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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 July 2009 to 18 November 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Diniobium pentaoxide
EC Number:
215-213-6
EC Name:
Diniobium pentaoxide
Cas Number:
1313-96-8
Molecular formula:
Nb2O5
IUPAC Name:
diniobium(5+) pentaoxidandiide

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix obtained from Wistar rats
Test concentrations with justification for top dose:
See details given below.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
The Cells
V79 cells in vitro were widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells were chosen because of their relatively small number of chromosomes (diploid number, 2n = 22), their high proliferation rate (doubling time of the BSL BIOSERVICE V79 done in stock cultures: 12 – 14 h) and the high plating efficiency of untreated cells (normally more than 50%). These facts were necessary for the appropriate performance of the study.
The V79 (ATCC, CCL-93) cells are stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking for mycoplasma infections was carried out before freezing.
For the experiment, thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 oC in a 5% carbon dioxide atmosphere (95% air). 5 x 105 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FCS (foetal calf serum) and subcultures were made every 3 - 4 days.

Mammalian Microsomal Fraction S9 Mix
An advantage of using in vitro cell cultures was the accurate control of the concentration and exposure time of cells to the test item under study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system was necessary (5). Many substances only develop mutagenic potential when they are metabolised by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
The S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with Phenobarbital (80 mg/kg bw) and β-Naphthoflavone (100 mg/kg bw).
The following quality control determinations were performed:
a) Biological activity in the Salmonella typhimurium assay
Sterility Test
A stock of the supernatant containing the microsomes was frozen in ampoules of 2 and 4.5 mL and stored at ≤-75 °C.
The protein concentration in the S9 preparation (Lot: 250609) was 31 mg/mL.

S9 Mix
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.
Evaluation criteria:
according to the guideline

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

 In an in vitro chromosome aberration assay, the test item Diniobium Pentaoxide was investigated for the potential to induce structural chromosomal aberrations in Chinese hamster V79 cells in the absence and presence of metabolic activation with S9 homogenate.


The selection of the concentrations used in experiment I and II was based on data from the solubility test and the pre-experiment which were performed according to the guidelines.


In experiment I, without metabolic activation 1000 μg/mL and with metabolic activation 2500 μg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations. In experiment II, without metabolic activation 500 μg/mL and with metabolic activation 2000 μg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations.


The chromosomes were prepared 20 h after start of treatment with the test item. The treatment intervals were 4 h with and without metabolic activation (experiment I) and 4 h with and 20 h without metabolic activation (experiment II). Two parallel cultures were set up. 100 metaphases per culture were scored for structural chromosomal aberrations.


The following concentrations were evaluated for microscopic analysis:


Experiment I:


With metabolic activation: 500, 1000 and 2500 μg/mL


Without metabolic activation: 250, 500 and 1000 μg/mL


Experiment II:


With metabolic activation: 500, 1000 and 2000 μg/mL


Without metabolic activation: 125, 250 and 500 μg/mL


Precipitation:


In experiment I and II, precipitation of the test item was noted with and without metabolic activation at all the concentrations evaluated.


 


Toxicity:


In experiment I, without metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at 500 μg/mL and higher (55% at 500 μg/mL and 44% at 1000 μg/mL).With metabolic activation a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at 2500 μg/mL (59%). The cell density was not decreased with and without metabolic activation.


In experiment II without metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at 500 μg/mL (65%).With metabolic activation, no biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at concentrations evaluated. No decrease of the cell density was noted with and without metabolic activation.


 


Clastogenicity:


In experiment I without metabolic activation the aberration rate of the negative control (2.5%) and all the dose groups treated with the test item (1.5% (250 μg/mL), 1.5% (500 μg/mL) and 2.0% (1000 μg/mL)) were within the historical control data of the testing facility (0.0% – 4.0%).With metabolic activation, the aberration rates of the negative control (2.5%) and all dose groups treated with the test item 500 μg/mL (0.5%), 1000 μg/mL (0.5%) and 2500 μg/mL (1.0%) were within the historical control data of the testing facility (0.0% – 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was observed.


In experiment II without metabolic activation the aberration rate of the negative control (1.0%) and all dose groups treated with the test item (1.5% (125 μg/mL), 1.0% (250 μg/mL) and 1.5% (500 μg/mL)) were within the historical control data of the testing facility (0.0% – 4.0%).With metabolic activation the aberration rates of the negative control (2.0%) and all dose groups treated with the test item (1.0% (500 μg/mL), 1.0% (1000 μg/mL), and 2.0 (2000 μg/mL)) were within the historical control data of the testing facility (0.0% – 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was observed.


Polyploid cells:


No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.


EMS (400 and 600 μg/mL) and CPA (0.83 μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.


 

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item Diniobium Pentaoxide did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item Diniobium Pentaoxide is considered to be non-clastogenic.