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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The GLP conform, in vitro mammalian cell gene mutation tests by Lloyd (2010) according to OECD 476 are considered key studies and will be used for classification. Representative vanadium candidates from all three valency groups (III, IV and V) did not induce mutation at the HPRT locus of L5178Y mouse lymphoma cells (Lloyd, 2010). Testing in bacteria reverse mutation assays, although of limited relevance for metals (HERAG, 2007), also yielded negative results. Negative as well as positive results were obtained in clastogenicity assays in vitro. Vanadium substances may express some clastogenicity in vitro, this occurs only at unphysiologically high concentrations and via mechanisms that appear to lack physiological relevance, and is not mirrored in corresponding in vivo (RL=1) assays.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Experimental work started on 16 February 2010 and was completed on 28 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed by The Department of Health of the Government of the United Kingdom (2010-06-23)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The master stock of L5178Y tk hprt +/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co.
- Type and identity of media: RPMI 1640 media were prepared with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Amphotericin B (2.5 µg/mL), Pluronic (0.5 mg/mL except for "RPMI 20" with 0 mg/mL) and heat inactivated horse serum (0%, 10% or 20% (v/v) for "RPMI A", "RPMI 10" or "RPMI 20", respectively).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; each batch of cells was confirmed to be mycoplasma free.
- Periodically checked for karyotype stability: yes; each batch of cells was checked for spontaneous mutant frequency.
- Periodically "cleansed" against high spontaneous background: yes

For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Second Range Finder:
- with and without S9-mix: 0.1953, 0.3906, 0.7813, 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL.

Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range Finder Experiment.

Experiment 1:
- without S9-mix: 1.5, 3, 4.5, 6, 7.5, 9, 10.5, 12, 13.5 and 15 µg/mL;
- with S9-mix: 3, 6, 8, 10, 12.5, 15, 17.5, 20, 25 and 30 µg/mL.

Experiment 2:
- with S9-mix: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg/mL;
- without S9-mix: 1, 2, 4, 5, 6, 7, 8, 9, 10 and 15 µg/mL.

Cultures selected for mutation assessment:
Experiment 1:
- without S9-mix: 3, 4.5, 6 and 7.5 µg/mL;
- with S9-mix: 3, 6, 8, 10 and 12.5 µg/mL.

Experiment 2:
- without S9-mix: 1, 2, 4, 5, 6, 7, 8, 9, 10 and 15 µg/mL;
- with S9-mix: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Test article solutions were prepared under subdued lighting by formulating Vanadium oxide sulfate in purified water (with the aid of vortex mixing, as required).
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that wanadium oxide sulfate was soluble in water for irrigation (purified water) at concentrations up to at least 34.23 mg/mL of anhydrous test article.
Untreated negative controls:
yes
Remarks:
untreated controls (UTC), in which the vehicle was replaced with an equal volume of culture medium
Negative solvent / vehicle controls:
yes
Remarks:
treatments with the vehicle 0.5% MC diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-1-oxide; 0.1 and 0.15 µg/mL (dissolved in DMSO)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation

Migrated to IUCLID6: 2 and 3 µg/mL (dissolved in DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours at 37°C with gentle agitation
After treatment cultures were centrifuged, washed and resuspended in RPMI 10. Cell densities were determined using a Coulter counter. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival (incubation for 7 days).
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed.
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and cells were plated for determination of viability and for 6TG resistance.
- Selection time (if incubation with a selection agent): At the end of the expression period, 6TG was added to the cell cultures with a final concentration of 15 µg/mL. Cell cultures were placed into each well of 4 x 96 well microtitre plates (384 wells at 2 x 10^4 cells/well). Plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air until scoreable (12 to 13 days) and wells containing clones were identified.
SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S9, was in duplicate (single cultures only used for positive control treatments).

EVALUATION: Wells containing clones were identified.

DETERMINATION OF CYTOTOXICITY
- Method: relative survival:
A maximum concentration of 1630 µg/mL was selected for the initial cytotoxicity Range Finder Experiment in order that treatments were performed up to a maximum of 10 mM. Complete toxicity was observed at all concentrations tested (ranging from 50.94 to 1630 µg/mL) in the first Range Finder Experiment, therefore a second Range Finder Experiment was conducted, using lower test article concentrations. Concentrations selected for the Mutation Experiments were based on the results of the second cytotoxicity Range Finder Experiment.
Treatment and post treatment dilution of cell cultures for the cytotoxicity Range Finder Experiments were as described for the Mutation Experiments. However, single cultures only were used and positive controls were not included. Following treatment, cells were centrifuged, washed with tissue culture medium and resuspended in 20 mL RPMI 10. Cells were plated into each well of a 96 well microtitre plate for determination of relative survival. The plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air for 7 days. Wells containing viable clones were identified by eye using background illumination and counted.


OTHER EXAMINATIONS:
- probable number of clones/well (P) = -ln (EW/TW);
- Plating efficiency (PE) = P/No of cells plated per well;
- Percentage relative survival (% RS) = [PE (test)/PE (control)] x 100;
- Mutant frequency (MF) = [PE (mutant)/PE (viable)] x 10^6
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p<0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p<0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In Experiments 1 and 2 no statistically significant increases in mutant frequency were observed following treatment with vanadium oxide sulfate at any concentration tested.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details refer to "additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment media were taken in the first cytotoxicity Range-Finder Experiment.
- Effects of pH and osmolality: No marked changes in osmolality were observed in the first cytotoxicity Range Finder Experiment. A decrease in pH (compared to the concurrent control value) of more than one unit was observed following treatment at 1630 µg/mL in the absence and presence of S9. However, no marked changes in pH were observed at concentrations of 815 µg/mL or below and the highest concentration tested in the main experiments was 30 µg/mL, therefore this did not affect the interpretation of the data in any way.

RANGE-FINDING/SCREENING STUDIES: In the first cytotoxicity Range Finder Experiment, 6 concentrations were tested in the absence and presence of S9, ranging from 50.94 to 1630 µg/mL (equivalent to 10 mM at the highest concentration tested). Complete toxicity (0% RS) was observed at all concentrations tested in the absence and presence of S9.
In the second cytotoxicity Range Finder Experiment, 10 concentrations were tested in the absence and presence of S9, ranging from 0.1953 to 100 µg/mL (limited by toxicity). The highest concentrations to provide >10% RS were 6.25 µg/mL in the absence of S9 and 12.5 µg/mL in the presence of S9, which gave 42% and 28% RS respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: comparison of controls with historical means of controls (positive and negative)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: Ten concentrations, ranging from 1.5 to 15 µg/mL, in the absence of S9 and from 3 to 30 µg/mL in the presence of S9, were tested. 7 days after treatment, the highest five concentrations tested in the absence of S9 (9 to 15 µg/mL) and in the presence of S9 (15 to 30 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest concentration in the absence of S9 (1.5 µg/mL) was not selected as the % RS value at this concentration was unacceptably low; the reasons for this are not clear but it is likely that there was a dilution error during survival plating. All other concentrations were selected. The highest concentrations selected were 7.5 µg/mL in the absence of S9 and 12.5 µg/mL in the presence of S9, which gave 13% and 20% RS, respectively.
Experiment 2: Ten concentrations, ranging from 1 to 15 µg/mL in the absence of S9 and from 2 to 20 µg/mL in the presence of S9, were tested. 7 days after treatment, all concentrations were selected to determine viability and 6TG resistance. The highest concentrations tested were 15 µg/mL in the absence of S9 and 20 µg/mL in the presence of S9, which gave 4% and 17% RS, respectively. In the absence of S9, no concentration gave 10-20% RS. However, cultures treated at 10 and 15 µg/mL gave 32% and 4% RS, respectively, and therefore both concentrations were analysed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that vanadium oxide sulfate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9).
Executive summary:

Vanadium oxide sulfate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two cytotoxicity Range-Finder Experiments followed by two independent experiments, each conducted in the absence and presence of metabolic activation (S9). The test article was formulated inpurified water.

A 3 hour treatment incubation period was used for all experiments. In the first cytotoxicity Range-Finder Experiment, 6 concentrations were tested, ranging from 50.94 to 1630 µg/mL (equivalent to 10 mM at the highest concentration tested). Complete toxicity (0% relative survival, RS) was observed at all concentrations tested in the absence and presence of S9.

In the second cytotoxicity Range-Finder Experiment, 10 concentrations were tested, ranging from 0.1953 to 100 µg/mL (limited by toxicity). The highest concentrations to provide >10% RS were 6.25 µg/mL in the absence of S9 and 12.5 µg/mL in the presence of S9, which gave 42% and 28% RS, respectively.

Accordingly, for Experiment 1 ten concentrations, ranging from 1.5 to 15 µg/mL, in the absence of S9 and from 3 to 30 µg/mL in the presence of S9, were tested. 7 days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 7.5 µg/mL in the absence of S9 and 12.5 µg/mL in the presence of S9, which gave 13% and 20% RS, respectively.

In Experiment 2 ten concentrations, ranging from 1 to 15 µg/mL, in the absence of S9 and from 2 to 20 µg/mL in the presence of S9, were tested. 7 days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 15 µg/mL in the absence of S9 and 20 µg/mL in the presence of S9, which gave 4% and 17% RS, respectively. In the absenceof S9, no concentration gave 10-20% RS. However, cultures treated at 10 and 15 µg/mL gave 32% and 4% RS, respectively, and therefore both concentrations were analysed.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment. Clear increases in mutation were induced by the positive control chemicals 4‑nitroquinoline 1-oxide (without S9) and benzo(a)pyrene (with S9).

In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with vanadium oxide sulfate at any concentration tested in the absence and presence of S9 and there were no significant linear trends. Although a concentration giving <10% RS was analysed in the absence of S9 in Experiment 2, there was no evidence of mutagenic activity, therefore this did not affect data interpretation.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2009-11-04 to 2010-01-29.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt locus (i.e. hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The master stock of L5178Y tk +/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance are stored as frozen stocks in liquid nitrogen.
- Type and identity of media: RPMI 1640 medium containing 100 units/mL Penicillin, 100 µg/mL Streptomycin, 2.5 µg/mL Amphotericin B, 0.5 mg/mL Pluronic (except for RPMI 20 with no Pluronic) and heat inactivated horse serum (0%, 10% or 20% (v/v) for RPMI A, RPMI 10 or RPMI 20, respectively).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; each batch of cells was checked that it was mycoplasma free.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes; each batch of cells was checked for spontaneous mutant frequency.
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Range-Finder (with and without S9-mix): 46.88, 93.75, 187.5, 375, 750 and 1500 µg/mL.
Experiment I (with and without S9-mix): 0.1953, 0.3906, 0.7813, 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL;
Experiment II (with and without S9-mix): 0.25, 0.50, 1, 2, 4, 6, 8, 15, 25 and 50 µg/mL.

Cultures selected for mutation assessment:
Experiment I (with and without S9-mix): 0, 0.1953, 0.3906, 0.7813, 1.563, 3.125 and 6.25 µg/mL;
Experiment II (with and without S9-mix): 0, 1, 2, 4, 6, 8, 15 and 25 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that divanadium trioxide was not soluble in dimethyl sulphoxide but formed a homogeneous suspension in purified water with warming to approximately 60ºC at 5 to 14 mg/mL which was considered suitable for dosing.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation

Migrated to IUCLID6: 2.0 and 3.0 µg/mL (dissolved in DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline 1-oxide; 0.1 and 0.15 µg/mL (dissolved in DMSO)
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours’ incubation at 37±1ºC with gentle agitation
After exposure, cells were centrifuged, washed and resuspended in 20 mL RPMI 10 medium. Cells were transferred to flasks for growth through the expression period or were diluted to be plated for survival (scored after 7 days incubation).
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. From observations on recovery and growth of the cultures during the expression period, the cultures were selected to be plated for
viability and 6TG resistance.
- Selection time (if incubation with a selection agent): 13 to 15 days; At the end of the expression period, the cells were placed into each well of 4 x 96-well microtitre plates. Plates were incubated at 37±1ºC in a humidified incubator gassed with 5% v/v CO2 in air until scoreable (13 to 15 days) and wells containing clones were identified and counted.

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Cultures were tested in duplicate.

DETERMINATION OF CYTOTOXICITY
- Method: relative survival:
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described above for the Mutation Experiments. However, single cultures only were used and positive controls were not included. Following treatment, cells were washed with tissue culture medium and then resuspended in 20 mL tissue culture medium. Cells were plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5% v/v CO2 in air for 7 days. Wells containing viable clones were identified by microscope and counted.

OTHER:
Probable number of clones/well (P) = -ln (EW/TW);
Plating efficiency (PE) = P/No of cells plated per well;
PE = P/1.6;
Percentage relative survival (%RS) = [PE (test)/PE (control)] x 100;
Adjusted %RS = Post-treatment cell concentration for test article treatment / Post-treatment cell concentration for vehicle control;
Mutant frequency (MF) = [PE (mutant)/PE (viable)] x 10^6.
Evaluation criteria:
For valid data, the test article would be considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05).
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05).
3. The effects described above were reproducible.
Results that only partially satisfy the assessment criteria described above were considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No statistically significant increases in mutant frequency were observed following treatment with divanadium trioxide at any concentration tested.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
for details see below in the field "additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No statistically significant increases in mutant frequency were observed following treatment with divanadium trioxide at any concentration tested.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details see below in the field "additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder Experiment at the highest concentration analysed (93.75 μg/mL), compared to the concurrent vehicle controls.
- Water solubility: Preliminary solubility data indicated that divanadium trioxide was soluble in purified water with warming to approximately 60ºC at 5 to 14 mg/mL.
- Precipitation: Due to the intense colouration of the test article, it was difficult to determine the presence of precipitate accurately, therefore all concentrations cited in this report may be considered nominal.
In Experiment I: After the 3 hour treatment incubation period, precipitate was observed at the highest 5 concentrations tested in the absence and presence of S9 (6.25 to 100 μg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S9 was retained and higher concentrations were discarded.
In Experiment II: After 3 hour treatment incubation period, precipitate was observed at the highest 2 concentrations tested in the absence and presence of S9 (25 and 50 μg/mL). The lower concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S9 was retained and the higher concentration was discarded.

RANGE-FINDING/SCREENING STUDIES: 6 concentrations were tested in the absence and presence of S9 ranging from 46.88 to 1500 μg/mL (equivalent to approximately 10 mM at the highest concentration tested). No precipitate was observed at the time of treatment but, after the 3 hour treatment incubation period, precipitate was observed at all concentrations tested. The lowest two concentrations at which precipitation was observed at the end of the treatment incubation period in the absence and presence of S9 were retained (to provide an estimate of toxicity, even though post-treatment precipitate was observed at all concentrations) and all higher concentrations were discarded. The highest concentrations to give >10% RS were 46.88 μg/mL in the absence of S9 and 93.75 μg/mL in the presence of S9, which gave 56% and 69% RS, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: Comparison of controls with historical means

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I 10 concentrations, ranging from 0.1953 to 100 μg/mL, were tested in the absence and presence of S9. 7 days after treatment, concentrations were selected to determine viability and 6TG resistance. The highest concentration selected, 6.25 μg/mL, gave 83% and 81% RS in the absence and presence of S9, respectively.
In Experiment II 10 concentrations, ranging from 0.25 to 50 μg/mL, were tested in the absence and presence of S9. 7 days after treatment, concentrations were selected to determine viability and 6TG resistance. The highest concentration selected was 25 μg/mL, which gave 12% and 2% RS in the absence and presence of S9, respectively. In the presence of S9, no concentration gave 10-20% RS (cultures treated at 15 and 25 μg/mL gave 65% and 2% RS, respectively, therefore both concentrations were analysed).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Experiment I
Conclusions:
Interpretation of results (migrated information):
negative

Divanadium trioxide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic and/or precipitating concentrations in two independent experiments in the absence or presence of a rat liver metabolic activation system (S9).
Executive summary:

Divanadium trioxide was assayed for mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation (S9).

In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S9, ranging from 46.88 to 1500 μg/mL with a treatment period of 3 hours. The highest concentrations to give >10% relative survival (RS) were 46.88 μg/mL in the absence of S9 and 93.75 μg/mL in the presence of S9, which gave 56% and 69% RS, respectively.

Accordingly, in Experiment I 10 concentrations, ranging from 0.1953 to 100 μg/mL, were tested in the absence and presence of S9. After the 3 hour treatment incubation period, precipitate was observed at the highest 5 concentrations tested in the absence and presence of S9 (6.25 to 100 μg/mL). 7 days after treatment, the highest concentration selected to determine viability and 6TG resistance was 6.25 μg/mL, which gave 83% and 81% RS in the absence and presence of S9, respectively.

In Experiment II 10 concentrations, ranging from 0.25 to 50 μg/mL, were tested in the absence and presence of S9. After the 3 hour treatment incubation period, precipitate was observed at the highest 2 concentrations tested in the absence and presence of S9 (25 and 50 μg/mL). 7 days after treatment, the highest concentration selected to determine viability and 6TG resistance was 25 μg/mL, which gave 12% and 2% RS in the absence and presence of S9, respectively. In the presence of S9, no concentration gave 10 -20% RS.

Vehicle and positive control treatments were included in each Mutation Experiment in the absence and presence of S9.

In Experiments I and II, no statistically significant increases in mutant frequency were observed following treatment with divanadium trioxide at any concentration tested in the absence and presence of S9. A statistically significant linear trend was observed in the presence of S9 in Experiment II but, in the absence of any marked increases in mutant frequency at any concentration tested in this experiment, this observation was not considered biologically relevant.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
Experimental work started on 15 March 2010 and was completed on 22 April 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed by The Department of Health of the Government of the United Kingdom (2010-06-23)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The master stock of L5178Y tk hprt +/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co.
- Type and identity of media: RPMI 1640 media were prepared with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Amphotericin B (2.5 µg/mL), Pluronic (0.5 mg/mL except for "RPMI 20" with 0 mg/mL) and heat inactivated horse serum (0%, 10% or 20% (v/v) for "RPMI A", "RPMI 10" or "RPMI 20", respectively).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; each batch of cells was confirmed to be mycoplasma free.
- Periodically checked for karyotype stability: yes; each batch of cells was checked for spontaneous mutant frequency.
- Periodically "cleansed" against high spontaneous background: yes

For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Range Finder:
- with and without S9-mix: 56.84, 113.7, 227.4, 454.8, 909.5 and 1819 µg/mL.

Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range Finder Experiment.

Experiment 1:
- with and without S9-mix: 0.25, 0.5, 1, 2, 4, 8, 16, 32, 48 and 64 µg/mL.

Experiment 2:
- with S9-mix: 2, 4, 8, 12, 16, 20, 25, 30, 35 and 40 µg/mL;
- without S9-mix: 0.5, 1, 2, 4, 8, 12, 14, 16, 20 and 30 µg/mL.

Cultures selected for mutation assessment:
Experiment 1:
- without S9-mix: 1, 2, 4, 8 and 16 µg/mL;
- with S9-mix: 1, 2, 4, 8, 16 and 32 µg/mL.

Experiment 2:
- without S9-mix: 2, 4, 8, 12, 14, 16 and 20 µg/mL;
- with S9-mix: 8, 12, 16, 20, 25 and 30 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The test article was formulated as a suspension (at 18.20 mg/mL) in 0.5% w/v methyl cellulose (0.5% MC).
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that divanadium pentaoxide was not soluble in vehicles normally used in this laboratory including dimethyl sulphoxide, acetone, ethanol, dimethyl formamide, tetrahydrofuran and purified water.
Untreated negative controls:
yes
Remarks:
untreated controls (UTC), in which the vehicle was replaced with an equal volume of culture medium
Negative solvent / vehicle controls:
yes
Remarks:
treatments with the vehicle 0.5% MC diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-1-oxide; 0.1 and 0.15 µg/mL (dissolved in DMSO)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation

Migrated to IUCLID6: 2 and 3 µg/mL (dissolved in DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours at 37°C
After treatment, cultures were centrifuged, washed and resuspended in RPMI 10. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival (incubation for 7 days at 37°C).
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed.
- Selection time (if incubation with a selection agent): At the end of the expression period, the cells from selected cultures were placed into each well of 4 x 96 well microtitre plates (384 wells at 2 x 10^4 cells/well) together with 6TG (15 µg/mL). Plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air until scoreable (12 days) and wells containing clones were identified and counted.

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S9, was in duplicate (single cultures only used for positive control treatments).

EVALUATION: Wells containing clones were identified.

DETERMINATION OF CYTOTOXICITY
- Method: relative survival:
A maximum concentration of 1819 µg/mL was selected for the cytotoxicity Range Finder Experiment in order that treatments were performed up to 10 mM. Single cultures only were used and positive controls were not included.
Following treatment, cells were centrifuged washed with tissue culture medium and resuspended in 20 mL RPMI 10. Cells were plated into each well of a 96 well microtitre plate for determination of relative survival. The plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air for 8 days. Wells containing viable clones were identified by eye using background illumination and counted.

