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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro and in vivo genotoxicity data are evaluated in a weight-of evidence analysis below.

Link to relevant study records

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Endpoint:
in vitro cytogenicity / micronucleus study
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Reasonably well described study with minor reporting deficiencies: no information on cytotoxicity, no individual results, purity of the test substance not reported.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Testing of 16 chemicals in the in vitro micronucleus assay in SHE cells.
GLP compliance:
not specified
Remarks:
publication
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
mammalian cell line, other: Syrian hamster embryo (SHE) cells
Details on mammalian cell type (if applicable):
For the test, the cells were seeded at 1x10^6 cells/T-25 flask for control, and chemically-treated cultures. Afetr approximately 24 hours, the cells were exposed to the test chemical and cytochalasin B (3 µg/mL in DMSO) for 24 hours.
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Cytochalasin B (3 µg/mL in DMSO)
Metabolic activation:
without
Metabolic activation system:
not applicable
Test concentrations with justification for top dose:
10, 15, 20, and 25 µg/mL
Dose levels were selected based on solubility and/or toxicity limits.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Positive control substance:
colchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours and 10 minutes: After determination of the toxicity (see below) remaining cells were suspended in 37°c 0.075M KCl for 5-10 minutes, before the cells were collected by centrifugation and fixed in at least two changes of cold 25:1 methanol/acetic acid.

STAIN (for cytogenetic assays): Cells were stained for 1-5 minutes in a 10% Giemsa solution in Gurr buffer.

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED: In each treatment group, 500 cells were analysed to determine the percentage of binucleated cells and 1000 binucleated cells were analysed to determine the number of micronucleated cells.

DETERMINATION OF CYTOTOXICITY
- Method: other: number of binucleated cells after 24-hour exposure
After a 24-hour treatment period the media was aspirated off and the cells were collected by trypsinisation. An aliquot of cells was counted to determine the number of live cells (determined by trypan blue exclusion) as a measure of toxicity.

OTHER EXAMINATIONS:
Only cells with distinct cytoplasm and distinct binucleation were analysed for the presence of micronuclei. Only micronuclei that were entirely inside the cytoplasm, separate from the main nucleus, less than approximately one-third the size of the main nuclei, and non-refractile were recorded.
Evaluation criteria:
no data
Statistics:
The number of micronucleated binucleated cells (MNBC) in the treated group was compared to the number of MNBC in the vehicle control group using a one-sided Fisher's exact test where p<0.05 was considered significant.
Species / strain:
mammalian cell line, other: SHE cells
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
No statisticaly significant increase in micronucleated binucleated cells (MNBC) was observed.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
(as evidenced by at least a 50% reduction in the relative cell number and/or in the percentage of binucleated cells)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No details are reported.
Conclusions:
Under the test conditions described the test substance, vanadium pentoxide, has no mutagenic potential in the in vitro micronuceus test in Syrian hamster embryo (SHE) cells.
Executive summary:

Vanadium pentoxide as one of 15 other chemical substances was in the in vitro micronucleus assay in SHE at the following concentrations: 10, 15, 20, and 25 µg/mL. Dose levels were selected based on solubility and/or toxicity limits. After a 24-hour treatment period an aliquot of cells was counted to determine the number of live cells (determined by trypan blue exclusion) as a measure of toxicity. Remaining cells were collected by centrifugation, fixed and stained for analysis. In each treatment group, 500 cells were analysed to determine the percentage of binucleated cells and 1000 binucleated cells were analysed to determine the number of micronucleated cells.

Under the test conditions described the test substance, vanadium pentoxide, has no mutagenic potential in the in vitro micronuceus test in Syrian hamster embryo (SHE) cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
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
Cytokinesis block (if used):
not applicable
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:
4-nitroquinoline-N-oxide
benzo(a)pyrene
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
Remarks:
Experiment 1
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
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
Experiment 2
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
True negative controls validity:
not examined
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).
Conclusions:
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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-02-16 to 2010-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed by The Department of Health of the Government of the United Kingdom (2010-06-23)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
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
Cytokinesis block (if used):
not applicable
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)(culture medium)
Negative solvent / vehicle controls:
yes
Remarks:
(0.5% MC diluted 10-fold in the treatment medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 4-nitroquinoline-1-oxide
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
True negative controls validity:
not examined
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.
Conclusions:
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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-03-15 to 2010-04-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed by The Department of Health of the Government of the United Kingdom (2010-06-23)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
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
Cytokinesis block (if used):
not applicable
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 (culture medium)
Negative solvent / vehicle controls:
yes
Remarks:
(0.5% MC diluted 10-fold in the treatment medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 4-nitroquinoline-1-oxide
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
True negative controls validity:
not examined
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.

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:
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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2009-11-30 to 2010-02-05.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: draft OECD guideline 487 (November 2009)
Version / remarks:
2009-11-02
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human (peripheral)
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
Cytokinesis block (if used):
Cytochalasin B (formulated in DMSO; 6 µg/mL)
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:
cyclophosphamide
mitomycin C
vinblastine
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: human (peripheral)
Remarks:
treatment 3 + 21 hours
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:
cytotoxicity
Remarks:
At the highest concentration analysed (30 µg/mL), 52% cytotoxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: human (peripheral)
Remarks:
treatment 3 + 21 hours
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:
cytotoxicity
Remarks:
At the highest concentration analysed (100 µg/mL), 40% cytotoxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: human (peripheral)
Remarks:
treatment 24 + 24 hours
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:
cytotoxicity
Remarks:
At the highest concentration analysed (16 µg/mL), 44% cytotoxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True 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.
Conclusions:
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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-02-08 to 2010-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: the current version of draft OECD guideline 487 and the most recent update, dated 2 November 2009
Version / remarks:
2009-11-02
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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: human (peripheral)
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
Cytokinesis block (if used):
Cytochalasin B (formulated in DMSO; 6 µg/mL (Range finder and experiment 1) or 3 µg/mL (Experiment 2 and 3))
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:
4-nitroquinoline-N-oxide
cyclophosphamide
mitomycin C
vinblastine
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: human (peripheral)
Remarks:
3 + 21 hours (Experiment 1)
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
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: human (peripheral)
Remarks:
3 + 21 hours (Experiment 1)
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
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: human (peripheral)
Remarks:
24 + 24 hours (Experiment 1)
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
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Remarks:
3 + 21 hours (Experiment 2)
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
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Remarks:
24 + 24 hours (Experiment 2)
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
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Remarks:
3 + 21 hours (Experiment 3)
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
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Remarks:
24 + 24 hours (Experiment 3)
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
True 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.

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:
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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-03-15 to 2010-06-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: the current version of draft OECD guideline 487 and the most recent update
Version / remarks:
2009-11-02
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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: human (peripheral)
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
Cytokinesis block (if used):
Cytochalasin B (formulated in DMSO; 6 µg/mL (Range finder and Experiment 1) and 3 µg/mL (Experiment 2))
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)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
vinblastine
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: human (peripheral)
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
True negative controls validity:
not examined
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
True 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 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.

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:
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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-09-28 to 2010-11-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: draft OECD guideline 487
Version / remarks:
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
Cytokinesis block (if used):
Cytochalasin B (formulated in DMSO; 6 μg/mL/culture)
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
cyclophosphamide
vinblastine
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
True 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
True 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.

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:
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 gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2011-02-15 to 2011-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2010-03-22
Type of assay:
bacterial reverse mutation assay
Target gene:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, prepared 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:
Plate incorporation (first test): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Preincubation (second test): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (purified in-house by reverse osmosis)
- Justification for choice of solvent/vehicle: vanadium carbide nitride was insufficiently soluble in vehicles compatible with the test system. Water (purified in-house by reverse osmosis) containing 0.15% agar (bacteriological grade) was, therefore, used as the vehicle (suspending agent) for this study.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control: without metabolic activation; 2 µg/plate for strains TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Positive control: without metabolic activation; 50 µg/plate for strain TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Positive control: without metabolic activation; 2 µg/plate for strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Positive control: without metabolic activation; 2 µg/plate for strain WP2 uvrA (pKM101)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
Positive control: with metabolic activation; 5 µg/plate for strains TA100 and TA1535; 10 µg/plate for strain WP2uvrA (pKM101)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water containing 0.15% agar
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Positive control: with metabolic activation; 5 µg/plate for strains TA98 and TA1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation; first test) and preincubation (second test)

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn.

ACCEPTANCE CRITERIA
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the study director. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL.

Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989)* and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the study director would use his/her scientific judgement.

