Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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): Bismutvanadat BiVO4 (Pigment Yellow 184)
- Physical state: solid
- Analytical purity: Bi 65.6 g/100 g, V 15.4 g/100 g, Mo 0.019 g/100 g
- Impurities (identity and concentrations): Mo 0.019 g/100 g
- Lot/batch No.: GV-34213/026
- Expiration date of the lot/batch: --

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: 29.2 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: Makrolon cages, type M I, single housing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days


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


IN-LIFE DATES: From: 11 Feb 2008 To: 03 Apr 2008

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: a homogenous suspension of the test substance was obtained and historical control data are available
- Concentration of test material in vehicle: 50 mg/mL - 200 mg/mL
Duration of treatment / exposure:
The animals were treated once intraperitoneally.
Frequency of treatment:
The animals were treated once intraperitoneally with a volume of 10 mL/kg body weight of the test substance, the vehicle and the positive control substances. 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 / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (males only)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPP); vincristine sulfate (VCR)
- Route of administration: intraperitoneally
- Doses / concentrations: CPP: 20 mg/kg bw, VCR: 0.15 mg/kg bw

Examinations

Tissues and cell types examined:
Bone Marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: in a pretest for the determination of the intraperitoneal toxicity, at 2 000 mg/kg body weight recommended as the highest dose according to the OECD Guideline all animals (male and female) survived. The distinct clinical signs observed were piloerection and hunched posture. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 2 000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1 000 mg/kg and 500 mg/kg body weight were administered as further doses.


DETAILS OF SLIDE PREPARATION:
- The two femora of the animals sacrificed by cervical dislocation was 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.
The slides were stained with eosin and methylene blue (modified May-Grünwald solution
or Wrights solution) for about 5 minutes.
- After briefly rinsing in purified water, the preparations were soaked in purified water for
about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL
purified water) for about 15 minutes.
- After rinsing twice in purified 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).
Evaluation 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
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: piloerection and hunched posture


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): none
- Ratio of PCE/NCE (for Micronucleus assay): no effect
- Appropriateness of dose levels and route: 2000 mg/kg bw is the highest recommended dose

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen here, the test substance Bismutvanadate BiVO4 (Pigment Yellow 184) 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.