cervical dislocation, and all soft parts were removed.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube in reciprocal directions using a cannula filled with fetal calf serum which was at room temperature (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was pipetted off except for a few drops, and the precipitate was resuspended.
- 1 drop of this suspension was dropped onto clean microscopic slides in each case using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

Staining:
- Stain in eosin and methylene blue solution for 5 minutes
- Rinse in aqua dest., then place in fresh aqua dest. for 2 or 3 minutes
- Stain in Giemsa solution for 15 minutes
After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Entellan.

METHOD OF ANALYSIS: microscopic examination
As a rule, 2000 polychromatic erythrocytes from each of the male animal of every test group were evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occurred, 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.
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:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
See - Details of tissue and slide preparation - CRITERIA FOR DOSE SELECTION


RESULTS OF DEFINITIVE STUDY
- Analytical investigations:
The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of 90% - 110% of the nominal concentration. Based on the recovery rates it has to be considered that the test substance was stable at room temperature in the vehicle deionized water at least over a period of 35 minutes.

- Clinical examinations:
The single oral administration of the vehicle in a volume of 10 mL/kg bw was tolerated by all animals without any clinical observations.

The administration of the test substance led to distinct clinical signs of toxicity at both top dose groups (600 mg/kg bw). 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 bw nor that of vincristine sulfate in a dose of 0.15 mg/kg bw caused any evident signs of toxicity.

- Microscopic evaluation:
The single oral administration of the vehicle deionized water in a volume of 10 mL/kg bw led to 0.9‰ polychromatic erythrocytes containing micronuclei either at 24-hour sacrifice interval or at 48-hour sacrifice interval. For 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.

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 had no detrimental impact on the validity and the outcome of this study.

After the single administration of the highest dose of 600 mg/kg bw , 2.3‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 2.1‰ after 48 hours. Both values were statistically significant increased (p<= 0.05)compared to the respective vehicle control group.

In the two lower dose groups, rates of micronuclei of 1.9‰ (300 mg/kg bw group) and 1.0‰ (150 mg/kg bw group) were detected at a sacrifice interval of 24 hours in each case. Besides, the rate of micronucleated cells at mid dose group was statistically significant increased (p<= 0.05) compared to the respective vehicle control group.

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 induced by the treatment of mice with Benzylamine was detected at a dose of 600 mg/kg bw at 48-hour sacrifice interval.

All values were clearly within the test fecility`s historical negative control data range (0.3 – 3.2‰ micronucleated erythrocytes) and, therefore, the observation has to be regarded as biologically irrelevant.

The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (15.6‰; p<= 0.01) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected. Vincristine sulfate, a spindle poison, produced a statistically significant increase (28.4‰; p<= 0.01) in the number of polychromatic erythrocytes containing micronuclei. A significant portion increase, 5.3‰ was attributable to large micronuclei.







Applicant's summary and conclusion