*Reference:
MAHON, G.A.T., GREEN, M.H.L., MIDDLETON, B., MITCHELL, I. de G., ROBINSON, W.D. and TWEATS, D.J. (1989) Analysis of data from microbial colony assays in:
KIRKLAND, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part III. Statistical Evaluation of Mutagenicity Test Data, pp.26-65. Cambridge University Press, Cambridge.
Statistics:
The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Species / strain:
E. coli WP2 uvr A pKM 101
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:
PLATE INCORPORATION (first test)
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to vanadium carbide nitride at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

PREINCUBATION (second test)
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to vanadium carbide nitride at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No evidence of toxicity was obtained following exposure to vanadium carbide nitride.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that vanadium carbide nitride showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data available
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
not applicable
Species / strain / cell type:
S. typhimurium TA 97
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
TA102, TA1535 and TA 97: 0, 0.03, 0.1, 0.3, 1.0, 3.0, 6.0, 10.0 and 33.0 µg/plate
TA100 and TA 98: 0, 0.1, 0.3, 1.0, 3.0, 6.0, 10.0, 33.0, 100.0 and 333.0 µg/plate
Vehicle / solvent:
No vehicle is reported, but in the results (summary table) of the reference an evidence is given that a vehicle was used: "0 μg/plate was the solvent control."
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
mitomycin C
other: 4-nitro-o-phenylenediamine & 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar; preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: Each trial consisted of triplicate plates.

DETERMINATION OF CYTOTOXICITY
- Method: no data, but the high dose concentration was limited by toxicity.

OTHER EXAMINATIONS:
no other examinations performed
Evaluation criteria:
In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination.
An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity.
A negative response is obtained when no increase in revertant colonies is observed following chemical treatment.
There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
Statistics:
not mandatory for this test system
Species / strain:
S. typhimurium, other: TA97, TA98, TA100, TA102 and TA1535
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:
not specified
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
No details are reported.
Conclusions:
Under the test conditions reported the test substance was determined to be negative mutagenic in strains TA97, TA98, TA100, TA102 and TA1535 with and without S9.
Executive summary:

The genetic toxicity of vanadium pentoxide was assessed by testing the ability of the chemical to induce mutations in various strains of Salmonella typhimurium. Vanadium pentoxide was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100, TA102, or TA1535, with or without induced rat or hamster liver S9 enzymes.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2010-03-22
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung (CHL/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:
cyclophosphamide
mitomycin C
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:
Chinese hamster lung (CHL/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
True 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
Conclusions:
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
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:
comparable to guideline study with acceptable restrictions
Remarks:
Reasonably well described study with minor restrictions. The following experimental deficiencies restrict the use for hazard assessment: exposure towards test substance commenced 24h after mitogenic stimulation (guideline foresees 48h). The authors did not state on potential precipitation, pH effects, and osmolality effects of the test material in the culture medium. Incubation temperature before and during treatment is not specified. Cell cycle length not determined. Purity of the test substance not reported. Positive control cultures were not included. Historical control data is not provided. Evaluation and scoring criteria are not specified.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, the clastogenicity of vanadium(IV) tetraoxide was evaluated in human peripheral blood lymphocytes using the chromosome aberration test (CA). Moreover, cytotoxicity was determined using the mitotic index (MI), and the replicative index (RI).
GLP compliance:
not specified
Remarks:
publication
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: from human donors
Details on mammalian cell type (if applicable):
The study was conducted with peripheral blood lymphocytes obtained by venipuncture from 3 healthy non-smoking males between 23 and 29 years of age.
- Type and identity of media: Some 0.5 mL of heparinised blood was cultured in 4.5 mL of RPMI-1640 culture medium with 5 µg/mL of phytohaemaglutinin. Cells were cultured for 24 hours.
No further details are reported.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
colchicine (4 µg/mL)
Metabolic activation:
without
Test concentrations with justification for top dose:
2, 4, 8 or 16 µg/mL
Vehicle / solvent:
- Solvent used: distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
no
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): After 48 hours the cultures were harvested, fixed and prepared on flame-dried slides.

SPINDLE INHIBITOR (cytogenetic assays): 4 µg/mL of colchicine were added to each culture 2 hours before the harvest.
STAIN (for cytogenetic assays): Slides were stained with Giemsa.

NUMBER OF REPLICATIONS: All cultures were set up in duplicate.

NUMBER OF CELLS EVALUATED: 200 well-spread metaphases for each treatment were analysed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index:
8000 cells were counted to estimate the mitotic index (MI).

OTHER EXAMINATIONS:
The type of CA scored included chromatid and chromosome-types as well as gaps.
No further details are given.
Evaluation criteria:
No data given.
Statistics:
The significant statistical differences for the MI were determined by the z-test; the statistical differences for the RI and CA were established by the qui-square test.
Species / strain:
lymphocytes: from human donors
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
The frequency of all aberrations types (i.e. acentric fragments, breaks, and exchanges) was higher in the treated groups than in the controls. The percentage of aberrant cells (without gaps) was significantly increased at all concentrations tested.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A significant decrease was noted in the frequency of mitoses detected. Also, a gradual decline appeared in the inhibition of MI percentage.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not examined
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No data are reported.

RANGE-FINDING/SCREENING STUDIES: No details are reported, but concentrations tested were selected on the basis of preliminary experiments.

COMPARISON WITH HISTORICAL CONTROL DATA: no

ADDITIONAL INFORMATION ON CYTOTOXICITY: no further details
Conclusions:
In the study by Rodriguez-Mercado et al. (2003), the clastogenicity of vanadium(IV)tetraoxide was evaluated in human peripheral blood lymphocytes using the chromosome aberration test (CA). Cytotoxicity was determined using the mitotic index (MI) and the replicative index (RI). Vanadium(IV) tetraoxide induced a clear dose-response in MI inhibitions and modifications in the RI. In the CA, the proportion of aberrant cells was statistically significantly increased in the combined donor analysis at all concentration levels tested. Reasonably well described study with minor restrictions (RL2). The following experimental deficiencies restrict the use for hazard assessment: exposure towards test substance commenced 24h after mitogenic stimulation (guideline foresees 48h). The authors did not state on potential precipitation, pH effects, and osmolality effects of the test material in the culture medium. Incubation temperature before and during treatment is not specified. Cell cycle length not determined. Purity of the test substance not reported. Positive control cultures were not included. Historical control data is not provided. Evaluation and scoring criteria are not specified. Under the given experimental conditions the test substance vanadium(IV) tetraoxide is capable of inducing chromosomal damage.
Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data available
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The publication presented herein shows some reporting deficiencies and deficiencies in the study design. The SCE methodology is only poorly described. The authors did not state on potential precipitation, pH effects, and osmolality effects of the test material in the culture medium. Incubation temperature before and during treatment is not specified. Cell cycle length not determined. Purity of the test substance not reported. Positive control cultures were not included. Soring and evaluation criteria are not reported. Historical control data is not provided. Scoring and evaluation criteria are not specified. The positive findings were not confirmed in an independent experiment as required by the OECD TG 479.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, the clastogenicity of vanadium(IV) tetraoxide was evaluated in human peripheral blood lymphocytes using the sister chromatid exchanges (SCE). Moreover, cytotoxicity was determined using the mitotic index (MI), and the replicative index (RI).
GLP compliance:
not specified
Remarks:
publication
Type of assay:
sister chromatid exchange assay in mammalian cells
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: from human donors
Details on mammalian cell type (if applicable):
The study was conducted with peripheral blood lymphocytes obtained by venipuncture from 3 healthy non-smoking males between 23 and 29 years of age.
- Type and identity of media: Some 0.5 mL of heparinised blood was cultured in 4.5 mL of RPMI-1640 culture medium with 5 µg/mL of phytohaemaglutinin. Cells were cultured for 24 hours.
No further details are reported.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
colchicine (4 µg/mL)
Metabolic activation:
without
Test concentrations with justification for top dose:
2, 4, 8 or 16 µg/mL
Vehicle / solvent:
- Solvent used: distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
Remarks:
distilled water
True negative controls:
not specified
Positive controls:
no
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 72 hours after beginning the cultures were harvested, fixed and prepared on flame-dried slides.

SPINDLE INHIBITOR (cytogenetic assays): 4 µg/mL of colchicine were added to each culture 2 hours before the harvest.
STAIN (for cytogenetic assays): Differential staining of sister chromatids was then carried out exactly as described by Roldán and Altamirano (1990)*.

NUMBER OF REPLICATIONS: All cultures were set up in duplicate.

NUMBER OF CELLS EVALUATED: Sister chromatid exchanges (SCE) were scored in 60 second mitotic division cells per treatment.