OTHER EXAMINATIONS:
- probable number of clones/well (P) = -ln (EW/TW);
- Plating efficiency (PE) = P/No of cells plated per well;
- Percentage relative survival (% RS) = [PE (test)/PE (control)] x 100;
- Mutant frequency (MF) = [PE (mutant)/PE (viable)] x 10^6
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p<0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p<0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with divanadium pentaoxide at any concentration tested.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details refer to "additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range Finder Experiment.
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range Finder in the absence and presence of S9 at the highest concentration selected (113.7 µg/mL), compared to the concurrent vehicle controls (individual data not reported).
- Precipitation:
Experiment 1: Upon addition of the test article to the cultures, precipitate was observed at the highest 3 concentrations tested in the absence and presence of S9 (32 to 64 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest 4 concentrations in the absence of S9 (16 to 64 µg/mL) and at the highest 3 concentrations in the presence of S-9 (32 to 64 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S9 was retained and higher concentrations were discarded.
Experiment 2: Upon addition of the test article to the cultures, precipitate was observed at the highest 2 concentrations in the absence of S9 (20 and 30 µg/mL) and at the highest 5 concentrations in the presence of S9 (20 to 40 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest 2 concentrations tested in the absence of S9 (20 and 30 µg/mL) and the highest 3 concentrations tested in the presence of S9 (30 to 40 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S9 was retained and higher concentrations were discarded.

RANGE-FINDING/SCREENING STUDIES: 6 concentrations were tested in the absence and presence of S9, ranging from 56.84 to 1819 µg/mL (equivalent to 10 mM at the highest concentration tested). Upon addition of the test article to the cultures, precipitate was observed at all concentrations (56.84 to 1819 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest 5 concentrations tested in the absence of S-9 (113.7 to 1819 g/mL) and at all concentrations tested in the presence of S 9 (56.84 to 1819 µg/mL). At the end of the treatment incubation period, the lowest 2 concentrations tested in the absence and presence of S9 were retained. The lowest concentration tested, 56.84 µg/mL, gave 0% and 2% RS in the absence and presence of S9, respectively. Although extreme toxicity (<10% RS) was observed at all concentrations analysed in the Range Finder Experiment, no further Range Finder Experiments were considered necessary.

COMPARISON WITH HISTORICAL CONTROL DATA: comparison of controls with historical means for controls (positive and negative)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: Ten concentrations, ranging from 0.25 to 64 µg/mL, were tested in the absence and presence of S9. 7 days after treatment, the lowest 2 concentrations in the absence and presence of S9 (0.25 and 0.5 µg/mL) were not selected to determine viability and 6TG resistance as there were sufficient non-toxic concentrations. All other concentrations were selected in the absence and presence of S9. The highest concentrations selected were 16 µg/mL in the absence of S9 and 32 µg/mL in the presence of S9, which gave 28% and 21% RS, respectively.
Experiment 2: Ten concentrations, ranging from 0.5 to 30 µg/mL in the absence of S9 and from 2 to 40 µg/mL in the presence of S9, were tested. 7 days after treatment, the lowest 2 concentrations in the absence of S9 (0.5 and 1 µg/mL) and the lowest concentration in the presence of S9 (2 µg/mL) were not selected to determine viability and 6TG resistance as there were sufficient non-toxic concentrations. In addition, one culture at the highest concentration remaining in the absence of S9 (20 µg/mL) was considered too toxic for selection and both cultures at 4 µg/mL in the presence of S9 and single cultures at 20 and 30 µg/mL were not selected due to contamination. All other concentrations were selected in the absence and presence of S9. An intermediate concentration (4 µg/mL) in the absence of S9 was later rejected from analysis due to excessive heterogeneity. The highest concentrations analysed were 20 µg/mL in the absence of S9 and 30 µg/mL in the presence of S9, which gave 10% and 20% RS, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

In Experiment 2, one culture at the maximum concentration analysed in the absence of S9 (20 µg/mL) was not plated for viability and mutation because the cell count was unacceptably low at the end of the expression period. In the replicate culture, no colonies were observed on the mutant plates and, as the calculation for mutant frequency is based on probability (involving a logarithmic component) the mutant frequency for this culture could not be evaluated. However, cells were available to be plated because colonies were observed on the viability plates for this culture and the cell suspension for the viability was prepared by dilution from the cell suspension for the mutant plates, therefore it may be assumed that the mutant frequency for this culture was theoretically “zero”.

Furthermore, the maximum concentration analysed in the absence of S9 in Experiment 1 was 16 µg/mL, limited by the appearance of post-treatment precipitate. A concentration of 16 µg/mL was also analysed in Experiment 2 and no evidence of mutagenic activity was observed at this concentration in either experiment. The concentration of 16 µg/mL may therefore be considered at (or very close to) the limit of solubility and the mutation data were considered acceptable and valid.

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that divanadium pentaoxide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to precipitating and toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9).
Executive summary:

Divanadium pentaoxidewas assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation (S9). The test article was formulated in 0.5% w/v methyl cellulose.

A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S9, ranging from 56.84 to 1819 µg/mL (equivalent to 10 mM at the highest concentration tested). The lowest concentration tested, 56.84 mg/mL, gave 0% and 2% relative survival (RS) in the absence and presence of S9, respectively. Post-treatment precipitate was observed at 56.84 µg/mL in the presence of S9 but not in the absence of S9.

Accordingly, in Experiment 1 ten concentrations, ranging from 0.25 to 64 µg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentrations selected to determine viability and 6TG resistance were 16 µg/mL in the absence of S9 and 32 mg/mL in the presence of S9 (both limited by the appearance of post-treatment precipitate), which gave 28% and 21% RS, respectively.

In Experiment 2 ten concentrations, ranging from 0.5 to 30 µg/mL in the absence of S9 and from 2 to 40 mg/mL in the presence of S9, were tested. 7 days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 20 µg/mL in the absence of S9 and 30 mg/mL in the presence of S9, which gave 10% and 20 % RS, respectively (both maximum concentrations were also limited by the appearance of post-treatment precipitate).

Negative (vehicle) and positive control treatments were included in each Mutation Experiment.

Clear increases in mutation were induced by the positive control chemicals 4‑nitroquinoline 1 -oxide (without S9) and benzo(a)pyrene (with S9).

In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with divanadium pentaoxide at any concentration tested in the absence and presence of S9 and there were no significant linear trends. In Experiment 2, one culture at the maximum concentration analysed in the absence of S9 (20 mg/mL) could not be plated for viability and mutation because the cell count was unacceptably low at the end of the expression period. In the replicate culture, no colonies were observed on the mutant plates and, as the calculation for mutant frequency is based on probability (involving a logarithmic component) the mutant frequency for this culture could not be evaluated. However, cells were available to be plated because colonies were observed on the viability plates for this culture and the cell suspension for the viability was prepared by dilution from the cell suspension for the mutant plates, therefore it may be assumed that the mutant frequency for this culture was theoretically “zero”.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Experimental work started on 8 February 2010 and was completed on 9 August 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study.
Qualifier:
according to
Guideline:
other: the current version of draft OECD guideline 487 and the most recent update, dated 2 November 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed by The Department of Health of the Government of the United Kingdom (2010-06-23)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: from two healthy donors (female)
Details on mammalian cell type (if applicable):
Blood from two healthy, non-smoking female volunteers was used for each experiment in this study. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 1.5 hours. An appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 8.1 mL HEPES buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 50 µg/mL gentamycin, so that the final volume following addition of S9 mix/KCl and the test article in its chosen vehicle was 10 mL.
The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide.
Blood cultures were incubated at 37°C for 48 hours and rocked continuously.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
TK6 cells, purchased from European Collection of Cell Cultures (ECACC), UK are maintained at Covance Laboratories Limited in tissue culture flasks containing RPMI 1640 medium with GlutaMAX-1 including 10% foetal calf serum and Penicillin/Streptomycin at 37°C, 5% CO2, 95% humidity.
Stocks of cells preserved in liquid nitrogen are reconstituted for each experiment to maintain karyotypic stability.
The cells are routinely screened for mycoplasma contamination.
Cells were subcultured at low to medium density (approximately 1x105 cells/mL) into 75 cm² vented tissue culture flasks. Cells were passaged once prior to treatment.
On the day prior to treatment, cells were subcultured into tubes at a density of approximately 1 x 10^5 cells/mL. Cells were maintained at 37°C, 5% CO2, 95% humidity prior to treatment.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Range Finder Experiment:
- without and with S9-mix (3+21 hours): 5.914, 9.856, 16.43, 27.38, 45.63, 76.05, 126.7, 211.2, 352.1, 586.8, 978.0 and 1630 µg/mL;
- without S9-mix (24+24 hours): 5.914, 9.856, 16.43, 27.38, 45.63, 76.05, 126.7, 211.2, 352.1, 586.8, 978.0 and 1630 µg/mL.
Concentrations selected for the Main Experiments were based on the results of this cytotoxicity Range Finder Experiment.

Experiment 1
- without S9-mix (3+21 hours): 10, 20, 30, 35, 40, 45, 50, 55, 60, 70, 80 and 100 µg/mL;
- with S9-mix (3+21 hours): 5, 10, 20, 25, 30, 35, 40, 45, 50, 60, 70 and 80 µg/mL;
- without S9-mix (24+24 hours): 1, 2, 4, 5, 6, 7, 8, 9, 10, 12.5, 15 and 20 µg/mL.

Experiment 2
- without S9-mix (3+21 hours): 10, 20, 30, 35, 40, 45, 50, 55, 60, 70, 80 and 100 µg/mL;
- with S9-mix (3+21 hours): 1, 2, 4, 5, 6, 7, 8, 9, 10, 12.5, 15 and 20 µg/mL;

Experiment 3
- without S9-mix (3+21 hours): 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125 and 150 µg/mL;
- without S9-mix (24+24 hours): 1, 2, 4, 5, 6, 7, 8, 9, 10, 12.5, 15 and 20 µg/mL.

Concentrations selected for analysis:
Experiment 1
- without S9-mix (3+21 hours): 20, 30, 40 and 50 µg/mL;
- with S9-mix (3+21 hours): 10, 20 and 35 µg/mL; µg/mL;
- without S9-mix (24+24 hours): 5, 8 and 12.5 µg/mL.

Experiment 2
- without S9-mix (3+21 hours): 70, 80 and 100 µg/mL;
- with S9-mix (3+21 hours): 5, 8 and 9 µg/mL;

Experiment 3
- without S9-mix (3+21 hours): 100, 125 and 150 µg/mL;
- without S9-mix (24+24 hours): 2, 4, 7 and 9 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that vanadium oxide sulphate was soluble in water for irrigation (purified water) at concentrations up to at least 34.23 mg/mL of anhydrous test article.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sterile purified water was added to cultures designated as negative controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation; Experiment 1

Migrated to IUCLID6: 0.6 and 0.8 µg/mL (dissolved in purified water)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sterile purified water was added to cultures designated as negative controls
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation; Experiment 1

Migrated to IUCLID6: 6.25 and 12.5 µg/mL (dissolved in DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sterile purified water was added to cultures designated as negative controls
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: vinblastine; 0.02, 0.04 and 0.08 µg/mL (dissolved in purified water)
Remarks:
without metabolic activation; Experiment 1
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sterile purified water was added to cultures designated as negative controls
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-1-oxide; 0.25 and 0.3 µg/mL (dissolved in DMSO)
Remarks:
without metabolic activation; Experiment 2 and 3
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sterile purified water was added to cultures designated as negative controls
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: vinblastine; 0.02, 0.03 and 0.04 µg/mL (dissolved in purified water)
Remarks:
without metabolic activation; Experiment 2 and 3
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:Cultures were treated either for 3 hours with a 21-hour recovery period, or for 24 hours with a 24-hour recovery period.
For removal of the test article, cells were pelleted by centrifugation. Cultures were washed with sterile saline (pre-warmed to approximately 37°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and antibiotics. At the appropriate times, Cytochalasin B, formulated in DMSO, was added to post wash-off culture medium.
- Fixation time (start of exposure up to fixation or harvest of cells): At the defined sampling times (21 hours after treatment or 24 hours after the end of the treatments), cultures were centrifuged, the supernatant removed and discarded and cells resuspended in 0.075 M KCl at 37°C for 4 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation and resuspension. This procedure was repeated as necessary until the cell pellets were clean.
Lymphocytes were centrifuged and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of suspension were gently spread onto multiple clean, dry microscope slides.

STAIN: After the slides had dried the cells were stained for 5 minutes in filtered 4% (v/v) Giemsa in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

NUMBER OF REPLICATIONS: In the Experiments 1, 2 and 3, at least duplicate cultures were tested.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells from each culture were analysed for MN. The number of cells containing MN and the number of MN per cell on each slide was noted.

DETERMINATION OF CYTOTOXICITY
- Method: replication index (RI):
A maximum concentration of 1630 µg/mL was selected for the initial cytotoxicity Range Finder Experiment in order that treatments were performed up to a maximum of 10 mM. Cultures were treated with the test article or vehicle control (1 mL/culture). Positive control treatments were not included.
Cultures were incubated at 37°C.
Slides were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 or 500 cells per concentration. From these data the replication index (RI) was determined.

OTHER EXAMINATIONS:
- replication index (RI) = number binucleate cells + 2(number multinucleate cells)/total number of cells in treated cultures;
- relative RI (%) = RI of treated cultures/RI of vehicle controls*100;
Cytotoxicity (%) is expressed as (100 – Relative RI).

- OTHER:
Post-treatment procedures: Experiment 2 and 3
Immediately prior to harvesting, samples were taken for quantification of Caspase activity and hence apoptosis.
At the defined sampling time, an aliquot was taken for determination of cell number using a Coulter Counter. The remaining suspension was stored at room temperature prior to slide preparation.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at high or very toxic concentrations.
Statistics:
Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories Harrogate (CLEH) standard operating procedures. The analysts were physically located remote from the CLEH facility, but were subject to CLEH management and GLP control systems (including QA inspection). All slides and raw data generated by the remote analysts were returned to CLEH for archiving on completion of analysis.

Treatment of data
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p<0.05 were accepted as significant. Additionally, the number of MN per binucleate cell were obtained and recorded.
Species / strain:
lymphocytes: from human donors
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Frequencies of MNBN cells were significantly higher (p≤0.001) than those observed in concurrent vehicle controls at the highest conc. analysed (40 and 50 µg/mL). The MNBN cell frequencies in both cultures exceeded the 95th percentile of the normal range.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: from human donors
Metabolic activation:
with
Genotoxicity:
other: The observed signif. increases in MNBN cell frequencies at all conc. analysed (10, 20 and 35 µg/mL) were not large and fell within the 95th percentile of the normal range, therefore these observations were considered of little or no biological relevance.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: from human donors
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
The observed signif. increase in MNBN cell frequencies in one culture at 8 µg/mL and in both cultures at 12.5 µg/mL exceeded the 95th percentile of the normal range and there was clear evidence of a concentration-related response.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Frequencies of MNBN cells were signif. higher than those observed in vehicle controls at all concentrations analysed (70, 80 and 100 µg/mL). The MNBN cell frequencies in both cultures at all 3 concentrations exceeded the 95th percentile of normal range.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Frequencies of MNBN cells were signif. higher than those observed in vehicle controls at all concentrations analysed (5, 8 and 9 µg/mL) The MNBN cell frequencies in both cultures at all 3 concentrations exceeded the 95th percentile of the normal range.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Frequencies of MNBN cells were signif. higher than those observed in vehicle controls at all concentrations analysed (100, 125 and 150 µg/mL). MNBN cell frequencies in both cultures at all 3 concentrations exceeded the 95th percentile of the normal range.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
MNBN CF were signif. higher than those in vehicle controls at the highest conc. analysed (4, 7 and 9µg/mL) but not at 2µg/mL. MNBN CF in both cultures at 7µg/mL but only in single cultures at 4 and 9µg/mL exceeded the 95th percentile of the normal range.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range Finder Experiment.
- Effects of pH and osmolality: No marked changes in osmolality were observed in the Range Finder Experiment at the highest concentration tested (1630 µg/mL), compared to the concurrent vehicle controls. A reduction in pH of >1 unit (compared to the concurrent controls) was observed at 1630 µg/mL following the 3+21 hour and 24+24 hour treatments in the absence of S9, but changes of lesser magnitude were observed at two lower concentrations at which pH was measured (586.8 and 978.0 µg/mL). The highest concentrations tested in Experiment 1 and 2 were 100 µg/mL (3+21 hour –S9 treatment) and 20 µg/mL (24+24 hour –S9 treatment), therefore no marked changes in pH would have been expected in the Main Experiments.

RANGE-FINDING/SCREENING STUDIES: For details see attched tables.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical vehicle control ranges for the human peripheral blood lymphocyte micronucleus assay; as well as historical vehicle control ranges for TK6 micronucleus assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY: For details please refer to attched tables.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: ; 3+21 hours (Experiment 1)

Experiment 1 (human lymphocyte cells) – Results summary

Treatment

Concentration (µg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency (%)

Historical Control Range (%) #

Statistical significance

3+21 hours

-S9

Vehicle

-

0.45

0.1-1.2

-

20

18

0.35

 

NS

30

41

0.50

 

NS

40

54

1.50

 

p<0.001

50

58

1.45

 

p<0.001

MMC, 0.80

ND

9.10

 

p<0.001

3+21 hours

+S9

Vehicle

-

0.05

0.0-1.2

-

10

13

0.40

 

p<0.05

20

33

0.30

 

p<0.05

35

52

1.00

 

p<0.001

CPA, 12.5

ND

1.90

 

p<0.001

24+24 hours

-S9

Vehicle

-

0.40

0.1-1.2

-

5

0

0.35

 

NS

8

32

1.00

 

p<0.05

12.5

57

2.25

 

p<0.001

VIN, 0.08

ND

9.60

 

p<0.001

#95thpercentile of the observed range

NS = Not significant 

ND = Not determined

 

Experiment 2 (TK6 cells) – Results summary

Treatment

Concentration (µg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency (%)

Historical Control Range (%) #

Statistical significance

3+21 hours

-S9

Vehicle

-

2.15

0.20 – 1.40

-

70

4

5.60

 

p<0.001

80

19

6.95

 

p<0.001

100

12

9.65

 

p<0.001

NQO, 0.30

ND

2.89

 

NS

24+24 hours

-S9

Vehicle

-

2.70

0.20 – 1.40

-

5

22

4.20

 

p<0.01

8

44

9.04

 

p<0.001

9

60

7.55

 

p<0.001

VIN, 0.004

ND

4.07

 

p<0.01

 

Experiment 3 (TK6 cells) – Results summary

Treatment

Concentration (µg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency (%)

Historical Control Range (%) #

Statistical significance

3+21 hours

-S9

Vehicle

-

0.15

0.20 – 1.40

-

100

13

3.65

 

p<0.001

125

20

4.31

 

p<0.001

150

19

7.11

 

p<0.001

NQO, 0.30

ND

2.04

 

p<0.001

24+24 hours

-S9

Vehicle

-

0.90

0.20 – 1.40

-

2

7

1.00

 

NS

4

58

2.63

 

p<0.01

7

47

7.32

 

p<0.001

9

46

4.49

 

p<0.001

VIN, 0.003

ND

1.38

 

NS

 

There were no marked changes in Caspase activity in treated cultures under described treatment condition,therefore apoptosis was not the primary mechanism of toxicity.

The concentrations analysed in Experiment 3 were higher than the those analysed under treatment condition of 3 +21 hours in the absence of S9 in Experiment 2 but the highest concentration analysed in Experiment 3 (150 µg/mL) gave a reduction in RI of only 19%.

Treatment for 24+24 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p < 0.01) than those observed in concurrent vehicle controls at the highest 3 concentrations analysed (4, 7 and 9 µg/mL) but not at 2 µg/mL. The MNBN cell frequencies in both cultures at 7 µg/mL but only in single cultures at 4 and 9 µg/mL exceeded the 95th percentile of the normal range and it was not possible to analyse 2000 binucleate cells at any concentration tested. The toxicity profile was very erratic by comparison with Experiment 2.

The concentrations analysed in Experiment 3 were similar to the concentrations analysed under this treatment condition in Experiment 2.

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation when tested for 3+21 hours and for 24+24 hours
negative with metabolic activation when tested up to toxic concentrations for 3+21 hours

It is concluded that vanadium oxide sulphate induced micronuclei in cultured human peripheral blood lymphocytes and human lymphoblastoid TK6 cells when tested for 3+21 hours and for 24+24 hours in the absence of S9.
Vanadium oxide sulphate did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the presence of S9.
Executive summary:

Vanadium oxide sulphate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Due to the nature of the results observed in this experiment, vanadium oxide sulphate was tested in two further in vitro Micronucleus Experiments using duplicate cultures of human lymphoblastoid TK6 cells. Treatments were performed in the absence and presence of metabolic activation (S9) in Experiment 1 and in the absence of S9 only in Experiments 2 and 3.

