Diniobium pentaoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation (OECD 471): Negative with and without metabolic activation

In vitro cytogenicity in mammalian cells (OECD 473): Negative with and without metabolic activation

In vitro gene mutation in mammalian cells (OECD 476): Negative with and without metabolic activation

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

not applicable

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.

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

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

 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.

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-10-11 to 2001-02-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurim histidine reversion system
E. coli tryptophan reversion system

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

water containing 0.15% agar

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene strains TA1535 and E.coli with S9; AF-2 E.coli without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;
DURATION
- Preincubation period: 30 minutes
- Exposure duration: at least 48 h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

Evaluation criteria:

The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the extract vehicle control. A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose group occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strain TA 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strain TA 98 TA 1535 TA 1537 the number of reversions is at least three times higher as high than the reversion rate of the extract vehicle control

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

E. coli WP2 uvr A pKM 101

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

Conclusions:

It is concluded that, under the test conditions employed, Nb2O5 Niobium Pentoxide Grade LN showed no evidence of mutagenic activity in this bacterial system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 2009 to 15 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix obtained from rat liver

Test concentrations with justification for top dose:

Experiment I
with and without metabolic activation: 39, 78, 156, 312, 625, 1250, 2500 and 5000 μg/mL
Experiment II
with metabolic activation: 60, 125, 250, 500, 1000, 2000, 3500 and 5000 μg/mL
and without metabolic activation: 39, 78, 156, 312, 625, 1250, 2500 and 5000 μg/mL

Vehicle / solvent:

Cell culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: no
- Exposure duration: 4 hours and in experiment II without metabolic activation 24 hours
- Expression time (cells in growth medium): 72 h and 48 h for the 24 h exposure experiment
NUMBER OF REPLICATIONS: 4 per dose level
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

According to guideline

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Precipitation:

Precipitation of the test item was noted in all dose groups in experiment I and II with and without metabolic activation.

 

Toxicity:

No biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation.

In experiment I with metabolic activation the relative total growth (RTG) was 106.95% for the highest concentration (5000 μg/mL) evaluated. The highest concentration evaluated without metabolic activation was 5000 μg/mL with a RTG of 84.41 %.

In experiment II with metabolic activation the relative total growth (RTG) was 115.75% for the highest concentration (5000 μg/mL) evaluated. The highest concentration evaluated without metabolic activation was 5000 μg/mL with a RTG of 69.97%.

 

Mutagenicity:

In experiment I with metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 84-268 mutants per 10E+6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies of the negative controls were found to be 117.37 and 144.96 mutants/10E+6 cells and in the range of 98.30 to 169.48 mutants/10E+6 cells with the test item, respectively. The highest mutation factor (compared to the negative control values) of 1.29 was found at a concentration of 1250 μg/mL with a RTG of 108.33%.

Without metabolic activation the mutant values found for the negative controls were within the historical control data of the test facility BSL BIOSERVICE (about 82-238 mutants per 10E+6 cells). In addition mutant values at concentrations up to 1250 μg/mL were within or slightly above the historical control data. The higher mutant values found at concentrations of 2500 μg/mL and 5000 μg/mL were regarded as not biologically relevant due to the lack of mutagenicity. In addition no dose-response relationship could be observed.

Mutation frequencies of the negative controls were found to be 164.40 and 195.58 mutants/10E+6 cells and in the range of 148.27 to 299.75 mutants/10E+6 cells with the test item, respectively. The highest mutation factor (compared to the negative control values) of 1.67 was found at a concentration of 5000 μg/mL with a RTG of 84.41%.

In experiment II with metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 84-268 mutants per 10E+6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative controls were found to be 148.61 and 157.27 mutants/10E+6 cells and in the range of 107.06 to 187.25 mutants/10E+6 cells with the test item, respectively. The highest mutation factor (compared to the negative control values) of 1.22 was found at a concentration of 1000 μg/mL with a RTG of 100.02%.

In experiment II without metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 82-238 mutants per 10E+6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies of the negative controls were found to be 100.54 and 135.45 mutants/10E+6 cells and in the range of 83.13 to 154.50 mutants/10E+6 cells with the test item, respectively. The highest mutation factor (compared to the negative control values) of 1.31 was found at a concentration of 156 μg/mL with a RTG of 90.15%.

EMS (500 and 200 μg/mL), MMS (10 μg/mL) and B[a]P (3.5 μg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

 

Relationship of large to small colonies:

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone), indicated by a low large/small colony ratio (ratio of the clastogenic controls MMS and/or B[a]P with a coefficient of 1.5), is an indication for potential clastogenic effects and/or chromosomal aberrations.

In experiment I with metabolic activation, the quotients of large/small colonies of the negative controls were found to be 7.45 and 10.71. The quotient of large/small colonies of the positive control was found to be 1.53. The quotients of the highest dose groups were found to be 6.07 (1250 μg/mL), 9.50 (2500 μg/mL) and 13.83 (5000 μg/mL). All dose groups were considered as not clastogenic.

Without metabolic activation, the quotients of large/small colonies of the negative controls were found to be 2.96 and 2.63. The quotient of large/small colonies of the positive control was found to be 0.47. The quotients of the highest dose groups were found to be 3.39 (1250 μg/mL), 3.77 (2500 μg/mL) and 3.78 (5000 μg/mL). All dose groups were considered as not clastogenic.

In experiment II with metabolic activation, the quotients of large/small colonies of the negative controls were found to be 3.48 and 5.72, the quotient of large/small colonies of the positive control was found to be 1.35. The quotients of the highest dose groups were found to be 4.14 (2000 μg/mL), 13.63 (3500 μg/mL) and 7.46 (5000 μg/mL). All dose groups were considered as not clastogenic.

Without metabolic activation, the quotients of large/small colonies of the negative controls were found to be 5.87 and 3.86, the quotient of large/small colonies of the positive control was found to be 0.88. The quotients of the highest dose groups were found to be 5.88 (1250 μg/mL), 8.25 (2500 μg/mL) and 6.23 (5000 μg/mL).All dose groups were considered as not clastogenic.

The positive controls MMS (10 μg/mL) and B[a]P (3.5 μg/mL) induced a significant increase of the mutant frequency and a biologically significant increase of small colonies, thus proving the ability of the test system to indicate potential clastogenic effects.

 

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Diniobium Pentaoxide is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Diniobium pentaoxide was negative, with and without metabolic activation, in an Ames test, in an in vitro chromosomal aberration assay and in an in vitro mouse lymphoma assay.

Justification for classification or non-classification

Diniobium pentaoxide was negative, with and without metabolic activation, in a full battery of in vitro genotoxicity tests. No classification is warranted for this endpoint.