DETERMINATION OF CYTOTOXICITY
- Method: replicative and mitotic index:
400 metaphases were classified as either first, second or third division cycles, according to the differential stain in order to calculate the replicative index (RI) and 8000 cells were counted to estimate the MI. All the slides were coded for analysis.

OTHER EXAMINATIONS:
No further details are given.

*References:
- Roldán, E., Altamirano, M., 1990. Chromosomal aberrations, sister chromatid exchanges, cell-cycle kinetics and satellites association in human lymphocytes culture exposed to vanadium pentoxide. Mutat. Res. 245, 61-65.
Evaluation criteria:
No data given.
Statistics:
The significant statistical differences for the MI were determined by the z-test; the statistical differences for the RI were established by the qui-square test and for the SCE by the ANOVA-Tukey test.
Species / strain:
lymphocytes: from human donors
Metabolic activation:
without
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not examined
Additional information on results:
SCE ASSAY
- Only slight increases in SCE/cell were observed at concentrations ≥ 4µg/mL with interindividual variations.
- Regression analysis of the combined data showed a positive concentration response relationship.
- Findings only in presence of cytotoxicity.

CYTOTOXICITY
- The MI was statistically significantly reduced at concentrations ≥ 2 µg/mL in a concentration dependent manner.
- The RI was slightly but statistically significantly decreased at ≥ 2 µg/mL.

TEST-SPECIFIC CONFOUNDING FACTORS
No data are reported.

RANGE-FINDING/SCREENING STUDIES: No details are reported, but concentrations tested were selected on the basis of preliminary experiments.

COMPARISON WITH HISTORICAL CONTROL DATA: no

ADDITIONAL INFORMATION ON CYTOTOXICITY: no further details
Remarks on result:
not determinable because of methodological limitations
Conclusions:
In the study by Rodriguez-Mercado et al. (2003), the clastogenicity of vanadium(IV) tetraoxide was evaluated in human peripheral blood lymphocytes using the sister chromatid exchanges (SCE). Cytotoxicity was determined using the mitotic index (MI), and the replicative index (RI). This substance induced a clear dose-response in MI inhibitions and modifications in the RI. Combined analysis of the SCE revealed slightly but statistically significantly increased SCEs per cell at concentration of and greater than 4 µg/mL. The response was concentration related. However, the positive findings were only observed in presence of statistically significant cytotoxicity and showed highly similar increments. Moreover, the individual donor analysis revealed high inter-individual variation in the SCE frequency. The publication shows some reporting deficiencies and deficiencies in the study design. The SCE methodology is only poorly described. The authors did not state on potential precipitation, pH effects, and osmolality effects of the test material in the culture medium. Incubation temperature before and during treatment is not specified. Cell cycle length not determined. Purity of the test substance not reported. Positive control cultures were not included. Soring and evaluation criteria are not reported. Historical control data is not provided. Scoring and evaluation criteria are not specified. The positive findings were not confirmed in an independent experiment as required by the OECD TG 479. No conclusion on chromosomal damage can be drawn based on the findings of the study (RL3).
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The publication presented herein shows some reporting deficiencies and deficiencies in the study design. The authors did not state on confounding factors, i.e. precipitation of the test material as well as pH and osmolality effects of the test material on the culture medium. The test material was only tested in a long-term treatment, whereas the test guidelines requires per default a short-term treatment, except in case positive findings were observed. The lymphocytes were not sufficiently characterised, since information on the normal cell doubling time and the age of the donor are not specified. The mitogen stimulation was slightly shorter than recommended (44 vs. ≥48 h) The type of negative control is not specified. Historical control data is not provided. Acceptability and evaluation criteria are not provided.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, vanadium(III) trioxide (V2O3) was assessed for its potential to induce structural chromosomal aberrations in human peripheral blood lymphocytes. The lymphocytes from a single donor were treated with V2O3 at concentration levels of 1, 2, 4, and 8 µg/mL for 28 hours. Afterwards, the cells were harvested, fixed and stained with Giemsa. The experiment was run in duplicates. A total of 100 metaphases per culture were scored for chromosomal aberrations. Moreover, the Mitotic Index (MI) was determined in order to evaluate cytotoxicity.
GLP compliance:
not specified
Remarks:
publication
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.

From these 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.
Cytokinesis block (if used):
colchicine (4 µg/mL)
Metabolic activation:
not specified
Test concentrations with justification for top dose:
1, 2, 4, or 8 µg/mL
Vehicle / solvent:
- Vehicle 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
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 with 5 μg/mL of phytohemagglutinin and treated with vanadium(V) pentoxide 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.

The test item was dissolved in distilled water and added to cultures at different concentrations, 44 hours after the cultures were started. 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:
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.
- 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.
Rationale for test conditions:
The concentrations for the test item were selected based on preliminary reports (Roldán and Altamirano, 1990; Rodríguez-Mercado et al., 2003)*.

* 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.
Evaluation criteria:
not specified
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
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True 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:
not determinable because of methodological limitations

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 CA

 

 

 

 

Ct

Cs

G

Total (A+A’)

 

 

Test substance (µg/mL)

Cells scored by culture

A

A’

A

A’

A

A’

Without G

With G

% aberrant cells without G

MI%±SD (inhibition %)

Negative control

100

2

1

0

0

1

2

3

6

1.5

2.64 ± 0.35

1

100

4

3

1

0

3

6

8

17

4.0

1.85 ± 0.15 (30)b

2

100

0

3

1

0

3

5

4

12

2.0

1.66 ± 0.15 (37)b

4

100

2

1

0

0

4

4

3

11

1.5

1.65 ± 0.31 (38)b

8

100

3

2

2

0

2

2

7

11

2.5

1.35 ± 0.15 (49)b

MMC

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, chromatid and isochromatid gaps; A, first culture and A´, duplicate culture; Regression lines MI: V2O3 y = 2.662 - 0.276x, r = 0.8121

Conclusions:
In this study, vanadium(III) trioxide (V2O3) was assessed for its potential to induce structural chromosomal aberrations in human peripheral blood lymphocytes. The lymphocytes from a single donor were treated with V2O3 at concentration levels of 1, 2, 4, and 8 µg/mL for 28 hours. Afterwards, the cells were harvested, fixed and stained with Giemsa. The experiment was run in duplicates. A total of 100 metaphases per culture were scored for chromosomal aberrations. Moreover, the Mitotic Index (MI) was determined in order to evaluate cytotoxicity.

The test substance induced no statistically significant increase in the number of structural chromosomal aberrations and proportion of aberrant cell under the conditions of the test. However, the test material induced statistically significant and concentration dependent cytotoxicity.

The publication presented herein shows some reporting deficiencies and deficiencies in the study design.

The authors did not state on confounding factors, i.e. precipitation of the test material as well as pH and osmolality effects of the test material on the culture medium. The test material was only tested in a long-term treatment, whereas the test guidelines requires per default a short-term treatment, except in case positive findings were observed. The lymphocytes were not sufficiently characterised, since information on the normal cell doubling time and the age of the donor are not specified. The mitogen stimulation was slightly shorter than recommended (44 vs. ≥48 h) The type of negative control is not specified. Historical control data is not provided. Acceptability and evaluation criteria are not provided.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The publication presented herein shows some reporting deficiencies and deficiencies in the study design. The effect found on the proportion of aberration cells were only slight when compared to the negative control cultures and historical control data from another test laboratory (≤5.5% vs. 0-4%). Moreover, the effects on chromosomal damage were only found in presence of marked cytotoxicity (32-74%) and the effect observed showed no strong concentration-response relationship. The authors did not state on confounding factors, i.e. precipitation of the test material as well as pH and osmolality effects of the test material on the culture medium. The test material was only tested in a long-term treatment, whereas the test guidelines requires per default a short-term treatment, except in case positive findings were observed. The lymphocytes were not sufficiently characterised, since information on the normal cell doubling time and the age of the donor are not specified. The mitogen stimulation was slightly shorter than recommended (44 vs. ≥48 h). The type of negative control is not specified. Historical control data is not provided. Acceptability and evaluation criteria are not provided. The cells were not tested in presence of a metabolic activation system.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, vanadium(IV) tetraoxide (V2O4) was assessed for its potential to induce structural chromosomal aberrations in human peripheral blood lymphocytes. The lymphocytes, obtained from a single donor, were treated with V2O4 at concentration levels of 1, 2, 4, and 8 µg/mL for 28 hours. Afterwards, the cells were harvested, fixed and stained with Giemsa. The experiment was run in duplicates. A total of 100 metaphases per culture were scored for chromosomal aberrations. Moreover, the Mitotic Index (MI) was determined in order to evaluate cytotoxicity.
GLP compliance:
not specified
Remarks:
publication
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.