The test article was formulated in purified water and the highest concentrations used in the Main Experiments (limited by toxicity) were determined following a preliminary cytotoxicity Range-Finder Experiment.

In the Micronucleus Experiments, micronuclei were analysed at 3 or 4 concentrations. Appropriate negative (vehicle) control cultures were included in the test system in each experiment. Vinblastine (VIN), Mitomycin C (MMC), 4‑nitroquinoline-1-oxide (NQO) and Cyclophosphamide (CPA) were employed as positive control chemicals.

Experiment 1: Human lymphocyte cells

Treatment of cells with vanadium oxide sulphate for 3+21 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p < 0.001) than those observed in concurrent vehicle controls at the highest 2 concentrations analysed (40 and 50 µg/mL). The MNBN cell frequencies in both cultures at 40 and 50 µg/mL exceeded the 95thpercentile of the normal range.

Treatment for 3+21 hours in the presence of S9 resulted in frequencies of MNBN cells that were significantly higher (p < 0.05) than those observed in concurrent vehicle controls at all concentrations analysed (10, 20 and 35 µg/mL) and there was evidence of a concentration-related response. However, the increases in MNBN cell frequencies in treated cultures were not large and fell within the 95th percentile of the normal range; therefore these observations were considered of little or no biological relevance.

Treatment for 24 +24 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p < 0.05) than those observed in concurrent vehicle controls at the highest 2 concentrations analysed (8 and 12.5 µg/mL). The MNBN cell frequencies in one culture at 8 µg/mL and in both cultures at 12.5 µg/mL exceeded the 95thpercentile of the normal range and there was clear evidence of a concentration-related response.

The criteria for a positive result were therefore fulfilled following 3+21 hour and 24+24 hour treatments in the absence of S9. Two further Micronucleus Experiments (designated Experiments 2 and 3) was therefore performed under these two treatment conditions using TK6 cells. Measurements of Caspase activity were taken in Experiments 2 and 3 to investigate whether the primary mechanism of toxicity was by apoptosis,which would not be taken into account by assessment of replication index alone.

Experiment 2: TK6 cells

Treatment for 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p < 0.001) than those observed in concurrent vehicle controls at all three concentrations analysed (70.00, 80.00 and 100.0 µg/mL). The MNBN cell frequencies in both cultures at all three concentrations exceeded the 95th percentile of the normal range.

Treatment for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p < 0.01) than those observed in concurrent vehicle controls at all three concentrations analysed (5.000, 8.000 and 9.000 µg/mL) The MNBN cell frequencies in both cultures at all three concentrations exceeded the 95th percentile of the normal range.

There were no marked changes in Caspase activity in treated cultures under either treatment condition; therefore apoptosis was not the primary mechanism of toxicity.

The criteria for a positive result were therefore fulfilled following 3+21 hour and 24+24 hour treatments in the absence of S9, confirming the conclusion reached for Experiment 1 under these treatment conditions.

The positive controls showed only weak induction of micronuclei under both treatment conditions, although a statistically significant increase was observed for the 24+24 hour –S9 treatment. However, marked increases in the frequency of MNBN cells were observed in test article-treated cultures under both treatment conditions, thus confirming the sensitivity of the testsystem. Furthermore, the MNBN cell frequencies in all vehicle control cultures exceeded the normal range; therefore a further experiment was performed under both treatment conditions.

Experiment 3: TK6 cells

Treatment for 3+21 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p < 0.001) than those observed in concurrent vehicle controls at all 3 concentrations analysed (100, 125 and 150 µg/mL). The MNBN cell frequencies in both cultures at all 3 concentrations exceeded the 95th percentile of the normal range.

The concentrations analysed in Experiment 3 were higher than the those analysed under this treatment condition in Experiment 2 but the highest concentration analysed in Experiment 3 (150 µg/mL) gave a reduction in RI of only 19%.

Treatment for 24+24 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p < 0.01) than those observed in concurrent vehicle controls at the highest 3 concentrations analysed (4, 7 and 9 µg/mL) but not at 2 µg/mL. The MNBN cell frequencies in both cultures at 7 µg/mL but only in single cultures at 4 and 9 µg/mL exceeded the 95th percentile of the normal range and it was not possible to analyse 2000 binucleate cells at any concentration tested. The toxicity profile was very erratic by comparison with Experiment 2.

The concentrations analysed in Experiment 3 were similar to the concentrations analysed under this treatment condition in Experiment 2.

There were no marked changes in Caspase activity in treated cultures under either treatment condition.

The criteria for a positive result were again fulfilled following 3+21 hour and 24+24 hour treatments in the absence of S9 (although less convincingly so for the 24+24 hour treatments by comparison with the 2 previous experiments).

One of the positive control replicates showed clear induction of micronuclei under both treatment conditions (the mean MNBN cell frequency also exceeded the normal range for the 3+21 hour treatment and was at the upper limit of the normal range for the 24+24 hour treatment). Marked increases in the frequency of MNBN cells were again observed in test article-treated cultures under both treatment conditions,confirming the sensitivity of the test system. Furthermore, the MNBN cell frequencies in vehicle control cultures fell within the normal range with one exception (which exceeded the normal range very marginally) and the mean MNBN cell frequencies of the vehicle controls under both treatment conditions were well within the normal range.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
Experimental work started on 15 March 2010 and was completed on 7 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study.
Qualifier:
according to
Guideline:
other: the current version of draft OECD guideline 487 and the most recent update, dated 2 November 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed by The Department of Health of the Government of the United Kingdom (2010-06-23)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: from two healthy donors
Details on mammalian cell type (if applicable):
Blood from two healthy, non-smoking male volunteers was used for each experiment in this study. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 1.5 hours. An appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 8.1 mL HEPES buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 µg/mL gentamycin, so that the final volume following addition of S9 mix/KCl and the test article in its chosen vehicle was 10 mL.
The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide.
Blood cultures were incubated at 37°C for 48 hours and rocked continuously.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
TK6 cells, purchased from European Collection of Cell Cultures (ECACC), UK are maintained at Covance Laboratories Limited in tissue culture flasks containing RPMI 1640 medium with GlutaMAX-1 including 10% foetal calf serum and Penicillin/Streptomycin at 37°C, 5% CO2, 95% humidity.
Stocks of cells preserved in liquid nitrogen are reconstituted for each experiment to maintain karyotypic stability.
The cells are routinely screened for mycoplasma contamination.
Cells were subcultured at low to medium density (approximately 1x105 cells/mL) into 75 cm² vented tissue culture flasks. Cells were passaged once prior to treatment.
On the day prior to treatment, cells were subcultured into tubes at a density of approximately 1 x 10^5 cells/mL (9.4 mL cell suspension per tube). Cells were maintained at 37°C, 5% CO2, 95% humidity prior to treatment. All cultures were incubated on a slope.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Range Finder Experiment:
- without and with S9-mix (3+21 hours): 6.599, 11.00, 18.33, 30.55, 50.92, 84.87, 141.4, 235.7, 392.9, 654.8, 1091 and 1819 µg/mL;
- without S9-mix (24+24 hours): 6.599, 11.00, 18.33, 30.55, 50.92, 84.87, 141.4, 235.7, 392.9, 654.8, 1091 and 1819 µg/mL.
Concentrations selected for the Main Experiments were based on the results of this cytotoxicity Range Finder Experiment.

Experiment 1
- without and with S9-mix (3+21 hours): 5, 10, 20, 30, 35, 40, 45, 50, 55, 60, 75 and 100 µg/mL;
- without S9-mix (24+24 hours): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5 and 20 µg/mL.

Experiment 2
- without and with S9-mix (3+21 hours): 5, 10, 20, 30, 35, 40, 45, 50, 55, 60, 75 and 100 µg/mL;
- without S9-mix (24+24 hours): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5 and 20 µg/mL.

Concentrations selected for analysis:
Experiment 1
- without S9-mix (3+21 hours): 20, 30 and 40 µg/mL;
- with S9-mix (3+21 hours): 30, 40 and 50 µg/mL;
- without S9-mix (24+24 hours): 6, 7, 8 and 10 µg/mL.

Experiment 2
- without S9-mix (3+21 hours): 20, 30, 55 and 60 µg/mL;
- with S9-mix (3+21 hours): 30, 45 and 60 µg/mL;
- without S9-mix (24+24 hours): 3, 4, 6 and 7 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The test article was formulated as a suspension (at 18.20 mg/mL) in 0.5% w/v methyl cellulose (0.5% MC).
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that divanadium pentaoxide was not soluble in commonly used vehicles including purified water, dimethyl sulphoxide (DMSO), acetone, dimethyl formamide, tetrahydrofuran and ethanol.
Untreated negative controls:
yes
Remarks:
untreated controls (UTC)
Negative solvent / vehicle controls:
yes
Remarks:
0.5% MC was added to cultures
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation; Experiment 1

Migrated to IUCLID6: 0.6 and 0.8 µg/mL (dissolved in purified water)
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
0.5% MC was added to cultures
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation; Experiment 1

Migrated to IUCLID6: 6.25 and 12.5 µg/mL (dissolved in DMSO)
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
0.5% MC was added to cultures
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: vinblastine; 0.02, 0.04 and 0.08 µg/mL (dissolved in purified water)
Remarks:
without metabolic activation; Experiment 1
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
0.5% MC was added to cultures
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-1-oxide; 0.25 and 0.3 µg/mL (dissolved in DMSO)
Remarks:
without metabolic activation; Experiment 2
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
0.5% MC was added to cultures
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation; Experiment 2

Migrated to IUCLID6: 6 and 8 µg/mL (dissolved in DMSO)
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
0.5% MC was added to cultures
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: vinblastine; 0.02, 0.03 and 0.04 µg/mL (dissolved in purified water)
Remarks:
without metabolic activation; Experiment 2
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:Cultures were treated either for 3 hours with a 21-hour recovery period, or for 24 hours with a 24-hour recovery period.
- Fixation time (start of exposure up to fixation or harvest of cells): For removal of the test article, cells were pelleted aby centrifugation, for 10 minutes. Cultures were washed twice with sterile saline (pre-warmed to approximately 37°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and antibiotics. At the appropriate times, Cytochalasin B, formulated in DMSO, was added to post wash-off culture medium.
Two slides were prepared per culture. The samples were centrifuged for 5 minutes. Slides were fixed by immersion in a bath of ice-cold 90% methanol for 9 minutes, air dried and stored at room temperature.

STAIN: Slides were stained by immersion in 125 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 seconds. Slides were then washed with PBS (with agitation) for a few seconds before transfer and immersion in a second container of PBS for approximately 8-10 minutes. The quality of the stain was checked. Slides were air-dried and stored in the dark at room temperature prior to analysis. Immediately prior to analysis a minimal volume of PBS was added to the slides before mounting with glass coverslips.

NUMBER OF REPLICATIONS: In the Experiments 1 and 2, at least duplicate cultures were tested.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells from each culture (2000 per concentration), were initially analysed for MN. In Experiment 1, a further 1000 binucleate cells per replicate were analysed from cultures treated with 30.00 µg/mL for the 3+21 hour treatment in the absence of S9 in order to aid data interpretation. The number of cells containing MN and the number of MN per cell on each slide was noted. All slides for analysis were coded.

DETERMINATION OF CYTOTOXICITY
- Method: replication index (RI):
A maximum concentration of 1819 µg/mL was selected for the cytotoxicity Range Finder Experiment in order that treatments were performed up to 10 mM. S9 mix or KCl (0.5 mL/culture) was added appropriately. Cultures were treated with the test article, vehicle or untreated control (1 mL/culture). Positive control treatments were not included. Cultures were incubated at 37°C for the designated exposure time.
At the defined sampling time, cultures were centrifuged, the supernatant removed and discarded and cells resuspended in 4 mL (hypotonic) 0.075 M KCl for 4 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation and resuspension. Several drops of suspension were gently spread onto microscope slides. After the slides had dried the cells were stained for 5 minutes in filtered 4% (v/v) Giemsa in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips. Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined.

OTHER EXAMINATIONS:
- replication index (RI) = number binucleate cells + 2(number multinucleate cells)/total number of cells in treated cultures;
- relative RI (%) = RI of treated cultures/RI of vehicle controls*100;
Cytotoxicity (%) is expressed as (100 – Relative RI).

Slides were examined, uncoded, for proportions of mono-, bi- and multinucleate cells to a minimum of 500 cells per culture.

- OTHER:
Post-treatment procedures: Experiment 2
Immediately prior to harvesting, samples were taken for quantification of Caspase activity and hence apoptosis. Analysis was either performed immediately (3+21 hour treatments in the absence and presence of S9) or samples were stored at -80°C overnight (24+24 hour treatment in the absence of S9) prior to analysis.
At the defined sampling time, an aliquot was taken for determination of cell number using a Coulter Counter. The remaining suspension was stored at room temperature prior to slide preparation.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at high or very toxic concentrations.
Statistics:
Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories Harrogate (CLEH) standard operating procedures. The analysts were physically located remote from the CLEH facility, but were subject to CLEH management and GLP control systems (including QA inspection). All slides and raw data generated by the remote analysts were returned to CLEH for archiving on completion of analysis.

Treatment of data
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p<0.05 were accepted as significant. Additionally, the number of MN per binucleate cell were obtained and recorded.
Species / strain:
lymphocytes: from human donors
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Treatment for 3+21 hours or 24+24 hours resulted in frequencies of MNBN cells that were significantly higher than those observed in concurrent vehicle controls, respectively.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Treatment for 3+21 hours or 24+24 hours resulted in frequencies of MNBN cells that were significantly higher than those observed in concurrent vehicle controls, respectively.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment.
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed at the highest concentration tested (1819 µg/mL) as compared to the concurrent vehicle controls (individual data not reported).
- Precipitation: For details refer to attached tables.

RANGE-FINDING/SCREENING STUDIES: The results of the cytotoxicity Range-Finder Experiment were used to select suitable maximum concentrations for the Main Experiments. A test concentration of 50.92 µg/mL resulted in a cytotoxicity of 50%, 64% or 100% for any of the three treatment conditions . For more details see attached tables.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical vehicle control ranges for the human peripheral blood lymphocyte micronucleus assay; as well as historical vehicle control ranges for TK6 micronucleus assay were used.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The highest concentration for MN analysis was one at which approximately 55% (typically 50%-60%) reduction in RI had occurred (where possible) or was the highest concentration tested.
Experiment 2: There were no marked changes in Caspase activity in treated cultures under any of the three treatment conditions, therefore apoptosis was not the primary cause of cytotoxicity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment 1 (human lymphocytes) - Result summary

Treatment

Concentration (µg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency (%)

Historical Control Range (%) #

Statistical significance

3+21 hours

-S9

Vehicle

-

0.15

0.10–0.90

-

20

17

0.30

 

NS

30

47

1.03

 

p<0.001

40

60

1.60

 

p<0.001

MMC, 0.08

ND

3.35

 

p<0.001

3+21 hours

+S9

Vehicle

-

0.20

0.0-0.7

-

30

7

0.40

 

NS

40

25

0.55

 

p<0.05

50

54

1.35

 

p<0.001

CPA, 6.25

ND

0.85

 

p<0.001

24+24 hours

-S9

Vehicle

-

0.15

0.10–0.90

-

6

11

0.90

 

p<0.001

7

25

0.50

 

p<0.05

8

56

2.40

 

p<0.001

10.00

44

1.70

 

p<0.001

VIN, 0.08

ND

2.00

 

p<0.001

#95thpercentile of the observed range

NS = Not significant

ND = Not determined

 

 Experiment 2 (TK6 cells) - Result summary

Treatment

Concentration (µg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency (%)

Historical Control Range (%) #

Statistical significance

3+21 hours

-S9

Vehicle

-

0.26

0.2-1.4

-

20

15

2.55

 

p<0.001

30

40

4.21

 

p<0.001

55

49

12.89

 

p<0.001

60

59

8.39

 

p<0.001

NQO, 0.30

ND

0.99

 

p<0.05

3+21 hours

+S9

Vehicle

-

0.30

0.3-1.4

-

30

0

1.61

 

p<0.001

45

21

4.97

 

p<0.001

60

46

4.67

 

p<0.001

CPA, 8.00

ND

0.45

 

NS

24+24 hours

-S9

Vehicle

-

0.41

0.2-1.4

-

3

13

1.30

 

p<0.01

4

28

2.15

 

p<0.001

6

45

3.61

 

p<0.001

7

43

5.52

 

p<0.001

VIN, 0.08

ND

0.65

 

NS

#95thpercentile of the observed range

NS = Not significant

ND = Not determined

 

Conclusions:
Interpretation of results (migrated information):
positive

It is concluded that Divanadium pentaoxide induced micronuclei in cultured human peripheral blood lymphocytes and human lymphoblastoid TK6 cells when tested up to toxic and/or precipitating concentrations for 3 hours in the absence and presence of S 9 and for 24 hours in the absence of S9.
Executive summary:

Divanadium pentaoxide was tested in an in vitro micronucleus (MN) assay using duplicate human lymphocyte cultures. Due to the nature of the results observed in this experiment, the test item was tested in a further in vitro MN assay using duplicate cultures of human lymphoblastoid TK6 cells.

In both experiments, different concentrations were tested both in the absence and presence of metabolic activation (S9). The test article was treated as a suspension in 0.5% w/v methyl cellulose (0.5% MC) and the highest concentrations used in the Main Experiments (limited by toxicity) were determined following a preliminary cytotoxicity Range-Finder Experiment.

Appropriate negative (vehicle and untreated) control cultures were included in the test system in each experiment. Vinblastine (VIN), mitomycin C (MMC), 4-nitroquinoline-1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive controls.

Experiment 1: Human lymphocyte cells

Treatment of cells with divanadium pentaoxide for 3+21 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle controls at the highest 2 concentrations analysed (30.00 and 40.00 µg/mL). The MNBN cell frequencies in one culture at 30.00 µg/mL and in both cultures at 40.00 µg/mL exceeded the 95th percentile of the normal range. Post treatment precipitate was observed at both concentrations at which the induction of MN was observed.

Treatment for 3 +21 hours in the presence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.05) than those observed in concurrent vehicle controls at the highest 2 concentrations analysed (40.00 and 50.00 µg/mL). The MNBN cell frequencies in both cultures at 40.00 µg/mL fell within the normal range but both cultures at 50.00 µg/mL exceeded the 95th percentile of the normal range.

Treatment for 24+24 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.05) than those observed in concurrent vehicle controls at all 4 concentrations analysed (6.000, 7.000, 8.000 and 10.00 µg/mL). The MNBN cell frequencies in one culture at 6.000 µg/mL and in both cultures at 8.000 and 10.00 µg/mL exceeded the 95thpercentile of the normal range. The criteria for a positive result were therefore fulfilled under all treatment conditions. A further Micronucleus Experiment (designated Experiment 2) was therefore performed using TK6 cells. Measurements of Caspase activity were also taken in Experiment 2 to investigate whether the primary mechanism of toxicity was by apoptosis,which would not be taken into account by assessment of replication index alone.

Experiment 2: TK6 cells

Treatment for 3+21 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle controls at all 4 concentrations analysed (20.00, 30.00, 55.00 and 60.00 µg/mL). The MNBN cell frequencies in both cultures at all 4 concentrations exceeded the 95thpercentile of the normal range.

Treatment for 3 +21 hours in the presence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle controls at all 3 concentrations analysed (30.00, 45.00 and 60.00 µg/mL). The MNBN cell frequencies in one culture at 30.00 µg/mL and in both cultures 45.00 and 60.00 µg/mL exceeded the 95th percentile of the normal range.

Treatment for 24+24 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.01) than those observed in concurrent vehicle controls at all 4 concentrations analysed (3.000, 4.000, 6.000 and 7.000 µg/mL) The MNBN cell frequencies in one culture at 3.000 µg/mL and inboth cultures at the other 3 concentrations exceeded the 95thpercentile of the normal range.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
Experimental work started on 30 November 2009 and was completed on 5 February 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Qualifier:
according to
Guideline:
other: draft OECD guideline 487 (November 2009)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: from human donors
Details on mammalian cell type (if applicable):
Blood from two healthy, non-smoking female volunteers was used for each experiment in this study.
No volunteer was suspected of any virus infection or any known exposure to radiation or genotoxic chemicals. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 1.5 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within two days of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 8.1 mL HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 μg/mL gentamycin, so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle was 10 mL.
The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37°C±1°C for 48 hours and rocked continuously.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Range-Finder:
- 3+21 hour treatment (without and with S9-mix): 5.442, 9.070, 15.12, 25.19, 41.99, 69.98, 116.6, 194.4, 324.0, 540.0, 900.0 and 1500 µg/mL;
- 24+24 hour treatment (without S9-mix): 5.442, 9.070, 15.12, 25.19, 41.99, 69.98, 116.6, 194.4, 324.0, 540.0, 900.0 and 1500 µg/mL.

