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Toxicological information

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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997-07-21
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)
Version / remarks:
1998-08
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid, brown

- Stability under test conditions: the stability of the test substance under storage conditions throughout the study period was guaranteed until 2012 as indicated by the sponsor, and the sponsor holds this responsibility. 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 preparations.
- 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: mice / Crl:NMRI from Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: 29.43 g
- Assigned to test groups randomly: yes
- Housing: single housing in Makrolon cages, type M II
- Diet (e.g. ad libitum): standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland); ad libitum
- Water (e.g. ad libitum): drinking water from bottles; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
Fully air-conditioned rooms with central air conditioning
- 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: corn oil
- Concentration of test material in vehicle: 50, 100 or 200 mg/ml, respectively for the low, mid and high dose groups
- 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 suspended in corn oil. To achieve the homogeneity of the test substance in the vehicle, the test substance preparation was stirred with a spatula and shaken thoroughly. All test substance formulations were prepared immediately before administration. For the determination of the test substance concentrations in the vehicle, 6 samples of each dose were taken from the test substance preparations, and the determination of the concentrations in the vehicle was carried out by means of UV/VIS spectrometer.
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
24 (all groups) or 48 hours (for additional 5 animals in in the vehicle control and in the high dose groups)
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 (10 in the vehicle control and in the high dose groups)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CCP; dissolved in deionized water, 2 mg/ml) and Vincristine (VCR; dissolved in deionized water, 0.015 mg/ml)
- Route of administration: orally for CCP or intraperitoneally for VCR, each 10 ml/kg bw
- Doses / concentrations: 20 mg/kg bw for CCP and 0.15 mg/kg bw for VCR

Examinations

Tissues and cell types examined:
Bone marrow; polychromatic erythrocytes, normochromatic erythrocytes,
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 bw recommended as the highest dose according to the OECD Guideline, all animals (male and female) tolerated without any signs of toxicity. Due to missing differences in clinical observations between male and female animals, males was used in the main experiment as requested by the current OECD Guideline 474. Therefore, a dose of 2000 mg/kg bw was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg bw were administered as further doses.

DETAILS OF SLIDE PREPARATION:
- The animals were anesthetized and 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; preheated up to 37°C; about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and centrifuged. The supernatant was removed and the precipitate was resuspended in 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.

METHOD OF ANALYSIS:
- Staining of the slides: the slides were stained with eosin and methylene blue, rinsing in deionized water, and then soaked in deionized water. The slides were subsequently stained with Giemsa solution, rinsed twice in deionized water, clarifying in xylene and mounted in Corbit-Balsam
- Microscopic evaluation: in general, 2000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded: (1) number of normochromatic erythrocytes; (2) number of normochromatic erythrocytes containing micronuclei; (3) ratio of polychromatic to normochromatic erythrocytes; (4) number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) [d = diameter of micronucleus, D = cell diameter].

OTHER:
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: (1) the quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2000 PCEs per animal and a clear differentiation between PCEs and NCEs. (2) The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected. (3) The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs. (4) 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: (1) Statistically significant and dose-related increase in the number of PCEs containing micronuclei. (2) 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: (1) 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: (1) * p ≤ 0.05; (2) ** p ≤ 0.01. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
no statistical significances / biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups or between the two sacrifice intervals (24 and 48 hours)
Toxicity:
no effects
Remarks:
discolored feces were observed from 24 hours after test substance administration onward
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): according to the results of the present study, there are thus no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (500 mg/kg, 1000 mg/kg and 2000 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) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range. Nor were large micronuclei (d ≥ D/4) observed either in the vehicle control group or in the three dose groups treated with the test substance.
- Ratio of PCE/NCE (for Micronucleus assay): 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 both for PCEs and for NCEs

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.

Any other information on results incl. tables

Table 1: Summary table – Induction of micronuclei in bone marrow cells

Test group (mg/kg bw)

Sacrifice interval (hours)

Number of animals

Micronuclei in polychromatic erothrocytes

Number of normochromatic erythrocytesc

Totala(‰)

Largeb(‰)

Vehicle control

24

5

1.5

0.0

3914

Test substance (500)

24

5

0.7

0.0

3369

Test substance (1000)

24

5

1.7

0.0

3957

Test substance (2000)

24

5

1.4

0.0

3264

Positive control (CCP; 20)

24

5

23.2**

0.0

4778

Positive control (VCR; 0.15)

24

5

45.2**

13.0**

4304

Vehicle control

48

5

1.6

0.0

4141

Test substance (2000)

48

5

1.0

0.0

2653

a: sum of small and large micronuclei;b: large micronuclei (indication for spindle poison effect);c: number of NCEs observed when scoring 10000 PCEs; **: = p ≤ 0.01

 

Applicant's summary and conclusion