From these 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.
Cytokinesis block (if used):
colchicine (4 µg/mL)
Metabolic activation:
not specified
Test concentrations with justification for top dose:
1, 2, 4, or 8 µg/mL
Vehicle / solvent:
- Vehicle 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
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 with 5 μg/mL of phytohemagglutinin 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.

The test item was dissolved in distilled water and added to cultures at different concentrations, 44 hours after the cultures were started. 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 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.

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:
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.
- 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:
Rationale for test conditions:
The concentrations for the test item were selected based on preliminary reports (Roldán and Altamirano, 1990); Rodríguez-Mercado et al., 2003)*.

* References:
ome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocytes cultures. Mutat Res 246:301–322.
- 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.
Evaluation criteria:
not specified
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
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
The proportion of aberrant cells (without gaps) and the number of structural aberrations (without gaps) were statistically significantly increased when compared to the negative control. Further, the results show a significant concentration-related decrease in the Mitotic Index (MI) in cultures treated with the test item (please refer to table 1 in the field "Any other information on results incl. tables" below).
Remarks on result:
not determinable because of methodological limitations

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 CA

 

 

 

 

Ct

Cs

G

Total (A+A’)

 

 

Test substance (µg/mL)

Cells scored by culture

A

A’

A

A’

A

A’

Without G

With G

% aberrant cells without G

MI%±SD (inhibition %)

Negative control

100

2

1

0

0

1

2

3

6

1.5

2.64 ± 0.35

1

100

2

0

0

1

1

1

3

5

1.5

2.59 ± 0.26 (2)

2

100

3

4

1

4

2

0

12a

14

5.5a

1.80 ± 0.24 (32)b

4

100

4

3

4

2

2

3

12a

17

5.5a

1.48 ± 0.39 (44)b

8

100

3

7

3

0

6

6

13a

25

5.0a

1.41 ± 0.27 (47)b

MMC

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, chromatid and isochromatid gaps; A, first culture and A´, duplicate culture; Regression lines MI: V2O4 y = 3.055 - 0.357x, r = 0.9008

Conclusions:
In this study, vanadium(IV) tetraoxide (V2O4) was assessed for its potential to induce structural chromosomal aberrations in human peripheral blood lymphocytes. The lymphocytes, obtained from a single donor, were treated with V2O4 at concentration levels of 1, 2, 4, and 8 µg/mL for 28 hours. Afterwards, the cells were harvested, fixed and stained with Giemsa. The experiment was run in duplicates. A total of 100 metaphases per culture were scored for chromosomal aberrations. Moreover, the Mitotic Index (MI) was determined in order to evaluate cytotoxicity.

According to the authors, the test substance induced a statistically significantly increased proportion of aberrant cells, and number of structural chromosome aberrations. However, the effects were not clearly concentration dependent and were observed only in presence of marked and statistically significant cytotoxicity. The cytotoxic effects were found to be concentration dependent.

The publication presented herein shows some reporting deficiencies and deficiencies in the study design.

The effect found on the proportion of aberration cells were only slight when compared to the negative control cultures and historical control data from another test laboratory (≤5.5% vs. 0-4%). Moreover, the effects on chromosomal damage were only found in presence of marked cytotoxicity (32-74%) and the effect observed showed no strong concentration-response relationship. The authors did not state on confounding factors, i.e. precipitation of the test material as well as pH and osmolality effects of the test material on the culture medium. The test material was only tested in a long-term treatment, whereas the test guidelines requires per default a short-term treatment, except in case positive findings were observed. The lymphocytes were not sufficiently characterised, since information on the normal cell doubling time and the age of the donor are not specified. The mitogen stimulation was slightly shorter than recommended (44 vs. ≥48 h). The type of negative control is not specified. Historical control data is not provided. Acceptability and evaluation criteria are not provided. The cells were not tested in presence of a metabolic activation system.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The publication presented herein shows some reporting deficiencies and deficiencies in the study design. The selection of the top concentration was not consistent with the criteria set out in the test guideline, since the test material was not tested up to the required cytotoxicity level (MI: 41% vs. 50%). Moreover, the authors did not state on confounding factors, i.e. precipitation of the test material as well as pH and osmolality effects of the test material on the culture medium. The test material was only tested in a long-term treatment, whereas the test guidelines requires per default a short-term treatment, except in case positive findings were observed. The lymphocytes were not sufficiently characterised, since information on the normal cell doubling time and the age of the donor are not specified. The mitogen stimulation was slightly shorter than recommended (44 vs. ≥48 h). The type of negative control is not specified. Historical control data is not provided. Acceptability and evaluation criteria are not provided. The cells were not tested in presence of a metabolic activation system.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, vanadium(V) pentoxide (V2O5) was assessed for its potential to induce structural chromosomal aberrations in human peripheral blood lymphocytes. The lymphocytes from a single donor were treated with V2O5 at concentration levels of 1, 2, 4, and 8 µg/mL for 28 hours. Afterwards, the cells were harvested, fixed, and stained with Giemsa. The experiment was run in duplicates. A total of 100 metaphases per culture were scored for structural chromosomal aberrations. Moreover, the Mitotic Index (MI) was determined in order to evaluate cytotoxicity.
GLP compliance:
not specified
Remarks:
publication
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.

From these 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.
Cytokinesis block (if used):
colchicine (4 µg/mL)
Metabolic activation:
not specified
Test concentrations with justification for top dose:
1, 2, 4, or 8 µg/mL
Vehicle / solvent:
- Vehicle 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
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 with 5 μg/mL of phytohemagglutinin and treated with vanadium(V) pentoxide 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.

The test item was dissolved in distilled water and added to cultures at different concentrations, 44 hours after the cultures were started. 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:
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.
- 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.
Rationale for test conditions:
The concentrations for the test item were selected based on preliminary reports (Roldán and Altamirano, 1990; Rodríguez-Mercado et al., 2003)*.

* 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.
Evaluation criteria:
not specified
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
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True 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:
not determinable because of methodological limitations

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 CA

 

 

 

 

Ct

Cs

G

Total (A+A’)

 

 

Test substance (µg/mL)

Cells scored by culture

A

A’

A

A’

A

A’

Without G

With G

% aberrant cells without G

MI%±SD (inhibition %)

Negative control

100

2

1

0

0

1

2

3

6

1.5

2.64 ± 0.35

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

MMC

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, chromatid and isochromatid gaps; A, first culture and A´, duplicate culture; Regression lines MI: V2O5 y = 2.671 - 0.239x, r = 0.8009.

Conclusions:
In this study, vanadium(V) pentoxide (V2O5) was assessed for its potential to induce structural chromosomal aberrations in human peripheral blood lymphocytes. The lymphocytes from a single donor were treated with V2O5 at concentration levels of 1, 2, 4, and 8 µg/mL for 28 hours. Afterwards, the cells were harvested, fixed and stained with Giemsa. The experiment was run in duplicates. A total of 100 metaphases per culture were scored for chromosomal aberrations. Moreover, the Mitotic Index (MI) was determined in order to evaluate cytotoxicity.

The test substance induced no statistically significant increase in the number of structural chromosomal aberrations and proportion of aberrant cell under the conditions of the test. However, the test material induced statistically significant and concentration dependent cytotoxicity.

The publication presented herein shows some reporting deficiencies and deficiencies in the study design.

The selection of the top concentration was not consistent with the criteria set out in the test guideline, since the test material was not tested up to the required cytotoxicity level (MI: 41% vs. 50%). Moreover, the authors did not state on confounding factors, i.e. precipitation of the test material as well as pH and osmolality effects of the test material on the culture medium. The test material was only tested in a long-term treatment, whereas the test guidelines requires per default a short-term treatment, except in case positive findings were observed. The lymphocytes were not sufficiently characterised, since information on the normal cell doubling time and the age of the donor are not specified. The mitogen stimulation was slightly shorter than recommended (44 vs. ≥48 h) The type of negative control is not specified. Historical control data is not provided. Acceptability and evaluation criteria are not provided.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006-09-04 till 2006-09-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
not applicable
Species / strain / cell type:
S. typhimurium TA 97
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
7, 21, 62, 185, 556 and 1667 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A 0.5%v aqueous solution of Na.carboxylmethylcellulose (CMC, Lot.No. 98H0328, Sigma) was wased as vehicle for the test substance and for the negative control group.
- Justification for choice of solvent/vehicle: The test substance was not soluble in water or DMSO. Therefore a suspension of the test substance in 0.5% CMC was prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene; 1 or 2µg (dissolved in diethylsulfoxide)
Remarks:
with metabolic activation; strains TA98, TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation, strain TA97a Migrated to IUCLID6: ; 10µg (dissolved in diethylsulfoxide)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxy-anthraquinone; 50µg (dissolved in diethylsulfoxide)
Remarks:
with metabolic activation, strain TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation; strain TA98 Migrated to IUCLID6: ; 2µg (dissolved in diethylsulfoxide)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation; strains TA100 and TA1535 Migrated to IUCLID6: ; 1 and 2µg (dissolved in sterile water)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine; 10µg (dissolved in diethylsulfoxide)
Remarks:
without metabolic activation; strain TA97a
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: t-Butyl-hydroperoxide; 50µg (dissolved in sterile water)
Remarks:
without metabolic activation; strain TA102
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Two test were performed.