Concentrations for the Main Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment.
Main Experiment, Trial 1:
- 3+21 hour treatment (without S9-mix): 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 20.0, 30.0, 50.0, 75.0, 100.0 and 200.0 µg/mL;
Main Experiment, Trial 2:
- 3+21 hour treatment (with S9-mix): 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 20.0, 30.0, 50.0, 75.0, 100.0 and 200.0 µg/mL;
- 24+24 hour treatment (without S9-mix): 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0, 20.0, 30.0 and 50.0 µg/mL.

Concentrations selected for analysis:
Main Experiment, Trial 1:
- 3+21 hour treatment (without S9-mix): 2.0, 8.0, 20.0 and 30.0 µg/mL;
Main Experiment, Trial 2:
- 3+21 hour treatment (with S9-mix): 10.0, 20.0, 30.0 and 100.0 µg/mL;
- 24+24 hour treatment (without S9-mix): 6.0, 10.0, 14.0 and 16.0 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that divanadium trioxide was not soluble in dimethyl sulphoxide (DMSO) but formed a homogeneous suspension in water for irrigation (purified water), with warming to approximately 80ºC, at 5 to 14 mg/mL which was considered suitable for dosing.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile purified water was added to cultures
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation; positive control for pulse treatments

Migrated to IUCLID6: 0.6 and 0.8 µg/mL (dissolved in purified water)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation

Migrated to IUCLID6: 6.25 and 12.5 µg/mL (dissolved in DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: vinblastine; 0.02, 0.04 and 0.08 µg/mL (dissolved in purified water)
Remarks:
without metabolic activation; positive control for continuous treatment
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours treatment (with 21 hours recovery period) in the presence or absence of metabolic activation or continuous 24 hours treatment (with 24 hours recovery period) in the absence of metabolic activation.
For removal of the test article, cells were pelleted by centrifugation, washed twice with sterile saline, and resuspended in fresh pre-warmed medium containing foetal calf serum and gentamycin.
At the appropriate times (51 hours or 72 hours after culture initiation), Cytochalasin B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 μg/mL/culture.
- Fixation time (start of exposure up to fixation or harvest of cells): At the defined sampling time (72 hours after initiation of the 3-hour treatments or 96 hours after initiation of the 24-hour treatment), cultures were centrifuged, the supernatant removed and discarded and cells resuspended in 0.075 M KCl at 37°C for 4 minutes to allow cell swelling to occur. Cells were fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation and resuspension. This procedure was repeated as necessary until the cell pellets were clean.
Slides were prepared and stained.

STAIN: The cells were stained for 5 minutes in filtered 4% (v/v) Giemsa in pH 6.8 buffer.

NUMBER OF REPLICATIONS: Test item and positive controls were tested in duplicates; 4 cultures were tested with the vehicle control for each treatment

NUMBER OF CELLS EVALUATED: 1000 binucleate cells from each culture (2000 per concentration) were analysed for micronuclei.

DETERMINATION OF CYTOTOXICITY
- Method: relative replication index:
Cultures were treated with the test article or vehicle control (1 mL/culture), positive control treatments were not included. Cultures were incubated at 37°C ± 1°C for the designated exposure time: 3 hours of treatment +21 hours recovery (with and without S9-mix) or 24 hours of treatment + 24 hours recovery (without S9-mix). Treatment conditions were the same as described above for the main experiment. Slides were examined, uncoded, for proportions of mono-, bi- and multinucleate cells to a minimum of 500 cells per culture. From these data the replication index (RI) was determined. Cytotoxicity (%) is expressed as (100 – Relative RI).

OTHER EXAMINATIONS:
RI = number binucleate cells + 2(number multinucleate cells) / total number of cells in treated cultures;
Relative RI (%) = RI of treated cultures / RI of vehicle controls * 100;
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at high or very toxic and/or precipitating concentrations.
Statistics:
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion
test.
The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.
Species / strain:
lymphocytes: from human donors
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
Frequencies of MNBN cells were similar to vehicle controls. An exception was observed at the highest conc. where the MNBN cell frequency was signif. higher than the controls, but within the 95th percentile of the normal range (not biologically relevant).
Cytotoxicity / choice of top concentrations:
other: At the highest concentration analysed (30 µg/mL), 52% cytotoxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: from human donors
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
Observ. indicate evidence of induction of MN, but the only conc. at which the MNBN cell frequency exceeded the 95th percentile of the normal range 100μg/mL) was well into the precipitating range, therefore observation is of questionable biolog. relevance.
Cytotoxicity / choice of top concentrations:
other: At the highest concentration analysed (100 µg/mL), 40% cytotoxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: from human donors
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Frequencies of MNBN cells were signif. higher than those observed in vehicle controls at the 3 highest conc.. The MNBN cell frequencies in both cultures at all 3 conc. exceeded the 95th percentile of the normal range and were considered biolog. relevant.
Cytotoxicity / choice of top concentrations:
other: At the highest concentration analysed (16 µg/mL), 44% cytotoxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment.
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder Experiment at the highest concentration tested (1500 μg/mL), compared to the concurrent vehicle controls.
- Water solubility: Divanadium trioxide formed a homogeneous suspension in water, with warming to approximately 80ºC, at 5 to 14 mg/mL.
- Precipitation: Due to the intense colouration of the test article, it was difficult to determine the presence of precipitate accurately, therefore all concentrations cited in this report may be considered nominal.

RANGE-FINDING/SCREENING STUDIES: For details see attached tables.

COMPARISON WITH HISTORICAL CONTROL DATA: yes; Historical vehicle control ranges for the human peripheral blood lymphocyte (micronucleus assay) are available.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The lowest concentration at which precipitate was observed post-treatment was 30.00 μg/mL but one higher precipitating concentration (100.0 μg/mL), giving 40% reduction in RI, was also analysed. For more details see attached tables.
Remarks on result:
other: other: treatment of cells with divanadium trioxide for 3+21 hours
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation treatment of cells with divanadium trioxide for 24+24 hours
ambiguous with metabolic activation treatment of cells with divanadium trioxide for 3+21 hours
negative without metabolic activation treatment of cells with divanadium trioxide for 3+21 hours

Divanadium trioxide did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the absence of S9. Divanadium trioxide showed evidence of inducing micronuclei when tested for 3+21 hours in the presence of S9 (but primarily at precipitating concentrations, therefore considered of questionable biological relevance). V2O3 induced micronuclei in cultured human peripheral blood lymphocytes when tested for 24+24 hours in the absence of S9. However, it is not evident that the positive responses in this assay are true effects based on V2O3 induced chromosome damage and not e.g. induction of apoptosis. Therefore, an ad-hoc experiment was initiated to measure the primary mechanism of toxicity (i.e. apoptosis) in the caspase assay in TK6 cells.
Executive summary:

Divanadium trioxide was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments were performed both in the absence and presence of metabolic activation (S9). Treatments were conducted 48 hours following mitogen stimulation by Phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of divanadium trioxide on the replication index (RI). In the Main Experiment, micronuclei were analysed at 4 concentrations.

Appropriate vehicle control cultures were included in the test system. Mitomycin C (MMC), Vinblastine (VIN) and Cyclophosphamide (CPA) were employed as positive controls.

Treatment of cells with divanadium trioxide for 3+21 hours in the absence of S9 resulted in frequencies of MNBN cells that were generally similar to those observed in concurrent vehicle controls at all concentrations analysed. The only exception was observed at 30.00 μg/mL (the highest concentration analysed), where the MNBN cell frequency was significantly higher (p ≤ 0.05) than the concurrent vehicle control values. However, the MNBN cell frequency in both cultures at this concentration and in all other cultures under this treatment condition fell within the 95th percentile of the normal range, therefore this isolated observation was not considered biologically relevant.

Treatment for 3+21 hours in the presence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.05) than those observed in concurrent vehicle controls at 3 of the 4 concentrations analysed (10.00, 30.00 and 100.0 μg/mL, but not at 20.00 μg/mL). The MNBN cell frequencies in single cultures at 10.00 and 30.00 μg/mL and both cultures at 100.0 μg/mL exceeded the 95th percentile of the normal range. These observations indicate evidence of induction of micronuclei under this treatment condition, but the only concentration at which the MNBN cell frequency exceeded the normal range in both concentrations (100.0 μg/mL) was well into the precipitating range, therefore this observation is of questionable biological relevance.

Treatment for 24+24 hours in the absence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle controls at the highest 3 concentrations analysed (10.00, 14.00 and 16.00 μg/mL). The MNBN cell frequencies in both cultures at all 3 concentrations exceeded the 95th percentile of the normal range, although there was no clear evidence of a concentration-dependent increase in MNBN cell frequency. These observations were considered biologically relevant.

However, it is not evident that the positive responses in this assay are true effects based on V2O3 induced chromosome damage and not e.g. induction of apoptosis. Therefore, an ad-hoc experiment was initiated to measure the primary mechanism of toxicity (i.e. apoptosis) in the caspase assay in TK6 cells.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
Experimental work started on 28 September 2010 and was completed on 26 November 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: draft OECD guideline 487 (November 2009)
Principles of method if other than guideline:
This study also included quantification of Caspase activity and hence apoptosis.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
TK6 cells, purchased from European Collection of Cell Cultures (ECACC), Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK, are maintained at Covance Laboratories Limited in tissue culture flasks containing RPMI 1640 Medium with GlutaMAXTM-1 including 10% Foetal Calf Serum, plus Penicillin/Streptomycin at 37°C, 5% CO2, 95% humidity
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, routinely
- Periodically checked for karyotype stability: yes, stocks of cells preserved in liquid nitrogen were reconstituted for each experiment so as to maintain karyotypic stability
- Periodically "cleansed" against high spontaneous background: yes, cells were subcultured at low density, and before overgrowth occurs, to maintain low aberration frequencies.
- Cells were maintained at 37°C, 5% CO2, 95% humidity prior to treatment.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Main Experiment, Micronucleus-TK6 cells
- 3+21 hour treatment (with S9-mix): 1.00, 2.00, 4.00, 6.00, 8.00, 10.00, 20.00, 30.00, 50.00, 75.00, 100.0, 200.0 µg/mL;
- 24+24 hour treatment (without S9-mix): 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0, 20.0, 30.0 and 50.0 µg/mL.

Concentrations selected for analysis:
- 3+21 hour treatment (with S9-mix): 20.00, 30.00, 100.0, 200.0 µg/mL;
- 24+24 hour treatment (without S9-mix): 6.0, 10.0, 14.0, 18.0 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Preliminary solubility data (performed as part of study number 8217823: k_Lloyd_2010 (MN)) indicated that divanadium trioxide was not soluble in dimethyl sulphoxide (DMSO) but formed a homogeneous suspension in water for irrigation (purified water), with warming to approximately 80ºC, at 5 to 14 mg/mL which was considered suitable for dosing.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation

Migrated to IUCLID6: 0.05, 0.075, 0.1 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation

Migrated to IUCLID6: 12.0, 16.0 µg/mL dissolved in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: vinblastine; 0.005, 0.0075, 0.01µg/mL, dissolved in purified water
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 3 hours treatment (with 21 hours recovery period) in the presence of metabolic activation or continuous 24 hours treatment (with 24 hours recovery period) in the absence of metabolic activation.
For removal of the test article, cells were pelleted by centrifugation, washed twice with sterile saline, and resuspended in fresh pre-warmed medium containing foetal calf serum and gentamycin.
At the appropriate times (51 hours or 72 hours after culture initiation), Cytochalasin B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 μg/mL/culture.
- Selection of concentrations for micronucleus analysis, slide analysis and anaylsis of results are described in k_Lloyd_2010 (MN), Lloyd, M. (2010), Induction of micronuclei in cultured human peripheral blood lymphocytes - Divanadium trioxide. Covance Laboratories Ltd, Otley Road, Harrogate, North Yorkshire HG3 1PY, UK. Report No. 8217823.

NUMBER OF REPLICATIONS: duplicates, except vehicle control wih 4 replicates

NUMBER OF CELLS EVALUATED:
- total binucleated cells scored: 1000 per replicate (4000 for the vehicle control, if possible 2000 for each evaluated concentration; range 701 - 2000)

DETERMINATION OF CYTOTOXICITY
- Method: replication index (RI)

OTHER EXAMINATIONS:
- Osmolality and pH measurements on post-treatment incubation medium were taken in the Micronucleus Experiment.

OTHER: Capase activity:
- Before harvest samples were taken for quantification of Caspase activity and hence apoptosis.
- The Caspase detection reagent was prepared as follows:
1) The Caspase-Glo substrate and Caspase-Glo buffer were removed from the freezer and allowed to thaw at room temperature before beginning.
2) The Caspase-Glo buffer was added to Caspase-Glo substrate to form the reagent. 100 μL of cell suspension was added in duplicate to corresponding wells of a 96-well Luminometer plate and brought to room temperature. 100 μL of Caspase Reagent was added to each sample in the 96-well plate and shaken for at least 30 seconds (300-500 rpm) and incubated at room temperature for 1.5 hours.
The reaction carries on for in excess of 3 hours allowing reading times to be adjusted as the same result is obtained at 30 minutes and 3 hours.
3) Luminescence was analysed using a Spectramax Gemini EM plate reader (set at 5 reads / plate.)
- Harvesting: At the defined sampling time cultures were centrifuged and medium was removed from each culture tube and the cell pellets resuspended in fresh complete medium. An aliquot of each cell suspension was added to Isoton solution and a cell count performed using a Coulter counter. The remaining cell suspension was stored at room temperature prior to slide preparation.
- Slide preparation: The cell concentration of each culture (per treatment concentration) was adjusted to approximately 6 x 10E+04 cells/mL for cytospin slide preparation. Two slides were prepared per culture. Slides were stained by immersion in Acridine Orange.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at high or very toxic and/or precipitating concentrations.
Statistics:
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion
test.
The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Divanadium trioxide induced micronuclei in cultured human lymphoblastoid TK6 cells when tested for 24+24 hours in the absence of S-9
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
37 % for 10.00 and 14.00 µg/mL and 65 % for 18.00 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
Divanadium trioxide showed evidence of inducing micronuclei when tested for 3+21 hours in the presence of S-9 but the observation was considered of questionable biological relevance.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
ANALYSIS OF DATA (for more details see attachment):
3+21 hours + S9:
- Treatment of TK6 cells with Divanadium trioxide for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle controls at the highest concentration analysed (200.0 μg/mL).
- However, the MNBN cell frequencies exceeded the 95th percentile of the normal range in only one of the two cultures analysed at this concentration. This cannot be explained on the basis of toxicity; because there was no reduction in RI compared to the mean vehicle control value.
- Furthermore, these results were not comparable with those observed in human lymphocytes under this treatment condition – no precipitate was observed at any concentration analysed (up to 200.0 μg/mL), whereas post-treatment precipitate was observed at 30.00 μg/mL and above in human lymphocyte cultures in Covance study Number 8217823 (k_Lloyd_2010 (MN)).
- Overall, 16 micronuclei were observed in a total of 701 binucleated cells scored, equating to 2.28% MNBN cells. This exceeded the normal range pro rata, indicating evidence of micronucleus induction. However, due to the low numbers of cells analysed in one replicate culture and poor reproducibility between replicate cultures at this concentration, the observation may be considered of questionable biological relevance.

24+24 hours -S9:
- Treatment for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.05) than those observed in concurrent vehicle controls at all four concentrations analysed (6.000, 10.00, 14.00 and 18.00 μg/mL).
- The MNBN cell frequencies in one culture at 6.000 μg/mL and in both cultures at the highest three concentrations exceeded the 95th percentile of the normal range and there was evidence of a concentration-related response.
- Furthermore, the concentrations analysed for micronuclei under this treatment condition were very similar to those analysed in human lymphocyte cells, therefore the data are comparable between the two cell lines.

- Overall, the toxicity and solubility profiles of Divanadium trioxide were similar in human lymphocytes and TK6 cells for the 24+24 hour –S-9 treatment but differed markedly between the two test systems for the 3+21 hour +S-9 treatment. This may provide an explanation for the disparity between micronucleus induction seen between the cell lines following the 3+21 hour +S-9 treatment.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Osmolality and pH were measured in the cytotoxicity Range-Finder Experiment. No marked changes in osmolality or pH were observed at the highest concentrations tested under both treatment conditions, compared to the concurrent vehicle controls.

RANGE-FINDING/SCREENING STUDIES: see K_Lloyd_2010 (MN)

VALIDITY OF STUDY:
1) The binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures (for MNBN cell frequency) for the 3+21 hour +S-9 treatment. For the 24+24 hour –S-9 treatment, significant heterogeneity (p ≤ 0.05) between replicate cultures was observed. Marked differences in MNBN cell frequencies (and binucleate cells analysed) were observed between replicate cultures at all concentrations analysed but clear increases in MNBN cell frequencies were observed at all concentrations that exceeded the normal range, therefore the observed heterogeneity did not affect the interpretation of data, which were considered valid.
2) The frequency of MNBN cells in vehicle controls fell within the normal ranges.
3) The positive control chemicals induced statistically significant increases in the proportion of MNBN cells.
4) A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in negative control cultures at the time of harvest following the 24+24 hour –S-9 treatment. For the 3+21 hour +S-9 treatment, only 41% of cells had gone through one or more cell divisions, but the data were considered acceptable because three of the four cultures were at (or very close to) 50%.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical vehicle control ranges for TK6 micronucleus assay were used.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

It may be noted that three trials of the Micronucleus Experiment were performed and data from the third trial are reported here. In the first two trials, the replication index values of the vehicle control cultures were unacceptably low and slides were not analysed for micronuclei. No data from the first two trials are reported.

Conclusions:
Interpretation of results (migrated information):
negative There were no marked changes in Caspase activity in treated cultures under either treatment condition.
ambiguous with metabolic activation Divanadium trioxide showed evidence of inducing micronuclei when tested for 3+21 hours in the presence of S-9 but the observation was considered of questionable biological relevance.
positive without metabolic activation Divanadium trioxide induced micronuclei in cultured human lymphoblastoid TK6 cells when tested for 24+24 hours in the absence of S-9

It is concluded that Divanadium trioxide did not induce apoptosis in cultured human lymphoblastoid TK6 cells, demonstrated by unaltered Caspase activity in all treated cultures.
Divanadium trioxide induced micronuclei in cultured human lymphoblastoid TK6 cells when tested for 24+24 hours in the absence of S-9. In the same test system, Divanadium trioxide showed evidence of inducing micronuclei when tested for 3+21 hours in the presence of S-9 but the observation was considered of questionable biological relevance.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2011-03-01 to 2011-04-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2010-03-22
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung (CHL) cells, strain IU
Details on mammalian cell type (if applicable):
Chinese hamster lung (CHL) cells, strain IU, were originally obtained from the European Collection of Cell Cultures (ECACC) Cell Bank and are regularly checked for mycoplasma contamination.
The cells were routinely grown and subcultured in Minimal Essential medium supplemented with 10% heat-inactivated foetal calf serum, 1% of a 100x Non-essential amino acids solution, 2.0 mM L-Glutamine and 0.1% of a 50 mg/mL Gentamycin solution. The cells were grown at 37°C in an atmosphere containing 5% carbon dioxide in 75 cm^2 tissue culture flasks. The doubling time of CHL cells, strain IU, is approximately 10 hours and they have a modal chromosome number of 25.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was obtained from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver
Test concentrations with justification for top dose:
First test: 21.54, 35.91, 59.84, 99.74, 166.23, 277.06, 461.76 and 769.60 µg/mL (absence or presence of S9)
Second test: 21.54, 35.91, 59.84, 99.74, 166.23, 277.06, 461.76 and 769.60 μg/mL (absence or presence of S9(1% v/v))
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 1% w/v methylcellulose (suspending agent)
- Justification for choice of solvent/vehicle: vanadium carbide nitride was insoluble in solution, with the exception of using low pH water (not compatible with the test system) where slight solubility was observed.
Based on the insolubility and density of vanadium carbide nitride, it was recommended using 1% w/v methylcellulose as the suspending agent to achieve a homogeneous suspension acceptable for dosing.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% v/v methylcellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Positive control: without metabolic activation; final concentrations: 0.1 and 0.2 µg/mL. The lowest concentration was selected for metaphase analysis.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1% v/v methylcellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Positive control: with metabolic activation; final concentrations: 5 and 10 μg/mL. The lowest concentration was selected for metaphase analysis.
Details on test system and experimental conditions:
Vanadium Carbide Nitride was added to cultures at 1% v/v (50 μL per 5 mL culture).