DURATION
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3 plates per dose; for the control groups 6-fold repetitions were run.

COUNTING OF COLONIES: The plates with less than about 50 revertant colonies, i.e. the plates of TA98 and TA1535 with the exception of the positive controls, were counted visually. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Different concentrations (21, 62, 185, 556, 1667 and 5000µ/plate) of the test substance suspensions were mixed with phosphate buffer or S9 mix, bacteria (TA100) and top-agar and spread over a plate with minimal agar. The plates were incubated at 37°C for 48 hours and the growth of the bacterial background and the density of revertant colonies was determined.
The test substance was toxic at 5000 and 1667 µg/petri dish. At 556 µg/plate the bacterial background was normal. There fore 1667 µg/plate was used as the highest concentration which could be toxic and 6 concentrations were tested. Each of the other 5 concentrations was 1/3 of the preceding one.

OTHER EXAMINATIONS:
no further details are reported
Evaluation criteria:
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2.5 fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1.67 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of the available historic data.
Statistics:
not madatory for this test system
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
There was no increase in the number of mutants at any of the tested concentrations. The addition of S9 mix did not change these results.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
The test substance was toxic to the bacteria at the highest concentration tested, resulting in a reduced number and size of the colonies.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
There was no increase in the number of mutants at any of the tested concentrations. The addition of S9 mix did not change these results.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
The test substance was toxic to the bacteria at the highest concentration tested, resulting in a reduced number and size of the colonies.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
There was no increase in the number of mutants at any of the tested concentrations. The addition of S9 mix did not change these results.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
The test substance was toxic to the bacteria at the highest concentration tested, resulting in a reduced number and size of the colonies.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
There was no increase in the number of mutants at any of the tested concentrations. The addition of S9 mix did not change these results.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
The test substance was toxic to the bacteria at the highest concentration tested, resulting in a reduced number and size of the colonies.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
There was no increase in the number of mutants at any of the tested concentrations. The addition of S9 mix did not change these results.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
The test substance was toxic to the bacteria at the highest concentration tested, resulting in a reduced number and size of the colonies.
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: 3.00 (1% suspension in deionised water, w/v, determined with a pH-Meter WTW pH 340).
- Water solubility: The test substance was not soluble.
- Precipitation: Test substance particles were visible in all of the concentration groups with increasing intensity when the test substance was mixed with the agar.

RANGE-FINDING/SCREENING STUDIES: In the preliminary test the test substance was toxic to the bacteria at 5000 and 1667µg/plate, resulting in a missing bacterial background lawn.

COMPARISON WITH HISTORICAL CONTROL DATA: yes; For the evaluation of the results threshold values were derived from the variations in the control samples of available historic data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no further information
Conclusions:
Interpretation of results (migrated information):
negative

According to the results obtained in this study, Sodiumpolyvanadate (SPV) is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolic activation system up to 1667µg/plate, wich is the limit of toxicity.
Executive summary:

The test item was tested for mutagenicity activity with the Ames test. The study was conducted in accordance with the OECD guideline 471. The test substance was suspended in 0.5% CMC. The following concentrations were tested: 7, 21, 62, 185, 556 and 1667 µg/plate with and without S9-mix.

The test substance was toxic to the bacteria at 1667 µg/plate. resulting in a reduced number and size of the colonies.

In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250% of the controls for strains TA98 and TA1535, 167% of the controls for strains TA97a, TA100 and TA102) was observed. Metabolic activation did not change these results.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006-09-05 till 2006-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
not applicable
Species / strain / cell type:
S. typhimurium TA 97
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I:
- without metabolic activation: 7, 21, 62, 185, 556 and 1667µg/plate
- with metabolic activation: 21, 62, 185, 556, 1667 and 5000µg/plate
Experiment II:
- without metabolic activation: 2.3, 7, 21, 62, 185 and 556µg/plate
- with metabolic activation: 7, 21, 62, 185, 556 and 1667µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A 0.5% aqueous solution of Na-carboxymethylcellulose (CMC, Lot No. 98H0328, Sigma) was used as vehicle for the test substance and for the negative control group (vehicle control, 100µl).
- Justification for choice of solvent/vehicle: The test substance was not soluble in water or DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene; 1 or 2µg (dissolved in DMSO)
Remarks:
with metabolic activation; strains TA98, TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation; strain TA97a Migrated to IUCLID6: ; 10µg (dissolved in DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxy-anthraquinone; 50µg (dissolved in DMSO)
Remarks:
with metabolic activation; strain TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation; strain TA98 Migrated to IUCLID6: ; 2µg (dissolved in DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation; strains TA100 and TA1535 Migrated to IUCLID6: ; 1 or 2µg (dissolved in sterile water)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine; 10µg (dissolved in DMSO)
Remarks:
without metabolic activation; strain TA97a
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: t-Butyl-hydroperoxide; 50µg (dissolved in sterile water)
Remarks:
without metabolic activation; strain TA102
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar
The test substance was tested with the plate incorporation method and the results were verified by a second independent experiment.

DURATION
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: Triplicate repetitions were run for each dose group; for the control groups 6-fold repetitions were run.

COUNTING OF COLONIES: The plates with less than about 50 revertant colonies, i.e. the plates of TA98 and TA1535 with the exception of the positive controls, were counted visually. The other plates were photographed and the picture files were scanned for colonies by a computer program.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth:
Different concentrations of test substance suspensions were mixed with phosphate buffer or S9 mix, bacteria (TA100) and top-agar and spread over a plate with minimal agar. The plates were incubated at 37°C for 48 hours and the growth of the bacterial background and the density of revertant colonies were determined.

OTHER EXAMINATIONS:
No further examinations.
Evaluation criteria:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2.5-fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1.67-fold of the amount of the spontaneous revertants.
Statistics:
Means and standard deviations were calculated for the number of mutants in every concentration group.
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.
Cytotoxicity / choice of top concentrations:
other: Slight toxicity to the bacteria was noted in the higher concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.
Cytotoxicity / choice of top concentrations:
other: Slight toxicity to the bacteria was noted in the higher concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.
Cytotoxicity / choice of top concentrations:
other: Slight toxicity to the bacteria was noted in the higher concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.
Cytotoxicity / choice of top concentrations:
other: Slight toxicity to the bacteria was noted in the higher concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.
Cytotoxicity / choice of top concentrations:
other: Slight toxicity to the bacteria was noted in the higher 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: pH 3.79 (1% suspension in deionised water, w/v, determined with a pH-Meter WTW pH 340)
- Water solubility: The test substance was not soluble.
- Precipitation: Test substance particles were visible in the higher concentration groups when the test substance was mixed with the agar.

RANGE-FINDING/SCREENING STUDIES: The test substance was toxic to the bacteria in the samples without metabolic activation at 5000 and 1667µg/plate and in the samples with metabolic activation at 5000µg/plate. Therefore 1667µg/plate was used as the highest concentration for the samples without metabolic activation and 5000 µg/plate as the highest concentration for the samples with metabolic activation which both could be toxic and 6 concentrations were tested each. Each of the other 5 concentrations was 1/3 of the preceding one.
The concentrations for the second experiment were changed due to the results of the first one: Toxicity was noted in the 1667 and 556µg/plate samples in the plates without metabolic activation and in the 5000 and 1667 µg/plate samples in the plates with metabolic activation. Therefore the concentrations were decreased one step each.

COMPARISON WITH HISTORICAL CONTROL DATA: Threshold values for evaluation of the results were derived from the variations in the control samples of available historical data of the Ames test.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Slight toxicity to the bacteria was noted in the higher concentrations: Although the bacterial background was normal in the higher concentrations, the colonies were reduced in number and size, or no growth of bacteria was noted at all.
Conclusions:
Interpretation of results (migrated information):
negative

According to the results obtained in this study, the test substance "Ammoniumpolyvanadate (APV)" is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolic activation system up to the limit of toxicity.
Executive summary:

"Ammoniumpolyvanadate (APV)" was tested for mutagenic activity with the Ames test. The study was conducted in accordance with the OECD guideline 471. The test substance was suspended in 0.5% CMC. The following concentrations were tested: 7, 21, 62, 185, 556 and 1667µg/plate without metabolic activation and 21, 62, 185, 556, 1667 and 5000µg/plate with S9 mix. In a second experiment the concentrations were changed as follows: 2.3, 7, 21, 62, 185 and 556µg/plate without S9 mix and 7, 21, 62, 185, 556 and 1667µg/plate with S9 mix.

The test was performed according to the "direct plate incorporation method". The bacterial strains TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included.

In the main test slight toxicity to the bacteria was noted in the higher concentrations: Allthough the bacterial background was normal in the higher concentrations, the colonies were reduced in number and size, or no growth of bacteria was noted at all.

In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250% of the controls for strains TA98 and TA1535, 167% of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.

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

Genetic toxicity in vivo

Description of key information

The Registrant has undertaken a thorough evaluation of all available data for the vanadium category substances and has compared the data against the classification criteria as laid down in the Guidance on the Application of the CLP criteria (ECHA, 2017) in a weight-of-evidence analysis. The outcome of this weight-of-evidence analysis can be summarised as follows:

            No evidence for in vitro mutagenicity in bacteria

            Equivocal evidence for in vitro clastogenicity/aneugenicity

            No evidence for in vitro mutagenicity in mammalian cells

            No evidence for in vivo mutagenicity in transgenic rodents

            No evidence for site of in vivo contact genotoxicity after inhalation

            No evidence for in vivo clastogenicity, positive findings stem largely from unreliable studies with unphysiological route of exposure

            Positive findings were largely obtained from studies published by one and the same working group of E. Rojas and M. A. Altamirano-Lozano (University of Mexico City), whose study design and reporting shows recurring deficiencies

The weight-of-evidence analysis of the entire genotoxicity database does not show any clear evidence of germ cell mutagenicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose for cross-reference:
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:
not specified
Remarks:
publication
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 or test system 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
Dose / conc.:
0.1 ppm (nominal)
Dose / conc.:
1 ppm (nominal)
Dose / conc.:
0.093 mg/m³ air (analytical)
Remarks:
4 week exposure
Dose / conc.:
1.178 mg/m³ air (analytical)
Remarks:
4 week exposure
Dose / conc.:
0.103 mg/m³ air (analytical)
Remarks:
8 week exposure
Dose / conc.:
1.041 mg/m³ air (analytical)
Remarks:
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:
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 somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-09-29 to 2010-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
minor
Qualifier:
according to guideline
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. QA statement)
Remarks:
signed 2010-03-02
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system 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
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
120 mg/kg bw/day
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:
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).

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 gavag eup to the maximum tolerated dose of 20 mg/kg/day, did not induce micronuclei in polychromatic erythrocytes of bone marrow.
Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)
Version / remarks:
2013-07-26
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:
not specified
Remarks:
publication
Type of assay:
transgenic rodent mutagenicity assay
Species:
mouse
Strain:
other: Big Blue (BB) B6C3F1
Sex:
male
Details on test animals or test system 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
Dose / conc.:
0.1 mg/m³ air (nominal)
Dose / conc.:
0.09 mg/m³ air (analytical)
Remarks:
SD: 0.018; 4 week exposure
Dose / conc.:
1.18 mg/m³ air (analytical)
Remarks:
SD: 0.194; 4 week exposure
Dose / conc.:
0.1 mg/m³ air (analytical)
Remarks:
SD: 0.017; 8 week exposure
Dose / conc.:
1.04 mg/m³ air (analytical)
Remarks:
SD: 0.200; 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:
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 / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997-07-21
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system 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
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
Dose / conc.:
1 mg/m³ air (nominal)
Dose / conc.:
2 mg/m³ air (nominal)
Dose / conc.:
3 mg/m³ air (nominal)
Dose / conc.:
8 mg/m³ air (nominal)
Dose / conc.:
16 mg/m³ air (nominal)
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:
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.

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-06-02 to 2010-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
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.

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
Hartmann, A. et al. (2003) Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis 18(1), 45-51.
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals or test system 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
Dose / conc.:
0.25 mg/m³ air (nominal)
Remarks:
group 2
Dose / conc.:
1 mg/m³ air (nominal)
Remarks:
group 3
Dose / conc.:
4 mg/m³ air (nominal)
Remarks:
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:
The GLP compliant study by Schuler (2010 and 2011) 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. 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 (0, 0.25, 1, 4 mg/m³). 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, receiving methylmethanesulfonate via oral route. 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 a 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, showing a structure typical for dead cells, complicating the macroscopic differentiation. Nevertheless, the validity of the study was not jeopardised, since the comet results showed a clear and statistically significant increase in DNA damage in cells treated with the positive control, after exclusion of the severely damaged cells (% tail intensity above 80%).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The endpoint genetic toxicity of the vanadium category substances is not addressed by substance-specific information but rather by read-across of data available for soluble tri-, tetra- and pentavalent vanadium substances as well as for insoluble vanadium substances with zero valency, such as vanadium carbide. 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 as well as in vivo, vanadium substances, should be considered void of genotoxicity. A detailed evaluation of the relevance, reliability and adequacy of each study is presented in the individual study records.

 

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. Thus, read-across of genetic toxicity data from soluble tetra- and pentavalent vanadium substances is justified. Further details on the read-across for systemic effects as well as local effects is given in the read-cross justification reports in section 13 of the technical dossier.

 

In vitro gene mutation assays

In vitro testing in bacteria reverse mutation assays (NTP 2002, Wolf 2006a and b, May 2011) were conducted in accordance with OECD 471 and under GLP. Both studies yielded negative results, as follows:

- vanadium pentaoxide was 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) (NTP 2002, reliable without restriction)

- sodium polyvanadate (Wolf 2006) 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 (Wolf 2006a, reliable with restriction)

- ammonium trivanadium octaoxide (NH4V3O8) 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 (Wolf 2006b, reliable with restriction)

- vanadium carbide nitride was not mutagenic in Salmonella typhimurium strain TA98, TA100, TA1535, TA1537 or E.coli WP2 uvr A pKM101, both in the presence as well as absence of metabolic activation (May 2011, reliable without restriction)

 

 

In a comprehensive testing programme, three guideline-conform in vitro mammalian cell gene mutation tests in the HPRT locus were conducted in accordance with OECD 476 and under GLP (Lloyd 2010a,b,c). In these studies, three representative and readily soluble tri-, tetra- and pentavalent vanadium substances were tested for the induction of mutation in the HPRT locus of L5178Y cells. Divanadium trioxide, vanadium oxide sulfate and divanadium pentoxide did not significantly increase the mutation frequency 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).

 

Zhong et al. (1994) evaluated the ability of vanadium pentaoxide (V2O5) to induce mutations at the hprt locus in Chinese hamster lung fibroblasts (V79). The cells were exposed to 1, 2, 3, and 4 µg/mL of V2O5. The relative survival (RS) was calculated to determine cytotoxicity. None of the 3 vanadium pentaoxide concentrations tested significantly increased the mutation frequency. The positive control, N-Methyl-N'-nitro-N-nitrosoguanidine showed a significantly increase in the mutation frequency, demonstrating the sensitivity of the test system. The relative survival proportions indicate that the tested concentrations of V2O5 were toxic to V79 cells, but not increasing the mutation frequency up to the highest concentration tested (4 µg/mL).The publication shows severe reporting deficiencies and deficiencies in the study design: Historical control data are neither shown nor discussed. Only three analysable concentrations were evaluated (at least four are recommended in OECD TG 476, 1984). Evaluation and scoring criteria are not specified. Confounding factors, such as precipitation, pH effects, and effects on osmolality, are not determined. The results are not discussed in correlation with the cytotoxicity observed. Information on cell line is insufficient, since data on mycoplasma contamination, karyotype stability, and cell doubling times are missing. Based on the above described deficiencies, the study by Zhong et al. is rated as “not reliable” (RL=3).

 

In vivo gene mutation assays

The lack of significant induction of cII mutant frequencies in the lungs of the transgenic Big Blue mice exposed to tumorigenic concentrations of divanadium pentaoxide by inhalation for up to 8 weeks suggests that divanadium pentaoxide (Manjanatha et al. 2015) 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 (Banda et al. 2015). 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 pre-existing mutation as being involved in the genesis of divanadium pentaoxide-induced mouse lung tumours.