TREATMENT OF CELLS WITH TEST SUBSTANCE - FIRST TEST (3 hour treatment and 12 hours recovery)
Duplicate cultures were prepared throughout for each 3 hour treatment in the absence and presence of S9 mix.
Absence and presence of S9 mix: 50 μL aliquots of vanadium carbide nitride were added to one set of duplicate cultures to give final concentrations of 21.54, 35.91, 59.84, 99.74, 166.23, 277.06, 461.76 and 769.60 μg/mL.
The solvent control and the positive controls (Mitomycin C: 0.1 and 0.2 µg/mL; cyclophosphamide: 5 and 10 µg/mL) were also tested in duplicate.
Three hours after dosing, the treated medium was removed and replaced with fresh medium. They were then incubated for a further 12 hours.

HARVESTIN AND FIXATION
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 μg/mL. After 2 hours incubation, the medium was removed from the flasks and saved in conical centrifuge tubes. Then, 2 mL of Accutase™ (pre-warmed at 37ºC) was added to each flask. After a minimum of five minutes, this was removed and placed in the appropriate conical centrifuge tube containing the saved medium.
An aliquot of the cell suspension (1 mL) was removed from all samples and added to 9 mL of Isoton®, an azide-free electrolyte balanced solution, and counted using an electronic particle counter (Coulter counter) to measure cell toxicity. The remaining cell suspensions were then centrifuged for 5 minutes at 200 g. The supernatant was discarded and the cells resuspended in 5 mL of a hypotonic solution, 0.075 M KCl (pre-warmed at 37ºC). After a 10 minute incubation at 37 ºC, 0.5 mL of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid) was gently added. The cell suspensions were then centrifuged for 5 minutes at 200 g and resuspended in 4 mL fixative, which was replaced a further three times.

SLIDE PREPARATION
The pellets were resuspended in a small volume of fresh fixative. A small amount of the cell suspensions were dropped onto pre-cleaned microscope slides, which were then allowed to air-dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and then mounted in DPX.

MICROSCOPIC EXAMINATION
Metaphase cells were identified using a low power objective and examined at a magnification of x1000 using an oil immersion objective. One hundred metaphase figures were examined from each culture. Chromosome aberrations were scored according to the classification of the ISCN (1985)*. Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. The vernier readings of all aberrant metaphase figures were recorded.

SECOND TEST (3 and 15 hour treatment)
In this second test a 15 hour continuous treatment was used in the absence of S9 mix. In the presence of S9 mix, a three hour treatment was used, as in the first test. The harvest time was at 15 hours for both parts of the test. Concentrations of vanadium carbide nitride were as follows:
In the absence of S9 mix: 21.54, 35.91, 59.84, 99.74, 166.23, 277.06, 461.76 and 769.60 μg/mL.
In the presence of S9 mix (1% v/v): 21.54, 35.91, 59.84, 99.74, 166.23, 277.06, 461.76 and 769.60 μg/mL.
Duplicate cultures were used for each treatment and two cultures were treated with the solvent control. Positive control cultures were treated as in the first test. Three hours after dosing, the cultures containing S9 mix, the treated medium was removed and replaced with fresh medium. They were then incubated for a further 12 hours. Cultures treated in the absence of S9 mix were incubated continuously for 15 hours.
All cultures were treated with Colcemid®, at a final concentration of 0.1 μg/mL, two hours before the end of the incubation period. They were then harvested, fixed and the slides prepared as previously described. The slides were then examined microscopically as previously described.

OTHER EXAMINATIONS:
- the osmolality and pH of the test substance in medium was tested at 769.6 μg/mL.

*Reference:
- ISCN (1985) An International System for Human Cytogenetic Nomenclature, HARNDEN, D.G. and KLINGER, H. P. (Eds). S. Karger AG, Basel.
Evaluation criteria:
An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
- statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
- the increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.
- the increases are reproducible between replicate cultures.
- the increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
- evidence of a concentration-response relationship is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.
Statistics:
The number of aberrant metaphase cells in each test substance group was compared with the solvent control value using the one-tailed Fisher exact test (Fisher 1973)*.
A Cochran-Armitage test for trend (Armitage, 1955)* was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.
D20s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002)*. The following model was used:
p = C + ((1-C) / 1+ exp(-ontercept - slopeln(conc)))
p is the proportion of cells with aberrations, conc is the concentration of test substance. C is a parameter estimating the control proportion of aberrations.

*References:
- FISHER, R.A. (1973) The Exact Treatment of 2 × 2 Table in: Statistical Methods for Research Workers. Hafner Publishing Company, New York.
- ARMITAGE, P. (1955) Tests for linear trends in proportions and frequencies. Biometrics, 11, 375-386. (Cochran-Armitage test).
- ARMITAGE, P., BERRY, G. and MATTHEWS, J.N.S. (2002) Statistical Methods in Medical Research, 4th edition. Blackwell Science, Oxford, UK.
Species / strain:
mammalian cell line, other: Chinese hamster lung (CHL) cells, strain IU
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: the osmolality and pH of the test substance in medium was tested at 769.6 μg/mL. No fluctuation in osmolality of more than 50 mOsm/kg and no change in pH of more than 1.0 unit were observed compared with the solvent control.

- Precipitation: precipitate (visible by eye) was present in the final culture medium at the end of treatment at all vanadium carbide nitride treatment concentrations. The highest three tested concentrations (277.06, 461.76 and 769.60 μg/mL) were selected for metaphase analysis, as precipitate did
not preclude analysis.

RESULTS - first test
- absence of S9 mix (3 hour treatment and 12 hours recovery): vanadium carbide nitride caused a reduction in the cell count to 76% of the solvent control value at 769.60 μg/mL, the highest tested concentration
- presence of S9 mix (3 hour treatment and 12 hours recovery): vanadium carbide nitride caused no significant reduction in cell count at 769.60 μg/mL, the highest tested concentration, when compared with the solvent control value.
- metaphase analysis: in both the absence and the presence of S9 mix, vanadium carbide nitride caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control.
- positive controls: both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

RESULTS - second test
- absence of S9 mix (15 hour continuous treatment): vanadium carbide nitride caused no reduction in the cell count at 769.60 μg/mL, the highest tested concentration, when compared with the solvent control.
- presence of S9 mix (3 hour treatment and 12 hours recovery): vanadium carbide nitride caused no reduction in the cell count at 769.60 μg/mL, the highest tested concentration, when compared with the solvent control.
- metaphase analysis: in both the absence and the presence of S9 mix, vanadium carbide nitride caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control.
- positive controls: both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

COMPARISON WITH HISTORICAL CONTROL DATA:
First test:
- all mean values for the solvent control (1% v/v methylcellulose), were within the laboratory historical control range, when taken at the 99% confidence limit.
- in both the absence and the presence of S9 mix, a number of mean values for vanadium carbide nitride treatment concentrations were marginally outside the 99% confidence limit. Although, these values exceeded the laboratory historical control range, with no observed statistical significance or evidence of a concentration-response, these apparent increases were considered not to be biologically relevant. All remaining mean values were within the laboratory historical control range, when taken at the 99% confidence limit.

Second test:
All mean values for the solvent control (1% v/v methylcellulose), and all vanadium carbide nitride treatment concentrations were within laboratory historical control range, when taken at the 99% confidence limit.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
From the cell count data of CHL cells treated with vanadium carbide nitride, no biologically significant cytotoxicity was observed (i.e. no significant reduction in mitotic index greater than 50%). Therefore, the maximum concentration tested (769.60 μg/mL) was the highest concentration selected for metaphase analysis.

POLYPLOID ANALYSIS
No statistically significant increases in polyploid metaphases were observed during metaphase analysis, in either test.

Please also refer for results to "Attached background material" below
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the test substance vanadium carbide nitride has shown no evidence of clastogenic activity in this in vitro cytogenetic test system, under the experimental conditions described.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Publication is reliable with restrictions - no exposure with metabolic activation - no stability data - significant cytotoxicity was observed for all concentrations evaluated - no data on solubility/precipitation, pH and osmolality - prolonged exposure time: 28 h
Qualifier:
no guideline followed
Principles of method if other than guideline:
Human peripheral lymphocyte cultures were exposed to 1, 2, 4, or 8 μg/mL of vanadium(V) pentoxide (V2O5). These cultures were then screened for structural chromosomal aberrations, and mitotic index measurements were made. Cytogenetic analysis followed the recommended protocols of Swierenga et al. (1991)* for chromosomal aberrations in mammalian cultures.
*Reference
Swierenga, S.H.H., Heddle, J.A., Sigal, E.A., Gilman, J.P.W., Brillinger, R.L., Duglas, G.R., et al. (1991). Recommended protocols based on a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocytes cultures. Mutat Res 246:301–322.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
Heparinized blood samples were obtained by venopuncture from a healthy nonsmoking male without a history of recent exposure to drugs and radiation.
Metabolic activation:
not specified
Test concentrations with justification for top dose:
1, 2, 4, or 8 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Untreated negative controls:
other: unspecific negative control (not clear what kind of negative control was used during the study.)
Negative solvent / vehicle controls:
other: unspecific negative control (not clear what kind of negative control was used during the study.)
True negative controls:
other: unspecific negative control (not clear what kind of negative control was used during the study.)
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Positive control: 0.4 µg/mL
Details on test system and experimental conditions:
CYTOGENETIC ANALYSIS, CULTURES, AND TREATMENTS
From the heparinized blood samples, 0.5 mL of blood was cultured in 4.5 mL of RPMI-1640 culture medium (Sigma, St. Louis, Missouri, USA) with 5 μg/mL of phytohemagglutinin (Sigma) and treated with vanadium salts for cytogenetic analysis following the recommended protocols of Swierenga et al. (1991)* for chromosomal aberrations in mammalian cultures, with the aim of reducing the spontaneous frequency of chromosomal aberration. When working with chemicals such as vanadium that delay the cell cycle, it is necessary to extend the incubation period to observe metaphase damage.

The test item was dissolved in distilled water and added to cultures at different concentrations, 44 hours after the cultures were started. The concentrations for the test item were selected based on preliminary reports (Roldán and Altamirano, 1990); Rodríguez-Mercado et al., 2003)*. All cultures were performed in duplicate and incubated at 37°C for 72 hours. Exposure duration was 28 hours.

HARVESTING
4 μg/mL of colchicine (Sigma) was added to each culture 1 hour before harvesting cells.
Cultures were harvested and fixed according to Roldán and Altamirano (1990)*. Briefly, cells were centrifuged at 1,200 rpm for 10 minutes, the culture medium was removed, and 5 mL of prewarmed (37°C) hypotonic KCl 0.075-M solution was added. Cells were resuspended and incubated in a water bath for 20 minutes. After incubation, the cell suspension was centrifuged and 5 mL of fixative (methanol-glacial acetic acid, 3:1; v/v) was added. Fixative was removed by centrifugation and changed twice. To prepare the slides for cytogenetic analysis, 5 drops of the fixed cell suspension were placed on clean glass slides, flame-dried, and subsequently stained for 20 minutes with a 0.04% Giemsa solution (Sigma).

CHROMOSOME ABERRATION EVALUATION
Two hundred metaphases from each concentration were examined for chromosomal aberration, and 4,000 cells were used to estimate the mitotic index, 100 metaphases, and 2,000 cells from each parallel culture, respectively. For the positive controls, an analysis of 100 metaphases was considered sufficient because of high toxicity. The structural chromosomal aberration was classified according to previous reports (Rodríguez-Mercado et al., 2003; Savage, 2004)*; the type of aberrations scored included chromatid and chromosome types as well as gaps. Only metaphases containing 46 ± 2 centromeres were analysed. Total number and type of aberrations, as well as the percentage of aberrant cells per treatment, were evaluated.

* References:
- Roldán, E., Altamirano, M. (1990). Chromosomal aberrations, sister chromatid exchanges, cell-cycle kinetics, and satellite association in human lymphocytes culture exposed to vanadium pentoxide. Mutat Res 245:61–65.
- Rodríguez-Mercado, J.J., Roldan-Reyes, E., Altamirano-Lozano, M. (2003). Genotoxic effects of vanadium(IV) in human peripheral blood cells. Toxicol Lett 144:359–369.
- Savage, J.R. (2004). On the nature of visible chromosomal gaps and breaks. Cytogen Gen Res 104:46–55.
*References:
Evaluation criteria:
no data
Statistics:
Statistical significance for differences in mitotic index were determined by the z-test, and chi-square tests were used for differences in chromosomal aberration.
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No differences were observed between duplicate cultures regarding the parameters evaluated in this study. Further, the results show a significant
concentration-related decrease in the frequency of mitoses in cultures treated with the test item (please refer to table 1 in the field "Any other information on results incl. tables" below).
The frequency of structural aberrations and the percentage of aberrant metaphases in cultures treated with the test item showed no significant increase. A statistically significant increase in chromosomal aberration frequency and aberrant cells was observed after lymphocytes were treated with mitomycin C.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Chromosomal aberrations (CAs) and mitotic index (MI) in human peripheral blood lymphocyte cultures treated with the test item and mitomycin for 28 hours

 

Distribution and total number of structural chromosomal aberration

 

 

 

Ct

Cs

G

Total (A + A’)

% aberrant cells without G

MI% ± SD (inhibition %)

Test substance (µg/mL)

Cells scored by culture

A

A’

A

A’

A

A’

Without G

With G

 

 

1

100

3

3

0

0

4

3

6

13

3.0

2.00 ± 0.27 (24)

2

100

2

4

0

0

4

3

6

13

3.0

1.90 ± 0.19 (28)a

4

100

1

2

0

0

3

3

3

9

1.5

1.57 ± 0.30 (41)b

8

100

3

3

0

0

3

3

6

12

3.0

1.66 ± 0.34 (38)b

0.4 µg/mL Mitomycin C

50

41

42

0

0

5

7

83c

95

48.0c

0.75 ± 0.15 (70)c

aP < 0.05;bP < 0.01;cP < 0.005, compared vs. control

SD, standard deviation; Ct, chromatid type: breaks, acentric fragments, and exchange figures; Cs, chromosome type: breaks, acentric fragments, dicentric, and exchange figures; G, chromatide and isochromatid gaps; A, first culture and A´, duplicate culture; Regression lines MI: V2O5 y = 2.671 – 0.239x, r = 0.8009.

Conclusions:
Interpretation of results (migrated information):
negative

The test item was tested negative for chromosomal aberrations, but positive for cytotoxicity.

Genetic toxicity in vivo

Description of key information

Vanadium substances may express some clastogenicity in vitro, this occurs only at unphysiologically high concentrations and via mechanisms that appear to lack physiological relevance, and is not mirrored in corresponding in vivo (RL=1) assays.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
no data available
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
minor
Qualifier:
according to
Guideline:
other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests, 1995 (European Agency for the Evaluation of Medicinal Products, 1995)
Deviations:
yes
Remarks:
minor
Principles of method if other than guideline:
Minor deviations without effecting the general outcome
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2010-03-02
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: out-bred young adult male Sprague Dawley rats were obtained from Charles River (UK) Ltd, Margate, UK or Harlan UK Ltd., Oxon, UK
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 219.3 - 273.7 g
- Assigned to test groups randomly: yes
- Housing: in groups of up to six in solid bottom, grid top cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%,
- Air changes: 15-20/hour
- Photoperiod: 12 hours dark/light cycle
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Dosing regime: Single dose.
Dose levels-range-finder: 90, 120, 170, 240, 300 mg/kg/day
Dose levels - MN study: 0, 30, 60, 120 mg/kg/day
Maximum dose: Maximum tolerated dose based on Range-Finder data.
Dose volume: 10 mL/kg
Animals per group: 6
Satellite animals per group: 3
Bone marrow sampled: 24 or 48 hour post final administration
Tissue sampled (satellite animals): 24 or 48 hour post final administration
Frequency of treatment:
single dose
Post exposure period:
Bone marrow sampled: 24 or 48 hour post final administration
No. of animals per sex per dose:
6 male rats per dose were exposed to vanadium pentoxide.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide (clastogenic positive control) 20 mg/kg, single oral administration.
- Vinblastine (aneugenic positive control) 0.5 mg/kg/day, two administrations via intraperitoneal injection.
Tissues and cell types examined:
- Bone marrow (femur) smears for micronucleus assessment
- Bone marrow samples (femur & humerus) and testes for bioanalysis
Details of tissue and slide preparation:
Bone marrow smears for micronucleus assessment:
- marrow from two femurs from each animal was flushed from the cavity with foetal bovine serum
- samples were filtered and centrifuged
- cell pellet was resuspended and smears were made
- two slides per animal were assigned to MN assessment and two to antikinetochore assessment

Tissue samples for bioanalysis:
- Bone marrow samples (femur and humerus) were scraped from the bone and samples pooled per animal.
- Both testes were sampled.
- Tissues were flash frozen in liquid nitrogen and held on dry ice prior to transfer to ≤-50°C for long term storage.

Slide analysis:
- Slides from all groups were independently coded and analysed blindly in a random design.
- Initially the relative proportions of PCE, seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed. Then at least 2000 PCE per animal were examined for the presence of micronuclei (MN).

- Calculation:
1. % PCE for each animal and the mean for each group. The group mean % PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity.
2. Frequency of MN PCE (i.e. MN per 2000 PCE) and % MN PCE for each animal and the group mean % MN PCE (± standard deviation). The MN PCE data from the vehicle control group(s) were compared with the laboratory's historical vehicle control ranges (individual animal distribution data and calculated group mean 95% confidence interval) to determine whether the assay was acceptable.

Evaluation criteria:
Acceptance criteria
The assay was considered valid if all the following criteria were met:
1. The vehicle control MN PCE data were comparable with the laboratory’s historical vehicle control ranges
2. At least five animals out of each group were available for analysis, and
3. The positive control chemical (CPA and VIN) induced a statistically significant increase in the frequency of MN PCE.

Evaluation criteria
For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data
3. The group mean MN PCE value at such a point exceeds the 95% calculated confidence interval for the mean historical vehicle control data
4. A dose-response trend in the proportion of MN PCE was observed (where more than two dose levels were analysed).

The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but
not essential in the evaluation of a positive result (Scott et al, 1990). Biological relevance was taken into account, for example consistency of response within and between dose levels.
Statistics:
For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square test (Lovell et al, 1989). The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square (Lovell et al, 1989). Probability values of p ≤ 0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
V2O5 did not induce micronuclei in polychromatic erythrocytes of bone marrow of male rats treated up to 120 mg/kg/day (an estimate of the maximum tolerated dose for this study).
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
Divanadium pentaoxide did not induce micronuclei in polychromatic erythrocytes of bone marrow of male rats treated up to 120 mg/kg/day (an estimate of the maximum tolerated dose for this study).
Executive summary:

The genetic toxicity of divanadium pentaoxide was assessed by testing the ability of V2O5 to induce an increase in the frequency of micronucleated erythrocytes in bone marrow of male rats. Vanadium tissue levels increased in all sampled tissues with increased V2O5 dosage levels. Thus, vanadium reached the target tissues bone marrow and testes in a dose-dependent manner. Divanadium pentaoxide, administered orally to male rats by gavageup to the maximum tolerated dose of 20 mg/kg/day, did not induce micronuclei in polychromatic erythrocytes of bone marrow.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
no data available
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented guideline study.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Mice were obtained from Taconic Farms, Inc. (Germantown, NY).
- Age at study initiation: 6 or 7 weeks old
- Weight at study initiation: range of mean body weights in the exposure groups: 25-26 g (males) and 20-21 g (females)
- Assigned to test groups randomly: yes
- Housing: individually
- Diet: ad libitum; except during exposure periods
- Water: ad libitum
- Acclimation period: 10 or 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9 +/- ca. 2
- Humidity (%): 55 +/- 15
- Air changes: 15/hour
- Photoperiod: 12 hours dark/light cycle
No further details are given.
Route of administration:
inhalation: aerosol
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- For the 3-month studies, vanadium pentoxide aerosol generation was based on the principle of pneumatic dispersion and consisted of two major components: a screw feeder (Model 310, Accurate, White Water, WI) that metered vanadium pentoxide powder at a constant rate and a Jet-O-Mizer jetmill (Fluid Energy Corp., Harfield, PA) that used compressed air to disperse the metered powder and form the aerosol.
- Aerosol leaving the jetmill passed through a one-stage impactor and a vertical elutriator to eliminate or deagglomerate the large particles before entering a plenum and manifold distribution system. The aerosol delivery system consisted of three holding chambers that diluted the aerosol in three stages. A metered amount of diluted aerosol was removed and mixed with conditioned air at the inlet to each exposure chamber to achieve the appropriate exposure concentration. The electrical charge buildup on the aerosol particles was neutralised by mixing the aerosol with high concentrations of bipolar ions, which were generated using a Pulse Gun (Static Control Services, Palm Springs, CA) air nozzle. A transvector air pump was installed at the aerosol inlet to each exposure chamber to provide additional control of the aerosol flow rate and improve stability of the chamber concentration.
- The stainless-steel inhalation exposure chambers (Lab Products, Inc., Maywood NJ), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERISATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week and monthly thereafter.
- A 10-stage Quartz Crystal Microbalance-based cascade impactor was used to separate the aerosol particles into sequential size ranges; the mass median aerodynamic diameter was calculated from the corresponding mass fraction of particles at each stage.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. Minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.
Duration of treatment / exposure:
3 months
Frequency of treatment:
6 hours plus T90 (15 minutes) per day, 5 days per week
Post exposure period:
1 day
Remarks:
Doses / Concentrations:
1 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
3 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
8 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
16 mg/m³
Basis:
nominal conc.
No. of animals per sex per dose:
Groups of 10 male and 10 female mice were exposed to vanadium pentoxide.
Control animals:
yes
Positive control(s):
no data
Tissues and cell types examined:
peripheral blood samples were obtained from male and female mice
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of nine or ten animals per exposure group.