 

 

In vitro clastogenicity assays

In a comprehensive testing programme, three guideline-conform in vitro mammalian micronucleus tests were conducted in accordance with OECD 487 and under GLP (Lloyd 2010d, e, f). In these studies, three representative and readily soluble tri-, tetra- and pentavalent vanadium substances were tested for the induction of micronuclei in TK6 cells. The results of the experiments were as follows:

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 (Lloyd 2010d). 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 (Lloyd 2011). 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.

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 (Lloyd 2010e). 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), 4nitroquinoline-1-oxide (NQO) and Cyclophosphamide (CPA) were employed as positive control chemicals.

Experiment 1 in 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 in 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 test system. 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 in 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.

 

Divanadium pentaoxide was tested assay using duplicate primary human lymphocyte cultures and human lymphoblastoid TK6 cells (Lloyd 2010f). 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 with 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 95th percentile 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 in 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 in both cultures at the other 3 concentrations exceeded the 95th percentile of the normal range.

It is not evident that the positive responses observed in the studies by Lloyd (2010) are true effects based on V2O3, VOSO4 or V2O5 induced chromosome damage at physiologically relevant concentrations since both cytotoxicity as well as precipitation were observed in treatment groups that tested positively. Negative as well as positive results were obtained in 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.

 

 

The chromosomal aberration frequency in Chinese hamster lung cells was evaluated after treatment with vanadium carbide nitride (VCN) in a study by Pritchard (2011). The chromosome damaging potential of vanadium carbide nitride was evaluated at concentration levels ranging from 21.54-769.60 µg/mL both in absence and presence of a S-9 metabolic activation system. The proportion of cells with chromosomal aberrations was not increased after vanadium carbide nitride exposure under any condition tested. Both positive control compounds, mitomycin C and cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. Thus, the efficacy of the S9 mix and the sensitivity of the test system were demonstrated. 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 of the test. This guideline and GLP compliant study is reliable without restrictions (RL1).

 

In this study by Rodriguez-Mercado et al. (2010), vanadium trioxide, vanadium tetraoxide, and vanadium pentaoxide were assessed for their potential to induce structural chromosomal aberrations in human peripheral blood lymphocytes. The lymphocytes from a single donor were treated with V2O5 at concentration levels of 1, 2, 4, and 8 µg/mL for 28 hours. Moreover, the Mitotic Index (MI) was determined in order to evaluate cytotoxicity.

Experiment 1 (vanadium trioxide): The test substance induced no statistically significant increase in the number of structural chromosomal aberrations and proportion of aberrant cell under the conditions of the test. However, the test material induced statistically significant and concentration dependent cytotoxicity. The publication presented herein shows some reporting deficiencies and deficiencies in the study design. The authors did not state on confounding factors, i.e. precipitation of the test material as well as pH and osmolality effects of the test material on the culture medium. The test material was only tested in a long-term treatment, whereas the test guidelines requires per default a short-term treatment, except in case positive findings were observed. The lymphocytes were not sufficiently characterised, since information on the normal cell doubling time and the age of the donor are not specified. The mitogen stimulation was slightly shorter than recommended (44 vs. ≥48 h) The type of negative control is not specified. Historical control data is not provided. Acceptability and evaluation criteria are not provided.

Experiment 2 (vanadium tetraoxide): According to the authors, the test substance induced a statistically significantly increased proportion of aberrant cells, and number of structural chromosome aberrations. However, the effects were not clearly concentration dependent and were observed only in presence of marked and statistically significant cytotoxicity. The cytotoxic effects were found to be concentration dependent. The publication presented herein shows some reporting deficiencies and deficiencies in the study design. The effect found on the proportion of aberration cells were only slight when compared to the negative control cultures and historical control data from another test laboratory (≤5.5% vs. 0-4%). Moreover, the effects on chromosomal damage were only found in presence of marked cytotoxicity (32-74%) and the effect observed showed no strong concentration-response relationship. The authors did not state on confounding factors, i.e. precipitation of the test material as well as pH and osmolality effects of the test material on the culture medium. The test material was only tested in a long-term treatment, whereas the test guidelines requires per default a short-term treatment, except in case positive findings were observed. The lymphocytes were not sufficiently characterised, since information on the normal cell doubling time and the age of the donor are not specified. The mitogen stimulation was slightly shorter than recommended (44 vs. ≥48 h). The type of negative control is not specified. Historical control data is not provided. Acceptability and evaluation criteria are not provided. The cells were not tested in presence of a metabolic activation system.

Experiment 3 (vanadium pentaoxide): The test substance induced no statistically significant increase in the number of structural chromosomal aberrations and proportion of aberrant cell under the conditions of the test. However, the test material induced statistically significant and concentration dependent cytotoxicity. The publication presented herein shows some reporting deficiencies and deficiencies in the study design: The selection of the top concentration was not consistent with the criteria set out in the test guideline, since the test material was not tested up to the required cytotoxicity level (MI: 41% vs. 50%). Moreover, the authors did not state on confounding factors, i.e. precipitation of the test material as well as pH and osmolality effects of the test material on the culture medium. The test material was only tested in a long-term treatment, whereas the test guidelines requires per default a short-term treatment, except in case positive findings were observed. The lymphocytes were not sufficiently characterised, since information on the normal cell doubling time and the age of the donor are not specified. The mitogen stimulation was slightly shorter than recommended (44 vs. ≥48 h), the type of negative control is not specified. Historical control data is not provided. Acceptability and evaluation criteria are not provided.

 

In this study (Roldán & Altamirano 1990), human peripheral lymphocytes were exposed to 2, 4, and 6 µg/mL of vanadium pentaoxide (V2O5). Cytotoxicity was evaluated via analysis of the average generation time (AGT) and the mitotic index (MI). The frequency of structural chromosomal aberrations was not increased in peripheral blood lymphocytes cultured in the presence of vanadium pentaoxide. However, statistically significant increases in the frequency of polyploid cells was observed after vanadium pentaoxide exposure at all concentration levels tested. The AGT was significantly higher (p<0.05) at 4 and 6 µg V2O5/mL. Correspondingly, the MI was significantly lower (p<0.05) at 4 and 6 µg V2O5/mL. The publication shows reporting deficiencies and deficiencies in the study design as follows: The item characterisation is insufficient, since information on the purity and source of the test material are not stated. Only a single treatment and harvesting time point was included (OECD TG 473, 1983 recommends treatment at various time points or alternatively single treatment with multiple harvesting time points). There is a complete lack of historical control data, which are not shown or discussed, likewise the evaluation and scoring criteria are not given. No positive control is used to demonstrate the sensitivity of the test system. The exposure duration is not unambiguously stated. The presence of the negative control is only stated as "control", without any further identification of the substance. No information on confounding factors, such as precipitation, pH effects, and effects on osmolality are provided. The determination of average generation time (AGT) and mitotic index (MI) is not sufficiently described. Only the total number of structural chromosomal aberrations is stated, without further specification on types and their occurrence. No information on statistical testing of differences in structural chromosomal aberration frequencies is available. Cytotoxicity is not discussed in correlation with the chromosomal aberrations observed. Based on the above-described deficiencies, the study by Roldan and Altamirano is considered not reliable (RL3).

 

In the study by Rodriguez-Mercado et al. (2003), the clastogenicity of vanadium(IV)tetraoxide was evaluated in human peripheral blood lymphocytes using the chromosome aberration test (CA). Cytotoxicity was determined using the mitotic index (MI) and the replicative index (RI). Vanadium(IV) tetraoxide induced a clear dose-response in MI inhibitions and modifications in the RI. In the CA, the proportion of aberrant cells was statistically significantly increased in the combined donor analysis at all concentration levels tested. Reasonably well described study with minor restrictions (RL2). The following experimental deficiencies restrict the use for hazard assessment: exposure towards test substance commenced 24h after mitogenic stimulation (guideline foresees 48h). The authors did not state on potential precipitation, pH effects, and osmolality effects of the test material in the culture medium. Incubation temperature before and during treatment is not specified. Cell cycle length not determined. Purity of the test substance not reported. Positive control cultures were not included. Historical control data is not provided. Evaluation and scoring criteria are not specified. Under the given experimental conditions the test substance vanadium(IV) tetraoxide is capable of inducing chromosomal damage.