OTHER: In addition, the ratio of polychromatic erythrocytes (PCEs) to NCEs among 1000 total erythrocytes was determined as a measure of bone marrow toxicity.
Evaluation criteria:
Statistical as well as biological factors are considered. For an individual assay, the statistical procedure for data analysis has been described in the preceding protocol. There have been instances, however, in which multiple aliquots of a chemical were tested in the same assay, and different results were obtained among aliquots and/or among laboratories. Results from more than one aliquot or from more than one laboratory are not simply combined into an overall result. Rather, all the data are critically evaluated, particularly with regard to pertinent protocol variations, in determining the weight of evidence for an overall conclusion of chemical activity in an assay.
Statistics:
The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups.
Sex:
male/female
Genotoxicity:
negative
Remarks:
No increase in the frequency of micronucleated NCEs was seen in peripheral blood samples from male or female mice exposed to vanadium pentoxide for 3 months by inhalation.
Toxicity:
no effects
Remarks:
Chemical exposure had no effect on the ratio of PCEs to NCEs in peripheral blood indicating no toxicity to the bone marrow by vanadium pentoxide.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Based on decreased survival in the 32 mg/m3 males and body weight decreases in 32 mg/m3 males and females, an exposure concentration of 32 mg/m3 was considered too high for use in a 3-month study. Therefore, the exposure concentrations selected for the 3-month inhalation study in rats were 0, 1, 2, 4, 8, and 16 mg/m3.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the frequency of micronucleated NCEs was seen in peripheral blood samples from male or female mice exposed to vanadium pentoxide for 3 months by inhalation.
- Ratio of PCE/NCE (for Micronucleus assay): Chemical exposure had no effect on the ratio of PCEs to NCEs in peripheral blood (data not presented), indicating no toxicity to the bone marrow by vanadium pentoxide.
Conclusions:
Interpretation of results (migrated information): negative
Vanadium pentoxide, administered for 3 months by inhalation to male and female mice, did not increase the frequency of micronucleated normochromatic erythrocytes in peripheral blood.
Executive summary:

The genetic toxicity of vanadium pentoxide was assessed by testing the ability of the chemical to induce an increases in the frequency of micronucleated erythrocytes in mouse peripheral blood. Mice were exposed 90 days to an vanadium pentaoxide aerosol by inhalation before blood was sampled and prepared for analysis.

Vanadium pentoxide, administered to male and female mice, did not increase the frequency of micronucleated normochromatic erythrocytes in peripheral blood.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
Experimental starting date was 02-June-2009. Experimental completion date was 12-June-2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: conducted according to GLP and according to guideline
Reason / purpose:
reference to same study
Qualifier:
no guideline available
Guideline:
other: This study was conducted according to the procedures indicated by the following recommendations: Tice, R.R.; et al. (200): Single Cell Gel/Comet Assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environmental and Molecular Mutagenesis
Guideline:
other: Hartmann, A. et al. (2003) Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis 18(1), 45-51.
Principles of method if other than guideline:
This experiment was performed to assess the potential of the test item divanadium pentaoxide to induce in vivo primary DNA breaks in individual cells of mouse lung tissue.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., 5190 Dominion Drive, Dublin, VA 24084 / USA
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: 16.9 to 23.2 g
- Assigned to test groups randomly: yes, under following basis: Computer-generated random algorithm.
- Fasting period before study:
- Housing: The mice were housed individually in Makrolon® type-2 cages with wire mesh tops and standard softwood bedding.
- Diet: Pelleted standard Kliba Nafag 3433 mouse maintenance diet was available ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30- 70%
- Air changes (per hr): 10- 15
- Photoperiod: There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.
Route of administration:
inhalation: aerosol
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a system similar to that originally described by Sachsse et al.. The mice are confined separately in restraint tubes which are positioned radially around the flow-past, nose-only exposure chamber described by Cannon et al.. The design of this chamber is based upon the fluid dynamic modeling of the test aerosol flow. The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube.
- Source and rate of air: The air for aerosol generation was delivered by a model 'Atlas Copco ZT30-8' oil-free, rotating gear-wheel compressor system'. Additionally, there was an air-cleaning filter system mounted at the entry in the exposure room.
- System of generating particulates/aerosols: A dust aerosol was generated from the test item using a rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a [63]Ni charge neutraliser. The [63]Ni charge neutralizer was used to electrostatically discharge the generated aerosol prior to arrival at the exposure chamber. An airvacuum dilution system was used to achieve the target aerosol concentration for groups 2 and 3.
- Temperature, humidity, pressure in air chamber:
- Air flow rate: The flow of air at each tube was 0.5 l/min, which was sufficient to minimize re-breathing of the test aerosol as it was more than twice the respiratory minute volume of a mouse.
- Method of particle size determination: The particle size distribution was determined gravimetrically and chemically.

TEST ATMOSPHERE
- Brief description of analytical method used: The aerosol concentrations of the test item determined gravimetrically and chemically, relative humidity, temperature and oxygen concentration were measured on test aerosol samples collected directly from the delivery tube in the breathing zone of the mice, at an empty port of the exposure chamber. Airflow rates were measured for the collection of samples for the determination of test item concentration and particle size using a dry-test meter (‘Schlumberger Industries SA’, City of Geneva) and/or a pressure gauge (Timeus & Co., Zürich), calibrated with a reference
dry-test meter.
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
16 days
Frequency of treatment:
daily, 6 hours per day
Post exposure period:
none
Remarks:
Doses / Concentrations:
0.25 mg/m³ air
Basis:
nominal conc.
group 2
Remarks:
Doses / Concentrations:
1.0 mg/m³ air
Basis:
nominal conc.
group 3
Remarks:
Doses / Concentrations:
4.0 mg/m³ air
Basis:
nominal conc.
group 4
No. of animals per sex per dose:
4 groups of 48 mice ( group 1 (control group and groups 2-4); 1 group of 6 mice (positive control: group 5)
Control animals:
yes
Positive control(s):
methylmethanesulfonate, 99% (Sigma-Aldrich Chemie GmbH)
- Justification for choice of positive control(s): The oral administration of MMS resulted in significantly increased percentage tail DNA intensities of the analysed lung cells in the lungs as demonstrated in Harlan CCR validation study 1048302 (group 5)
- Route of administration: oral (gavage)
- Doses / concentrations: 200 mg/kg b.w. (single dose)
Tissues and cell types examined:
Lung samples were analysed for DNA strand breaks using the comet assay.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The target aerosol concentrations were proposed by the Sponsor and are based on the results obtained within the previous National Toxicology Program (Toxicology and Carcinogenesis Studies of Vanadium Pentoxide (CAS No. 1314-62-1) in F344/N Rats and B6C3F1 Mice (Inhalation Studies)). NTP Technical Report Series No. 507. NIH Publication No. 03-4441. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, NTP, Research Triangle Park, NC.

TREATMENT AT SAMPLING TIME: As soon as feasible after the last exposure (groups 1-4) and 4 hours after the treatment of the positive controls (group 5), the following procedures were performed:
- Perfusion of lung tissue: After anaesthetizing the mice with 46% Ketamin, 23% Xylazin and 31% Midazolan (approx. 2 ml/kg body weight) the lung were perfused through the right ventricle with saline. Afterwards the lungs were intubated via the trachea and lavaged with approx. 20 ml mincing buffer (20 mM EDTA, 10 % DMSO in HBSS pH 7.4-7.6) for collection of BAL cells (Bronchio Alveolar Lavage cells). The lungs were then further perfused under resuscitation. Excised lung lobes were then minced in 1 ml ice-cold mincing buffer using fine scissors to obtain a single cell suspension containing minced lung tissue cells.
- Preparation of BAL cells: The cells isolated from lung lavage were centrifuged and resuspended in 0.7% agarose and afterwards brought onto slides.
- Preparation of minced lung tissue: The minced cells were filtered through a 40 µm cell strainer. The cell suspension was centrifuged and resuspended in 0.7% agarose and brought onto slides.

DETAILS OF SLIDE PREPARATION: 3 slides per minced lung tissue cells and one slide per BAL cells per animal were prepared with 10% cell suspension and 90% of a 0.7% (w/v) agarose (low melting point agarose) solution. 100 µl were applied per slide. The slides were cooled before being submerged in lysis buffer. The following steps of protocol were performed with the slides:
- Lysis: 1 hour up to 7 days in Lysis buffer pH 10 at 2-8°C in the dark.
- Alkaline treatment: 20 minutes in electrophoresis buffer, pH >13 at 2-8°C in the dark.
- Electrophoresis: 30 minutes in electrophoresis buffer 25 V, 300 mA, at 2-8°C in the dark.
- Neutralisation: about 11 minutes in neutralisation buffer.
- Dehydration: approximately 2 minutes in 99% ethanol.
After dehydration the slides were air-dried and stored protected from dust and light until evaluation.

METHOD OF ANALYSIS: The DNA of the cells was stained with the fluorescence dye ethidium bromide (20 µg/ml; 40 µl per slide), immediately before evaluation. Where possible all mice per test group, 100 cells per preparation and per animal (BAL: 100 cells from one slide, minced lung tissue cells: 50 cells per slide) were evaluated on coded slides with a fluorescence microscope.
The damage of each nucleus were measured and recorded by an image analysis programme (Comet Assay IV, Perceptive Instruments).
An increasing extent of DNA migration detected with the Comet Assay results in an increase of the mean of tail % intensity of one test group compared to the vehicle control. Tail % intensity is expressed as a percentage of the Comet's total intensity. Additionally, the number of nuclei from apoptotic or necrotic cells per 500 total nuclei was determined.
The following criteria are used for analysis of slides:
- Only clearly defined non-overlapping cells are scored.
- Nuclei from dead/apoptotic cells are not scored (% tail intensity above 80%).
- Cells with unusual staining artefacts are not scored.
- All other normal cells, 100/animal, are scored where possible.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a biologically relevant increase in the tail % intensity in a single dose group as compared to the historical control range.
Statistics:
The experimental unit of exposure for in vivo studies is the animal, and all analysis will be based on individual animal response.
Values obtained from each parameter will be treated as follows:
- The median is calculated for each slide.
- For each animal the mean of the medians are calculated, if possible.
- The mean of the medians and standard error of the means is calculated for each group.
Statistical methods will be used as an aid in evaluating the results. Normally distributed data will be analysed using a one-tailed Student's t-test. An F-test is first used to test for homogeneity of variances for pairwise analysis. However, the primary point of consideration will be the biological relevance of the results.
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No slides prepared from animals treated with any dose level of V2O5 showed an remarkable increase in number of dead cells indicating a good slide preparation.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
no data

The analysis of cells obtained from bronchioalveolar lavage (BAL cells) or mincing of the lung tissue showed that neither the treatment of the animals with the vehicle nor the treatment of the animals with the indicated doses of V2O5 induced DNA damage. The obtained values for the treated animals were near to those treated with air control. The mean % tail intensities of the animals treated with 0.25, 1.0 and 4.0 mg/m³ air were 0.307, 2.343 and 0.288 for BAL cells and 0.615, 0.210 and 0.428 for minced lung tissue cells, respectively. The variations in the values between the dose groups were not significant. All obtained values were within the historical control group.

The number of nuclei from apoptotic or necrotic cells per 500 total nuclei was determined for each sample to indicate the quality of the slide preparation. Neither the slides prepared from animals treated with the negative control nor from those treated with any dose level of V2O5 showed an remarkable increase in number of dead cells indicating a good slide preparation. However, the amount of dead cells could not be determined for the positive control groups. There, the cells showed a structure very close to dead cells complicating the macroscopic differentiation. Nevertheless, the validity of the study was not jeopardised, because the comet results where dead cells (% tail intensity above 80%) could be excluded showed a clear and statistically significant increase in DNA damage in cells treated with the positive control.

Conclusions:
Interpretation of results (migrated information): negative
The analysis of BAL cells or pulmonary cells did not demonstrate any DNA damage in the in vivo Comet assay induced by divanadium pentaoxide.
Executive summary:

In this toxicity study, divanadium pentaoxide was administered by nose-only inhalation to female B6C3H1/Hsd mice for a period of 16 days (6 hours per day) at 3 target concentrations. Mice were allocated to 4 groups of 48 mice each. The mice of Group 1 served as air controls. One additional group of 6 mice (group 5) was used as positive controls for a comet assay test.

After completion of treatment the animals of each sub group were subjected to lung preparation to investigations on DNA lesions in the lungs and/or to a comet assay with BAL and pulmonary cells.

The analysis of BAL cells or pulmonary cells did not demonstrate any DNA damage in the in vivo Comet assay induced by divanadium pentaoxide.

In groups 3 and 4, DNA modifications were noted, as were histopathological findings that may have been the result of incomplete particle clearance from the lung and sustained oxidative stress induced by the oxide.

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well-documented
Reason / purpose:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
This study investigated whether Kras mutation is an early event in the development of lung tumours induced by inhalation of particulate vanadium pentoxide aerosols. This study sought to: 1) characterize any Kras mutational response with respect to vanadium pentoxide exposure concentration, and 2) investigate the possibility that amplification of preexisting Kras mutation is an early event in vanadium pentoxide-induced mouse lung tumorigenesis. Male Big Blue B6C3F1 mice (6 mice/group) were exposed to aerosolized particulate vanadium pentoxide by inhalation, six hours/day, five days/week for four or eight weeks, using vanadium pentoxide exposure concentrations of 0, 0.1, and 1 mg/m³. The levels of two different Kras codon 12 mutations [GGT→GAT (G12D) and GGT→GTT (G12V)] were measured in lung DNAs by Allele-specific Competitive Blocker PCR (ACB-PCR).
GLP compliance:
no
Type of assay:
other: Kras mutation in the development of lung tumours
Species:
mouse
Strain:
other: Big Blue (BB) B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BioReliance, Rockville, MD (supplied through Taconic Farms, Germantown, New York)
- Age: eight weeks old
- Weight: control: 27.1 ± 1.02 g; 0.1 mg/m³ dose: 27.3 ± 1.01 g; 1.0 mg/m³ dose: 27.6 ± 1.53 g
- Housing: during the quarantine period (prior to group assignment), animals were housed in whole body stainless steel mouse cages used in Hazleton-2000 chambers. After randomization, animals were housed in Hazleton-2000 chambers as racks for the whole body stainless steel 40 compartment mouse cages.
- Diet (ad libitum, except during inhalation exposures): certified irradiated NTP-2000 Diet (Zeigler Brothers, Inc. Gardeners, PA)
- Water (ad libitum, except during inhalation exposures): drinking water (City of Chicago)
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 20-29ºC
- Relative humidity: 17-70%
- Air changes: minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

The study employed currently acceptable practices of good animal husbandry (Guide for the Care and Use of Laboratory Animals; National Research Council,2011) and all animal care and use procedures used in the study were approved by IIT Research Institute Institutional Animal Care and Use Committee.

Route of administration:
inhalation: aerosol
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: animals were restrained in holding tubes (CH Technologies, Westwood, NJ, USA) connected to the exposure chamber ports. Animals were acclimated to the tubes over the course of three days (1.5, 3 and 4.5 hours/day, respectively) prior to the beginning of the test period.
- System of generating particulates/aerosols: the vanadium pentoxide test atmosphere was generated by aerosolization of the test substance using compressed air-operated Wright Dust Feeder Aerosol Generation systems (BGI Incorporated, Waltham, MA), which were positioned over each chamber. The test substance was weighed and packed into a reservoir using a hydraulic shop press, forming a cake. A thin film of material was removed from the cake by a scraper rotating at a constant speed and dispersed as an aerosol with the aid of compressed air. The dispersed material was mixed with filtered, breathable quality compressed air in a mixing plenum to the appropriate vanadium pentoxide concentration prior to introduction to the inhalation chambers.
- Method of particle size determination: aerosol particle size distribution in the inhalations chambers were measured at preselected intervals using quartz crystal microbalance (QCM) cascade impactor (California Measurements Inc., Sierra Madre, CA) equipped with 10 stages to collect size-segregated samples.

TEST ATMOSPHERE
- Brief description of analytical method used: test material concentration in the exposure atmosphere was determined by collecting samples on mixed cellulose ester filters followed by chemical analysis to determine vanadium content.
Duration of treatment / exposure:
4 and 8 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0.1 and 1.0 ppm V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.093 and 1.178 mg/m³ V2O5
Basis:
analytical conc.
4 week exposure
Remarks:
Doses / Concentrations:
0.103 and 1.041 mg/m³ V2O5
Basis:
analytical conc.
8 week exposure
No. of animals per sex per dose:
6 male mice per dose and exposure duration
Control animals:
yes, sham-exposed
Positive control(s):
none
Tissues and cell types examined:
Please refer to the field "Details of tissue and slide preparation" below.
Details of tissue and slide preparation:
Sham-exposed and vanadium pentoxide-exposed mice were sacrificed, then lungs were removed and snap-frozen in liquid nitrogen. The left lobe of each mouse lung was placed on dry ice until ACB-PCR analysis of Kras mutation.

Clinical signs were evaluated twice a day and any abnormal findings were recorded.

ISOLATION OF LUNG DNA:
- lung tissues were minced
- lung tissue sample were homogenized in 1 mL of extraction buffer (0.5 mg/mL proteinase K, 20 mM NaCl, 1 mM CaCl2, pH 8.0, and 10 mM Tris pH 8.0).
- samples were incubated for 3 hours at 37°C
- samples were extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), and ethanol-precipitated.
- samples were resuspended in 400 μL of RNase buffer (10 mg/mL RNase A, 600 units/mL Ribonuclease T1, 100 mM sodium acetate, and 50 mM Tris-HCl (pH 8))
- samples were incubated ~16 hours at 37°C, then re-extracted with phenol/chloroform/isoamyl alcohol as described above.
- each ethanol-precipitated sample was resuspended in 50 μL of TE buffer (5 mM Tris, 0.5 mM EDTA, pH 7.5).
- DNA was digested with HindIII according to the manufacturer’s instructions (New England Biolabs).
- DNA was phenol/chloroform/isoamyl alcohol extracted and ethanol-precipitated, as described above, then resuspended in 20 μL of TE buffer.
- DNA concentrations were measured spectrophotometrically, diluted to ~0.5 μg/uL, and then the diluted DNA concentration was measured again.

PREPARATION OF STANDARDS AND UNKNOWNS BY FIRST-ROUND PCR AMPLIFICATION:
- first-round PCR products were prepared from standards and unknowns as previously described [Parsons, 2013]*.
- first-round PCR amplification, employing the PfuUltra hotstart high-fidelity DNA polymerase amplified a Kras gene sequence from linearized plasmid DNAs, in order to synthesize the mutant frequency standards.
- similarly, HindIII-digested, lung DNA samples were used to amplify Kras gene sequence from each DNA sample.
- optimized PCR conditions were used to ensure any PCR-induced errors would occur at a level below that measurable by ACB-PCR.
- HindIII-digested, lung DNA was used for first-round PCR amplification of a 170 bp gene segment encompassing part of the 5’ untranslated region, exon 1, and part of intron 1 (NC_000072 Region: 29,950 to 30,119).
- 200 μL PCR reaction contained: 1 μg genomic DNA, 200 nM primer TR67 (TR67, 5’-TGGCTGCCGTCCTTTACAA-3’), 200 nM primer TR68 (TR68, 5’-GGCCTGCTGAAAATGACTGAGTATAAACTTGT-3’), 200 nM dNTPs, 1X PfuUltra reaction buffer, and 10 units PfuUltra hotstart high-fidelity DNA Polymerase.
- cycling conditions were 94°C for 2 minutes, followed by 28 cycles of 94°C for 1 minute, 58°C for 2 minutes, 72°C for 1 minute, followed by a 7 minutes extension at 72°C.
- primers were purchased from Integrated DNA Technologies.

PURIFICATION OF AND QUANTIFICATION OF PCR PRODUCTS:
- PCR products (standards and unknowns) were purified by ion-pair reverse phase chromatography using a WAVE Nucleic Acid Fragment Analysis System.
- PCR products were complexed with 0.1 M triethylammonium acetate (Buffer A: 0.1M TEAA)
- PCR products were bound to a DNASep column (containing C18 alkylated PS/DVB polymer).
- PCR products, input template, unincorporated nucleotides, and primers were eluted using a gradient of increasing acetonitrile concentration (Buffer B: 0.1 M TEAA, 25% acetonitrile), thereby separating nucleic acids by size/column retention time.
- threshold collection method was used to collect the 170 bp PCR products based on their absorbance at 260 (using a UV detector at the appropriate retention time) into individual tubes in a chilled fraction collector.
- PCR products were evaporated to dryness using a Savant Speed-VacConcentrator (Model ISS110).
- PCR products were resuspended in TE buffer
- multiple 2-μl aliquots were prepared (stored at -80°C).
- multiple aliquots were repeatedly quantified using an Epoch Micro-Volume Spectrophotometer System with a Take3 Microplate Reader, until three measurements that varied by <10% from the group mean were obtained.

ACB-PCR QUANTIFICATION OF Kras CODON 12 GAT AND GTT MUTANT FREQUENCIES:
- purified mutant and wild type first-round PCR products (generated using plasmid templates) were mixed to generate standards with mutant frequencies of 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, and 0.
- duplicate mutant frequency standards and a no-DNA control were analysed in parallel with equal numbers of copies of first-round PCR products generated from mouse lung DNA samples.
- ACB-PCR analysis of both mutations: a total of 5 x 10^8 copies were analysed in 50 μL reactions performed in 96-well plates using a DNA Engine Tetrad 2.

1) Quantification of Kras codon 12 GAT mutation:
- each ACB-PCR reaction contained: 1X Standard Taq (Mg-free) reaction buffer, 0.1 mg/mL gelatin, 1 mg/mL Triton X-100, 40 μM dNTPs, 1.6 mM MgCl2, 150 nM mutant-specific primer (TR76, 5’-fluorescein-CTTGTGGTGGTTGGAGCTAA-3’), 520 nM blocker primer (TR77, 5’-CTTGTGGTGGTTGGAGCTAdG-3’), and 150 nM upstream primer (TR73, 5’-TCGTAGGGTCGTACTCATC-3’).
- each reaction was initiated with the addition of 1.2 units of Extreme Thermostable Single-stranded DNA Binding Protein, 0.33 mUnits of PerfectMatch PCR Enhancer, and 70 mUnits of Hemo KlenTaq DNA polymerase.
- cycling conditions were 2 minutes at 94°C, followed by 36 cycles of 94°C for 30 seconds, 45°C for 45 seconds, and 72°C for 1 minute.
- Kras codon 12 GAT ACB-PCR product is 91 bp in length.

2) Quantification of Kras codon 12 GTT mutation:
- each ACB-PCR reaction contained: 1X Standard Taq (Mg-free) reaction buffer, 0.1 mg/mL gelatin, 1 mg/mL Triton X-100, 40 μM dNTPs, 1.5 mM MgCl2, 400 nM mutant-specific primer (TR87, 5’-fluorescein-CTTGTGGTGGTTGGAGCTAT-3’), 440 nM blocker primer (TR113, 5’-CTTGTGGTGGTTGGAGCTTG-3’-phosporylation), and 400 nM upstream primer (TR110, 5’-TCGTAGGGTCATACTCATC-3’).
- each reaction was initiated with the addition of 160 mUnits of PerfectMatch PCR Enhancer (Stratagene), and 80 mUnits of Hemo KlenTaq DNA polymerase.
- cycling conditions were 2 minutes at 94°C, followed by 36 cycles of 94°C for 30 seconds, 41°C for 45 seconds, and 72°C for 1 minute.
- Kras codon 12 GTT ACB-PCR product is 91 bp in length.

GEL ELECTROPHORESIS AND QUANTIFICATION OF ACB-PCR PRODUCTS.
- following ACB-PCR, 10 μL of bromophenol blue/xylene cyanol-containing 6X ficol loading dye were added to each well of the 96-well plate.
- 15 μL of each Kras codon 12 GAT ACB-PCR reaction or 10 μL of each Kras codon 12 GTT ACB-PCR reaction were analysed on 8% nondenaturing polyacrylamide gels.
- fluorescent DNA length marker was used to confirm the identity of the 91-bp products.
- fluorescent bands were visualized using a PharosFX scanner with an external blue laser.
- pixel intensities of the bands were quantified using Quantity One® software and a locally-averaged background correction.

*Reference:
- B.L. Parsons, M.G. Manjanatha, M.B. Myers, K.L. McKim, S. D. Shelton, Y. Wang, B.B. Gollapudi, N.P. Moore, L.T. Haber, M.M. Moore, Temporal Changes in K-ras Mutant Fraction in Lung Tissue of Big Blue B6C3F1 Mice Exposed to Ethylene Oxide, Toxicological Sciences, 136 (2013) 26-38.
Evaluation criteria:
no data
Statistics:
The pixel intensities determined for the mutant frequency standards were plotted against their mutant frequencies on log-log plots. A trend line (power function) was fitted to the data and the formula of the function was used to calculate the mutant frequency in each unknown sample based on its pixel intensity. The arithmetic average of the three independent mutant frequency measurements was calculated.The mean of the three independent measurements and the standard error of the mean were calculated and plotted using GraphPad Prism Version 5. The average mutant frequency in each lung DNA sample was log-transformed and the average log-transformed mutant frequency for the six mice in each treatment group was calculated. This value is the geometric mean mutant frequency for each treatment group.
Because a significant portion of the ACB-PCR measurements was below the limit of accurate ACB-PCR quantification (10^-5), the statistical significance of treatment effects was performed by comparing the distribution of samples above and below 10^-5 among treatment groups, using a Fisher’s exact test. The Spearman correlation coefficient was used to determine whether the Kras codon 12 GAT and GTT mutant frequencies within particular mice were correlated. Relationships between these variables were also visualized using linear regression analyses. All statistical analyses were performed using GraphPad Prism Version 5 (GraphPad Software, Inc., La Jolla, CA), two-sided tests, and a significance level of 0.05.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
- wet inguinal fur was seen in all mice including controls, likely resulting from being restrained in the nose-only exposure tubes. No other clinical signs were observed and all animals survived the 4 and 8 week exposure periods.
- a statistically significant increase (P ≤ 0.05) in the lung weights was observed in the mice exposed to vanadium pentoxide at the 1 mg/m³ level after 4 and 8 weeks; however, no effect on lung weights was apparent in the 0.1 mg/m³ animals at either time [Manjanatha, in press].

- following DNA isolation, first-round PCR products encompassing Kras exon 2 sequences were generated from the 36 study samples, which were produced with similar yields.
- three independent ACB-PCR measurements were collected on each sample.

- average correlation coefficient found were as follows:
Kras codon 12 GAT standard curves: 0.9496 (range 0.9435 to 0.9528).
Kras codon 12 GTT standard curves: 0.9594 (range 0.9579 to 0.962)

- coefficient of determination were as follows:
for triplicate GAT mutant frequency measurements: 0.71.
for the triplicate GTT mutant frequency measurements: 0.82.

- all ACB-PCR mutant frequency data were log-transformed.

- 8-week Kras codon 12 GAT and 4- and 8-week Kras codon 12 GTT datasets included measurements below the limit of accurate ACB-PCR quantification (10^-5). Therefore, the significance of potential treatment effects within the datasets was analysed by comparing the number of samples with mutant frequencies greater than and less than 10^-5, using Fisher’s exact test. No significant differences were observed among treatment groups.

- a correlation analysis was performed to determine whether the levels of the two Kras mutations within individual mice were correlated. Only seven samples had measureable levels of both mutations (i.e., >10^-5). The two mutations were correlated at the 0.1, but not the 0.05, confidence level (Spearman r = 0.7143, P = 0.0881).

*Reference:
- M.G. Manjanatha, S.D. Shelton, L.T. Haber, B. Gollapudi, M. J.A., N. Rajendran, M.M. Moore, Evaluation of cII mutations in lung of male Big Blue mice exposed by inhalation to vanadium pentoxide for up to 8 weeks, Mutation Research - Genetic Toxicology and Environmental Mutagenesis, (in press).
Conclusions:
Interpretation of results (migrated information): negative
Inhalation of aerosols of particulate divanadium pentaoxide for 4 or 8 weeks did not result in significant changes in levels of Kras codon 12 GAT or GTT mutation. The data support the idea that the accumulation of additional Kras mutants is not an early event, and/or that the proliferative advantage of Kras mutant clones requires either longer expression times or larger cumulative divanadium pentaoxide exposures. Furthermore, the data do not provide support for either a direct genotoxic effect of divanadium pentaoxide on Kras in the context of the exposure conditions used, or early amplification of preexisting mutation as being involved in the genesis of divanadium pentaoxide-induced mouse lung tumours.
Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well-documented
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 488: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays
Principles of method if other than guideline:
In this study vanadium pentoxide was tested whether it has mutagenic potential in vivo in tumor target tissue (lungs). Groups of six male transgenic Big Blue mice were exposed to particulate aerosols containing 0, 0.1 or 1 mg/m³ (tumorigenic concentration) vanadium pentoxide for 4 or 8 weeks (6 hours/day, 5 days/week) and cII mutant frequencies were evaluated in the right lungs.
GLP compliance:
no
Type of assay:
transgenic rodent mutagenicity assay
Species:
mouse
Strain:
other: Big Blue (BB) B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BioReliance, Rockville, MD (supplied through Taconic Farms, Germantown, New York)
- Age: eight weeks old
- Weight: control: 27.1 ± 1.02 g; 0.1 mg/m³ dose: 27.3 ± 1.01 g; 1.0 mg/m³ dose: 27.6 ± 1.53 g
- Housing: during the quarantine period (prior to group assignment), animals were housed in whole body stainless steel mouse cages used in Hazleton-2000 chambers. After randomization, animals were housed in Hazleton-2000 chambers as racks for the whole body stainless steel 40 compartment mouse cages.
- Diet (ad libitum, except during inhalation exposures): certified irradiated NTP-2000 Diet (Zeigler Brothers, Inc. Gardeners, PA)
- Water (ad libitum, except during inhalation exposures): drinking water (City of Chicago)
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 20-29ºC
- Relative humidity: 17-70%
- Air changes: minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

The study employed currently acceptable practices of good animal husbandry (Guide for the Care and Use of Laboratory Animals; National Research Council,2011) and all animal care and use procedures used in the study were approved by IIT Research Institute Institutional Animal Care and Use Committee.

Route of administration:
inhalation: aerosol
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: animals were restrained in holding tubes (CH Technologies, Westwood, NJ, USA) connected to the exposure chamber ports. Animals were acclimated to the tubes over the course of three days (1.5, 3 and 4.5 hours/day, respectively) prior to the beginning of the test period.
- System of generating particulates/aerosols: the vanadium pentoxide test atmosphere was generated by aerosolization of the test substance using compressed air-operated Wright Dust Feeder Aerosol Generation systems (BGI Incorporated, Waltham, MA), which were positioned over each chamber. The test substance was weighed and packed into a reservoir using a hydraulic shop press, forming a cake. A thin film of material was removed from the cake by a scraper rotating at a constant speed and dispersed as an aerosol with the aid of compressed air. The dispersed material was mixed with filtered, breathable quality compressed air in a mixing plenum to the appropriate vanadium pentoxide concentration prior to introduction to the inhalation chambers.
- Method of particle size determination: aerosol particle size distribution in the inhalations chambers were measured at preselected intervals using quartz crystal microbalance (QCM) cascade impactor (California Measurements Inc., Sierra Madre, CA) equipped with 10 stages to collect size-segregated samples.

TEST ATMOSPHERE
- Brief description of analytical method used: test material concentration in the exposure atmosphere was determined by collecting samples on mixed cellulose ester filters followed by chemical analysis to determine vanadium content.
Duration of treatment / exposure:
4 and 8 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0.1 and 1.0 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.09 ± 0.018 and 1.18 ± 0.194 mg/m³
Basis:
analytical conc.
4 week exposure
Remarks:
Doses / Concentrations:
0.10 ± 0.017 and 1.04 ± 0.200 mg/m³
Basis:
analytical conc.
8 week exposure
No. of animals per sex per dose:
6 male mice per dose and exposure duration
Control animals:
other: sham air exposed
Positive control(s):
none
Tissues and cell types examined:
Please refer to the field "Details of tissue and slide preparation" below.
Details of tissue and slide preparation:
Control and test substance exposed mice were sacrificed and lungs were removed and snap-frozen in liquid nitrogen and kept on dry ice until subsequent DNA isolation and mutation analyses. The right lobe was used for cII mutation analysis, and the left lobe was used for allele-specific competitive blocker-polymerase chain reaction (ACB-PCR) analysis for K-ras mutations [Banda et al., 2015]*, thereby enabling comparisons of a neutral transgene mutant frequency (MF) with the endogenous K-ras mutant fraction.

At the time of processing lung tissue for DNA extraction, right lungs were weighed and their weights were recorded.

ISOLATION OF LUNG DNA AND cII MUTATION ASSAY:
High-molecular weight genomic DNA was extracted from the frozen mouse right lung using the RecoverEase DNA Isolation Kit, according to the manufacturer's instruction manual; the DNA was stored at 4°C until DNA packaging was performed. Packaging, plating, and the determination of mutant frequencies were performed in a blocked manner as described previously in Manjanatha et al., [2015]* so as to minimize bias from day-to-day variations in the procedure using the ʎ Select-cII Mutation Detection System for BB Rodents, according to Stratagene's protocol. The ʎ shuttle vectors containing the cII target gene were recued from the genomic DNA, using Transpack packaging extract. E. coli host strain G1250 was used in plating. The packaging and plating was repeated for DNA samples until at least 2.5 x 10^5 plaque forming units (PFU) were scored for each data point. The number of packaged phages screened was calculated using G1250 bacteria mixed diluted phage solution, plated on TB1 plates, and incubated overnight at 37°C (nonselective conditions). cII mutants were measured by growing infected G1250 bacteria on TB1 plates at 24°C for approximately 42 hours. During incubation at 24°C, infected phages with a wild-type cII gene undergo lysogenization and the infected bacteria become part of the developing lawn, while phages with a mutated cII gene undergo a lytic cycle, giving rise to plaques. At 37°C, all phages undergo the lytic cycle and form plaques regardless of whether they carry a wild-type or mutant cII gene. CII mutant frequencies were determined by the number of mutant plaques divided by the total number of plaques screened.

SEQUENCE ANALYSIS OF cII MUTANTS:
Mutant plaques from controls and 0.1 mg/m³ vanadium pentoxide treated animals were replated at low density to verify the mutant phenotype. Confirmed mutant plaques were selected and transferred into a 96-well plate containing 100 µL of autoclaved distilled water in each well. The plate containing mutant plaques was heated at 99°C for 10 minutes in an ABI 9700 Gene Amp PCR System (Foster City, CA), and centrifuged at 2000 x g for 5 minutes. The cII target DNA for sequencing was amplified by PCR with primers F1 (5'-AAAAAGGGCATCAAATTAAACC-3') and R1 (5'-CCGAAGTTGAGTATTTTTGCTG-3'). The PCR reaction mixture included 10 µL of the heated mutant phage supernatant and 10 mL of PCR Reaction Mix (Invitrogen, Carlsbad, CA), and made up such that the final concentrations of the reagents were 0.5 U/µL Platinum Taq polymerase, 1X PCR buffer (pH 8.5), 1.5 mM MgCl2, 200 mM of each dNTP, and 0.2 mM of each primer. The PCR reaction included a 3 minute denaturation at 95°C, followed by 30 cycles of 1 minute at 95°C, 1 minute at 60°C, and 1 minute at 72°C, with a final extension of 10 minutes at 72°C. The PCR products were purified using a MinEluteTM 96 UF PCR purification kit (Qiagen, Valencia, CA), and the purified DNA containing the entire cII gene was cycle-sequenced as described previously [Manjanatha et al., 2015]* using the cII-F1 PCR primer listed above and a CEQ Dye Terminator Cycle Sequencing (DTCS) with Quick Start Kit, according to the manufacturer's protocol (Beckman Coulter, Fullerton, CA). The sequencing reactions were analysed on a Beckman-Coulter CEQ 8000 Genetic Analysis System, following the procedure outlined by Beckman. All alterations in DNA sequence were verified at least once.

OTHER:
Exposure to varying concentration of vanadium pentoxide was confirmed by measuring the lung burden of vanadium at designated necropsies. Lungs of wild type mice that were exposed simultaneously with BB mice in the same exposure chambers from each of the exposure groups were removed at necropsy, weighed, homogenized and analysed for vanadium content using an ICP/MS method.

*References:
- M. Banda, K.L. McKim, L.T. Haber, J.A. MacGregor, B.B. Gollapudi, and B.L. Parsons, Quantification of K-ras mutant fraction in the lung DNA o mice exposed to aerosolized particulate vanadium pentoxide by inhalation. Submitted
- M.G. Manjanatha, L. Li-Wu, S.D. Shelton, D.R. Doerge, Acrylamide-induced carcinogenicity in mouse lung involves mutagenicity: cII gene mutations in the lung of Big Blue mice exposed to acrylamide and glycidamide for up to 4 weeks, Environmental Molecular Mutagenesis, (2015) in press.
Evaluation criteria:
no data
Statistics:
The cII mutant frequencies in lung tissues following control or vanadium pentoxide exposures were measured for each mouse and the values represent the total number of mutants observed in two to five packaging reactions per mouse, divided by the total number of PFUs examined across the packaging reactions. Mutant frequencies for the cII gene as well as lung weights from BB mice were analysed as a function of concentration X time by Proc GLM and the Tukey-Kramer (for unbalanced data) test to evaluate the differences in mutant frequencies and lung weights among groups. A logarithmic transformation was performed before conducting the analyses [Manjanatha et al., 2015]*. A value of P ≤0.05 was considered significant. Chi square analysis and the statistical test described by Cariello et al. [1994]* were used to analyse and compare cII mutational spectra from a small group of vanadium pentoxide-exposed animals.

*References:
- M.G. Manjanatha, L. Li-Wu, S.D. Shelton, D.R. Doerge, Acrylamide-induced carcinogenicity in mouse lung involves mutagenicity: cII gene mutations in the lung of Big Blue mice exposed to acrylamide and glycidamide for up to 4 weeks, Environmental Molecular Mutagenesis, (2015) in press.
- N.F. Cariello, W.W. Piegorsch, W.T. Adams, T.R. Skopek, Computer program for the analysis of mutational spectra: application to p53 mutations. Carcinogenesis 15 (1994) 2281 - 2285.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
inflammatory changes and histiocytosis in the lung
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not examined
Additional information on results:
TEST SUBSTANCE CONCENTRATION AND PARTICLE DISTRIBUTION:
- X-ray diffraction spectra of test substance sample (pre and post aerosolization): aerosolozation process did not alter the test substance composition or its characteristics.
- between-port variation was within 3.5%.
- aerosol concentration determined by chemical analysis (based on measurement of vanadium levels in the filter samples) was reproducible over the course of 4- and 8-week exposure periods.
- MMAD: 1.35 to 1.45 µm (GSD: 1.9 to 2.8)

VANADIUM LUNG LEVELS AND LUNG WEIGHTS:
- vanadium lung levels increased from exposure to the test item whereas levels in the control animals were all below the practical quantification limit, (PQL), of 1 - 2.5 µg/g lung tissue.
- increasing exposure time from 4 to 8 weeks did not results in a further increase in the concentration of vanadium in the lung.
- a concentration-dependent rise in right lung weight was observed in BB mice exposed to vanadium pentoxide which was significantly increased (P ≤ 0.05) in the 1 mg/m³ V2O5 exposure group at both the 4 and 8 week time points.
- average lung weight (control): 102 ± 3 mg and 111 ± 9.2 mg for 4 and 8 week exposure periods, respectively
- average lung weight (0.1 mg/m³): 112 ± 4.7 mg and 116 ± 10.3 mg for 4 and 8 week exposure periods, respectively (not significant)
- average lung weight (1.0 mg/m³): 138 ± 3 mg and 143 ± 8.3 mg for 4 and 8 week exposure periods, respectively (significant; P≤0.05)
- statistically significant increase in lung weights provided a measure of toxicity from exposure in the 1 mg/m³ group.

LUNG cII MUTANT FREQUENCIES:
- lung mutant frequencies (control): 30 ± 4.2 x 10^-6 and 29 ± 3.4 x 10^-6 for 4 and 8 week exposure periods, respectively.
- lung mutant frequencies (0.1 mg/m³ exposure): 39 ± 7.9 x 10^-6 and 48 ± 14.3 x 10^-6 for 4 and 8 week exposure periods, respectively (not significant; P ≥ 0.08).
- lung mutant frequencies (1.0 mg/m³ exposure): 24 ± 4.4 x 10^-6 and 17.0 ± 2.8 x 10^-6 for 4 and 8 week exposure periods, respectively (not significant; P ≥ 0.12).
- the slightly higher mean cII mutant frequencies in the groups exposed to the 0.1 mg/m³ concentration of test item were driven by a few animals ( animals exposed for 4 weeks; 2 animals exposed for 8 weeks). Further analysis indicate that the slight increase was not due to clonal expansion of mutants or test item treatment.

DNA SEQUENCE DATA (from 53 cII mutant selected from 12 control and 87 cII mutants selected from 4 mice exposed to 0.1 mg/m³ vanadium pentoxide for 4 and 8 weeks)
- 53 independent mutations from 12 control mice
- 76 independent mutations from 4 mice exposed to 0.1 mg/m³ vanadium pentoxide for 4 - 8 weeks.
- cloncal expansion of mutants ranged from 8 -15% for individual mice.
- statistical analysis of the mutational spectra showed no significant difference between the vanadium pentoxide-treated and the corresponding control spectra (P ≥ 0.1).
Conclusions:
Interpretation of results (migrated information): negative
The lack of significant induction of cII mutant frequencies on the lungs of BB mice exposed to tumorigenic concentrations of divanadium pentaoxide by inhalation for up to 8 weeks suggests that divanadium pentaoxide is unlikely to act via a mutagenic mode of action.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reasonably well descrtibed study. No GLP is reported.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan s.r.l. (Italy)
- Assigned to test groups randomly: yes
- Diet: ad libitum; laboratory rodent diet
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15
- Photoperiod: 12 hours dark/light cycle
No further details are given.
Route of administration:
oral: drinking water
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
No further details are reported.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Tap water was used to dilute VOSO4 stock solutions and administered as such to control animals.

DIET PREPARATION
not applicable
Duration of treatment / exposure:
5 weeks
Frequency of treatment:
daily
Post exposure period:
no data
Remarks:
Doses / Concentrations:
2 mg/L
Basis:
nominal in water
Remarks:
Doses / Concentrations:
10 mg/L
Basis:
nominal in water
Remarks:
Doses / Concentrations:
100 mg/L
Basis:
nominal in water
Remarks:
Doses / Concentrations:
500 mg/L
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1000 mg/L
Basis:
nominal in water
No. of animals per sex per dose:
8 to 10 male mice were randomly assigned to each treatment group.
Control animals:
yes, concurrent vehicle
Positive control(s):
methylmethanesulfonate
- Doses / concentrations: 80 mg/kg body weight
No further details are given.
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: preliminary test results

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): no further details

DETAILS OF SLIDE PREPARATION: Bone marrow cells were obtained by flushing both femours with phosphate buffer saline (PBS). After centrifugation and resuspension in PBS, the cell suspension was used for micronucleus assay.
Some drops of bone marrow cell suspension were spread on slides. For each animal 4 slides were prepared. Air-dried smears were then fixed in absolute methanol at room temperature for 5 minutes and stained with 5% solution of Giemsa in 0.01M phosphate buffer at pH 6.8 for 20 minutes to differentiate bone marrow polychromatic (PCE) from normochromatic erythrocytes (NCE).

METHOD OF ANALYSIS: Slides were coded and blind scored using a brightfield microscope. In the first experiment the frequency of micronucleated PCEs was evaluated analysing 2000 cells/animal (1000 cels each of two scorers); 1000 cells/animal were analysed in the positive control group.

OTHER: To assess bone marrow toxicity, the percentage of PCEs was evaluated over 1000 total erythrocytes (PCEs + NCEs).
Evaluation criteria:
No details are reported.
Statistics:
Means of different experimental groups were compared by two-tailed Student's t-test. The limit of statistical significance was set at p=0.05.
Sex:
male
Genotoxicity:
negative
Remarks:
No increase in the incidence of MnPCEs was observed in the mice treated with doses from 10 to 1000 mg/L.
Toxicity:
no effects
Remarks:
No treatment related changes in the PCE/NCE ratio.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
No details are reported, but the top dose was selected as maximum tolerated dose on the basis of the results of a preliminary range-finding experiment and of literature data (ciranni et al. 1995).

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the incidence of micronucleated polychromatic erythrocyte (MnPCEs) was observed in bone marrow cells of mice treated with vanadyl sulphate in a range of doses from 10 to 1000 mg/L. The negative result obtained in the first experiment was confirmed in the second experiment.
- Ratio of PCE/NCE (for Micronucleus assay): The determination of the PCE/NCE ratio did not show dose-related deviations in this parameter indicating bone marrow toxicity in treated mice.

General toxicity: Daily water consumption was significantly reduced in the two high dose groups (1000 and 500 µg/L), possibly because of the effect of vanadyl sulphate on the palatability of drinking water. This led to a less than proportional increase in vanadium intake in the high dose groups. Consequently, in the second experiment an increased water consumption was observed in the low dose group (2 mg/L). No significant differences in food consumption were observed among experimental groups. Treatment did not affect body weight gain in the 5 weeks of exposure.

Vanadium distribution: In the first experiment a dose-related, linear increase of vanadium content was observed in bone tissues (R²=0.95 and R²=0.96 in femours and in tibias, respectively). Slightly higher vanadium concentrations were measured in bone in the second experiments at the dose of 10 mg/L: however, when data from the two experiments were compared by t-test, no statistical significant differences at 0.05 level were observed.
Conclusions:
Interpretation of results (migrated information): negative
Under the given test conditions reported, vanadyl sulphate pentahydrate is not mutagenic in the bone marrow micronucleus assay.
Executive summary:

In this reference, the genotoxic effects induced in vivo by subacute oral exposure to vanadyl sulphate (VOSO4) were investigated. To this aim male CD1 mice were administered with VOSO4 in drinking water over the dose range 2 -1000 mg/L for 5 weeks. At the end of treatment, micronuclei in bone marrow polychromatic erythrocytes were determined. Tissue distribution of vandaium at sacrifice was determined by atomic absorption spectrometry.

The analysis of micronuclei did not reveal treatment related effects. Under the given test conditions reported, vanadyl sulphate pentahydrate is not mutagenic in the bone marrow micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The endpoint genetic toxicity is not addressed by substance-specific information but rather by read-acrossof data available for soluble tri-, tetra- and pentavalent vanadium substances.This is based on the assumption that once inorganic vanadium compounds become bioavailable, this will be in tetra- or pentavalent vanadium forms. Based on the available weight-of-evidence, and considering guideline-conform studies conducted under GLP - both in vitro and in vivo, vanadium substances, including vanadium oxide sulphate, should be considered void of genotoxicity.

Read-across: The read-across approach based on dissolved vanadium is based on the assumption that once inorganic vanadium compounds dissolve or become bioavailable, this will be in tetra- or pentavalent vanadium forms. In bioaccessibility tests of tetra- and pentavalent vanadium substances, tetra- and pentavalent forms dissolved completely within 2h in various media selected to simulate relevant human-chemical interactions (i.e. PBS mimicking the ionic strength of blood, artificial lung, lysosomal, and gastric fluid as well as artificial sweat). Pentavalent vanadium substances are released and retained as pentavalent forms in physiological media, with the exception of artificial lysosomal fluid in which tetravalent V dominates after 2h and is the only form present after 24h.Thus, it can be assumed that vanadium speciation in body fluids is controlled by the conditions of the respective medium but not by the vanadium source.

Further, the registrant is of the opinion that the toxicity of vanadium oxide sulphateis driven by the vanadium moiety and that the sulfate anion does not contribute to the overall toxicity of the substance vanadium oxide sulphate toany relevant extent, for the following reasons:

Sulfate anions are abundantly present in the human body in which they play an important role for the ionic balance in body fluids. Sulfate is required for the biosynthesis of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) which in turn is needed for the biosynthesis of many important sulfur-containing compounds, such as chondroitin sulfate and cerebroside sulfate.TheJoint FAO/WHO Expert Committee on Food Additives (JECFA) concludes that the few available studies in experimental animals do not raise any concern about the toxicity of the sulphate ion in sodium sulphate. Sodium sulphate is also used clinically as a laxative. In clinical trials in humans using 2-4 single oral doses of up to 4500 mg sodium sulphate decahydrate per person (9000 – 18000 mg per person), only occasional loose stools were reported. These doses correspond to 2700 - 5400 mg sulphate ion per person. High bolus dose intake of sulphate ion may lead to gastrointestinal discomfort in some individuals. No further adverse effects were reported (JECFA 2000, 2002). This position was adopted by the European Food Safety Authority (EFSA2004) without alteration.

Based on the above information, one can therefore safely assume that the sulfate anion in vanadium oxide sulphate does not contribute to the overall toxicity of vanadium oxide sulphate. Itis concluded that only the effect of “vanadium” is further considered in the human health hazard assessment of vanadium oxide sulphate. Thus, read-across of genetic toxicity data from soluble tetra- and pentavalent vanadium substances is justified.

In vitro gene mutation assays

Testing in bacteria reverse mutation assays (May, 2011; NTP, 2002; Wolf, 2006), although of limited relevance for metals (HERAG, 2007), yielded negative results with pentavalent vanadium substances:

- vanadium pentaoxide is not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100, TA102, or TA1535, both in the presence as well as absence of metabolic activation (rat or hamster liver S9 enzymes)

- sodium polyvanadate (Na2V6O16) is non-mutagenic in Salmonella typhimurium strain the TA97a, TA98, TA100, TA102 and TA1535, both in the presence as well as absence of metabolic activation up to the limit of toxicity.

 

Further, guideline-conform in vitro mammalian cell gene mutation tests according to OECD 476 conducted by Lloyd (2010a,b,c) under GLP are considered the key studies and will be used for classification. In these studies, soluble tri-, tetra- and pentavalent vanadium substances did not induce any mutations at the HPRT locus of L5178Y mouse lymphoma cells when tested under the conditions employed in these studies, including treatments up to toxic and/or precipitating concentrations in two independent experiments in the absence or presence of a rat liver metabolic activation system (S9).

 

n vivogene mutation assays

The lack of significant induction of cII mutant frequencies in the lungs of BB mice exposed to tumorigenic concentrations of divanadium pentaoxide by inhalation for up to 8 weeks suggests that divanadium pentaoxide is unlikely to act via a mutagenic mode of action.

Inhalation of aerosols of particulate divanadium pentaoxide for 4 or 8 weeks did not result in significant changes in levels of Kras codon 12 GAT or GTT mutation. The data support the idea that the accumulation of additional Kras mutants is not an early event, and/or that the proliferative advantage of Kras mutant clones requires either longer expression times or larger cumulative divanadium pentaoxide exposures. Furthermore, the data do not provide support for either a direct genotoxic effect of divanadium pentaoxide on Kras in the context of the exposure conditions used, or early amplification of preexisting mutation as being involved in the genesis of divanadium pentaoxide-induced mouse lung tumours.

In vitro clastogenicity assays

Trivalent vanadium substance:

In the key study by Lloyd (2010):

- V2O3did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the absence of S9

- V2O3showed evidence of inducing micronuclei when tested for 3+21 hours in the presence of S9 (but primarily at precipitating concentrations, therefore considered of questionable biological relevance)

- V2O3induced micronuclei in cultured human peripheral blood lymphocytes when tested for 24+24 hours in the absence of S9

 

Tetravalent vanadium substance

In the key study by Lloyd (2010):

- VOSO4did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the presence of S9

- VOSO4induced micronuclei in cultured human peripheral blood lymphocytes and human lymphoblastoid TK6 cells when tested for 3+21 hours and for 24+24 hours in the absence of S9

Rodriguez-Mercado et al. (2003) also examined the chromosome aberration and sister chromatid exchange in human lymphocytes after V2O4exposure; both experiments suggest a clastogenic effect. However the toxicological significance could not be properly assessed due to reporting and experimental deficiencies.

 

Pentavalent vanadium substance

In the key study by Lloyd (2010):

- V2O5showed evidence of inducing micronuclei in cultured human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the absence and presence of S9 (at the two highest concentrations with observed precipitation and cytotoxicity levels > 25%, therefore considered of questionable biological relevance) and for 24+24 hours in the absence of S9 (at cytotoxicity levels > 10%).

- V2O5showed evidence of inducing micronuclei in cultured human lymphoblastoid TK6 cells when tested up to toxic concentrations for 3+21 hours in the absence and presence of S9 and for 24+24 hours in the absence of S9 (with observed precipitation and at cytotoxicity levels > 15%, and therefore considered of questionable biological relevance).

V2O5was also tested for a 24-hour treatment period in thein vitromicronucleus assay in Syrian hamster embryo (SHE) cells at the following concentrations: 10, 15, 20, and 25 µg/mL. In this test, vanadium pentaoxide did not show any genotoxic potential (Gibson, 1997).

It is not evident that the positive responses observed in the studies by Lloyd (2010) are true effects based on V2O3, VOSO4or V2O5induced chromosome damage at physiologically relevant concentrations since both cytotoxicity as well as precipitation were observed in treatment groups that tested positively.

In sum, negative as well as positive results were obtained in various clastogenicity assays in vitro with soluble vanadium substances. However, these in vitro studies of effects upon eukaryotic cells in vitro employed high concentrations of soluble vanadium producing significant levels of cytotoxicity and only weak genotoxic responses. A central issue that requires resolution is whether such in vitro results have physiological relevance by virtue of the mechanisms involved or the concentrations required to produce effects.

 

In vivo induction of micronuclei

Altogether, there are several reliable and unreliable studies that report the in vivo induction of micronuclei as well as the lack thereof and that are summarized in the following table:

In vivo genotoxicity test

Route - exposure

Substance - Valency

Dose

(mg V / kg bw d)

Result - Reliability

Reference

Micronuclei in bone marrow - rats

p.o. – once, examined after 24h and 48h

Divanadium pentaoxide - V

i) 16.8

ii) 33.6

iii) 50.4

iV) 67.2

Negative – RL = 1

Beevers, 2011

Micronuclei in peripheral blood - mice

Inhalation – 3 mo

Divanadium pentaoxide - V

0.5 – 9 mg/m3

Negative – RL = 1

NTP, 2002

DNA damage in BAL or pulmonary cells (Comet assay)

Inhalation – 16 d

Divanadium pentaoxide - V

0.1 – 2.2 mg/m3

Negative – RL = 1

Schuler, 2010

Schuler et al, 2011

Micronuclei in peripheral blood lymphocytes & bone marrow - mice

 p.o. via drinking water - 5 wk

Vanadyl sulphate - IV

0.4 – 30

0.1 – 0.4

Negative – RL = 2

Villani et al, 2007

BrdU-incorporation assay

i) Aneuploidy in sperm

ii) Micronuclei in bone marrow – mice

i.p. – once

i) examined after 22 d

ii) examined after 24h

 

Sodium orthovanadate - V

i) 1.4 - 7

ii) 0.3 - 7

i) Positive –RL = 2

ii) Negative – RL = 2

Attia et al, 2005

Micronuclei in peripheral blood lymphocytes & bone marrow - mice

 p.o. via drinking water - 5 wk

Sodium orthovanadate - V

0.1 – 5.5

20.8– 33

Negative – RL = 3

Positive – RL = 3

Leopardi et al, 2005*

Micronuclei in bone marrow - mice

i.p. – once, examined after 30h

Ammonium metavanadate - V

10.2

Negative – RL = 3

Wronska-Nofer et al, 1999*

Sister chromatid exchange

i.p. – once, examined after 24h

Divanadium pentaoxide - V

3.2 – 12.9

Negative – RL = 3

Altamirano-Lozano et al, 1993*

Micronuclei in bone marrow - mice

i.p. – once, examined after 18h

Sodium orthovanadate - V

1.4 – 6.9

Positive – RL = 3

Mailhes et al, 2003*

Micronuclei in bone marrow - mice

p.o. – once, examined after 4-48 h

i) Ammonium metavanadate – V

ii) Sodium orthovanadate – V

iii) Vanadyl sulphate - IV

i) 21.8

ii) 20.8

iii) 31.2

Positive – RL = 3

Ciranni et al, 1995*

 mice

p.o. – once, examined after 24-36 h

i) Ammonium metavanadate – V

ii) Sodium orthovanadate – V

iii) Vanadyl sulphate - IV

i) 21.8

ii) 20.8

iii) 31.2

Positive – RL = 3

Ciranni et al, 1995*

* These references were rated unreliable based on the Klimisch score. Please find the list with all acquired (evaluated according to Klimisch et al.: A systematic approach for evaluating the quality of experimental and ecotoxicological data, Reg.Tox. and Pharm. 25, 1-5 (1997) and sorted by REACH Annex VII – X endpoints) in Chapter 13.

 

Whereas all reliable in vivo data generated in realistic exposure scenarios (inhalation or oral administration of vanadium substance) unequivocally indicate a negative potential for tetravalent and pentavalent V substances, data generated in unrealistic exposure scenarios (i.p. administration) and unreliable in vivo data report contradictory, i.e. in part positive and negative, findings.

The in vivo genetic toxicity of divanadium pentaoxide was assessed within the framework of an NTP (2002) programme by testing the ability of the chemical to induce increases in the frequency of micronucleated erythrocytes in mouse peripheral blood. Female and male mice were exposed for 90 days to a divanadium pentaoxide aerosol by inhalation (at 0.5 – 9 mg V /m3) before blood was sampled and analysed. As a result, divanadium pentaoxide, administered to male and female mice, did not increase the frequency of micronucleated normochromatic erythrocytes in peripheral blood.

In a follow-up of the NTP study, female mice were exposed for 16 days to a vanadium pentaoxide aerosol by inhalation (at 0.2 – 2.2 mg V /m3); the COMET assay conducted on exposed bronchioalveolar lavage (BAL) cells and pulmonary cells revealed unequivocal absence of DNA damage.

Furthermore, the genetic toxicity of divanadium pentaoxide was assessed via oral exposure by testing the ability of V2O5 to induce an increase in the frequency of micronucleated erythrocytes in bone marrow of male rats. While, vanadium tissue levels increased in all sampled tissues with increased V2O5dosage levels, and thus, vanadium reached the target tissues bone marrow and testes in a dose-dependent manner, V2O5administered orally by gavage to male rats, did not induce micronuclei in polychromatic erythrocytes of bone marrow treated up to the maximum tolerated dose of 120 mg/kg/day (Beevers, 2011).

However, reliable (RL=2) in vivo results of one study also indicate a positive threshold effect, but in this study, the toxicological significance could not be properly assessed due to unrealistic (i.p.) exposure and reporting and experimental deficiencies.

Thus, for pentavalent V, reliable (RL=1) and GLP-conform in vivo data indicate a negative potential for genetic toxicity.

 

References:

HERAG (2007) Fact sheet 05 - Mutagenicity. EBRC Consulting GmbH / Hannover /Germany.August 2007.[www.metalsriskassessment.org]

Justification for classification or non-classification

Representative tri-, tetra- and pentavalent vanadium substances have been verified unequivocally in vitro to be void of gene mutation activity both in bacterial as well as mammalian in-vitro cell systems. Testing in bacteria reverse mutation assays, although of limited relevance for metals (HERAG, 2007), also yields negative results. Negative as well as positive results were obtained in clastogenicity assays in vitro. However, these in vitro studies of effects upon eukaryotic cells in vitro employed high concentrations of soluble vanadium producing significant levels of cytotoxicity and only weak genotoxic responses. A central issue that requires resolution is whether such in vitro results have physiological relevance by virtue of the mechanisms involved or the concentrations required to produce effects. For example, induction of genotoxic effects in cultured cells at dissolved vanadium concentrations in the µM or mM range would have limited relevance to in vivo exposures wherein the concentration of vanadium available for transfer to the soft tissues is in the nM range or lower.

In vitro assays for cytogenetic changes conducted with metals are quite often positive, even with some indications that aneuploidy induction may be common. Conversely, upon in vivo testing, most metals with very few exceptions yield negative results, leading to the conclusion that the in vitro results should not be over-interpreted since unphysiologically high concentrations as obtainable in in vitro test systems do not mirror in vivo physiological conditions. This is verified by (i) a study conducted by NTP (2002) involving in vivo exposure to a V2O5aerosol for 3 months, which failed to elicit any clastogenic effects in mice, and (ii) another reliable GLP-conform study, in which V2O5, administered orally by gavage to male rats up to the maximum tolerated dose of 120 mg/kg/day, did not induce micronuclei in polychromatic erythrocytes of the bone marrow. 

Vanadium substances may express some clastogenicity in vitro, this occurs only at unphysiologically high concentrations and via mechanisms that appear to lack physiological relevance, and is not mirrored in corresponding in vivo (RL=1) assays via the oral and the inhalation route.

Based on the available weight-of-evidence, and considering guideline-conform studies conducted under GLP both in vitro as well as in vivo, vanadium should be considered void of genotoxicity. Similarly, classification of vanadium oxide sulphate with respect to mutagenic potential does not appear to be supported. Thus, according to EC Regulation No. 1272/2008, vanadium oxide sulphate should not be considered to have a mutagenic potential, and hence no classification or labelling is required.