 

In the study by Rodriguez-Mercado et al. (2003), the clastogenicity of vanadium(IV) tetraoxide was evaluated in human peripheral blood lymphocytes using the sister chromatid exchanges (SCE). Cytotoxicity was determined using the mitotic index (MI), and the replicative index (RI). This substance induced a clear dose-response in MI inhibitions and modifications in the RI. Combined analysis of the SCE revealed slightly but statistically significantly increased SCEs per cell at concentration of and greater than 4 µg/mL. The response was concentration related. However, the positive findings were only observed in presence of statistically significant cytotoxicity and showed highly similar increments. Moreover, the individual donor analysis revealed high inter-individual variation in the SCE frequency. The publication shows some reporting deficiencies and deficiencies in the study design. The SCE methodology is only poorly described. The authors did not state on potential precipitation, pH effects, and osmolality effects of the test material in the culture medium. Incubation temperature before and during treatment is not specified. Cell cycle length not determined. Purity of the test substance not reported. Positive control cultures were not included. Soring and evaluation criteria are not reported. Historical control data is not provided. Scoring and evaluation criteria are not specified. The positive findings were not confirmed in an independent experiment as required by the OECD TG 479. No conclusion on chromosomal damage can be drawn based on the findings of the study (RL3).

 

In the study by Zhong et al. (1994), the genotoxicity of vanadium pentaoxide (V2O5) was evaluated in Chinese hamster lung fibroblasts (V79) using the sister chromatid exchange (SCE) assay. The cells were exposed to vanadium pentaoixde at concentration levels of 1, 2, 3, and 4 µg/mL for 24 hours. Cytotoxicity in the SCE assay was evaluated via the determination of the replicative index (RI). The highest vanadium pentaoxide concentration level (4 µg/mL) was highly cytotoxic and hampered the SCE analysis. According to the authors, no significant increase in the SCE frequency was observed with the vanadium pentoxide concentrations tested. The replicative index showed a concentration-related decrease. The publication shows severe reporting deficiencies and deficiencies in the study design: The evaluation criteria for SCE scoring are partially missing and deviate from the OECD test guideline (i.e., only cells with 46 ± 2 centromeres of the modal chromosome number vs cells with 20-23 distinguishable and well-spread chromosomes, see OECD TG 479). Historical control data are not included. Potential confounders, i.e. pH effects, osmolality effects, and test material precipitation, were neither examined nor discussed. Moreover, the cell line used is insufficiently described, since information on mycoplasma contamination, karyotype stability, and cell doubling times are missing. The number of chromosomes and number of SCE per chromosome is not stated, the number of cells treated is unclear since this information is lacking. The concentration is specified in µg/mL, which is pointless for adherent cell cultures. The statistically significant increase of the SCE frequency at the intermediate concentration is not discussed or was not tested in an independent experiment. Based on the above described deficiencies, the study by Zhong et al. is rated as “not reliable” (RL=3).

 

Vanadium pentaoxide was tested for a 24-hour treatment period in the in vitro micronucleus 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 et al., 1997). The publication is well documented, meets generally accepted scientific principles and is considered acceptable for assessment (RL2)

 

In the study by Zhong et al. (1994), the genotoxicity of vanadium pentaoxide was evaluated in Chinese hamster lung fibroblasts (V79) using the in vitro micronucleus (MN) test. The cells were exposed to vanadium pentaoxide at concentration levels of 1, 2, and 3 µg/mL. Cytotoxicity in the MN test was evaluated via the determination of the nuclear division index (NDI). Additionally, in an isolated experiment, the effect of vanadium pentaoxide on mitosis was analysed via the determination of the number of mono- and binucleated cells after test substance treatment and subsequent cytokinesis block. The MN frequency was statistically significantly higher (p< 0.01) in all treatment groups and the dose-response relationship was statistically significant by the trend test (Z=2.894, p<0.005). The frequency of kinetochore positive MN was significantly higher in all V2O5 treated cells and showed a concentration related response. No increase in the number of kinetochore negative MN was not in V2O5 treated cells. The nuclear division index (NDI) was not found decreased in V2O5 treated cells. The publication shows severe reporting deficiencies and deficiencies in the study design: Historical control data are not included. The evaluation and scoring criteria are not specified. Cytotoxicity, as indicated by the replication index (RI), was not determined. Further, the cytotoxicity/cytostasis, as indicated by the cytokinesis-block proliferation index (CBPI), was only determined in an isolated experiment, using an equation different from the OECD 487 test guideline, in which the intermediate concentration (2 µg/mL) was not tested. Confounding factors such as precipitation, cytotoxicity, pH and osmolality are not determined or discussed, the cytochalasin B concentration is not specified. Information on cell line is insufficient, since data on mycoplasma contamination, karyotype stability, and cell doubling times are missing. Based on the above described deficiencies, the study by Zhong et al. is rated as “not reliable” (RL=3).

 

The authors (Ramírez et al., 1997) investigated the potential of vanadium pentoxide (V2O5) to induce aneuploidy in human lymphocytes. The cells were treated at V2O5 concentrations of 0.001, 0.01, and 0.1 µM. Aneuploidy was analysed using chromosome 1 and 7 specific probes in a fluorescent in situ hybridisation (FISH). Moreover, the effect of V2O5 on the microtubule organisation of mitotic spindles was investigated in human lymphocytes via immunocytochemistry. Moreover, tubulin de-/polymerisation was assessed in vitro using tubulin isolated form mice brain. The authors concluded that vanadium pentaoxide can induce aneuploidy in human lymphocytes which may be due to a disruption of microtubule function. The publication by Ramirez et al. (1997) shows major reporting and methodological deficiencies as well as deficiencies in the study design. First, the study solely investigates the presence of aneuploidy in cultured peripheral blood lymphocytes of two donors via FISH assay. However, this investigation is commonly used in in vitro micronucleus assays in cases when positive findings are received, to further differentiate between a clastogenic and aneugenic effect. Consequently, the study does not comply with a standard in vitro micronucleus test (OECD 487) and can therefore not be rated for reliability due to a complete lack of compliance with accepted guidelines for comparison. In addition to the (formal) non-guideline compliance, the study shows additional shortcomings: The age of the donors is not specified and it is unclear if and how the samples were pooled, since results are reported for 2 donors, although the methods section reports 3 (2 females, 1 male). It is further known that female donors show a higher clastogenic background level than males, which was not controlled for. The authors used colchicine as positive control substance for aneuploidy, which is very difficult to dose as positive control due to its very narrow therapeutic index, which is why vincristine is usually used as positive control for aneuploidy. The percentage of numerical aberration in chromosome 1 and 7 appears excessively high (approx. 10-15% in negative control and 14-20% in positive control for both hyper- and hypoploidy). Test material is insufficiently characterised (purity, physical appearance not specified), procedure on preparation of the test material is not described (unknown vehicle/data). Evaluation and scoring criteria are not given, vehicle control data and cytotoxicity were not measured or are not reported and authors do not provide historical control data to validate the findings. No information on hybridisation overlaps, potentially resulting in false negatives/positives. Confounding factors (such as precipitation, pH, osmolality) are not discussed and measurements were performed on single cultures as no replicates were used. Results are given without standard deviation or confidence interval. Based on the above described shortcomings, the results of this publication cannot be rated for their quality, due to a missing guideline and serious experimental and reporting deficiencies. Consequently, this reported study is rated as not reliable (RL=3) and disregarded for the weight-of-evidence assessment.

 

in vitro assays on DNA damage

In accordance with regulation (EC) 1272/2008, Annex I: 3.5.2.3.3 test systems assessing heritable alterations of DNA (such as in vitro gene mutation in the hprt locus) and systems investigating the induction of unspecific and transient DNA damage (such as comet assay) should be assessed differently: “Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests, preferably as described in Regulation (EC) No 440/2008 adopted in accordance with Article 13(3) of Regulation (EC) No 1907/2006 (‘Test Method Regulation’) such as those listed in the following paragraphs.”. In vitro DNA damage tests are neither validated nor were these tests conducted in accordance with an accepted guideline. The information gained from such systems should therefore be evaluated with great care and regarded to contribute to the overall evidence to a much lesser extent than information gained from tests using standardised, validated and accepted guidelines.

 

In vivo clastogenicity studies

The in vivo genetic toxicity of V2O5 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 vanadium pentaoxide aerosol by inhalation (at 0.5 – 9 mg/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.

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. While, vanadium tissue levels increased in all sampled tissues with increased V2O5 dosage levels, and thus, vanadium reached the target tissues bone marrow and testes in a dose-dependent manner, V2O5 administered 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).

 

Leopardi, P. et al. (2005) evaluated the clastogenic and aneugenic potential of sodium orthovanadate (Na3VO4) by its effect on frequency of micronuclei formation in bone marrow erythrocytes and peripheral blood reticulocytes of male CD-1 mice. Groups of five mice received sodium orthovanadate at concentration levels of 0.75, 7.5, 75, 750, and 1500 mg/L via drinking water over a period of 35 days. Untreated control animals were run concurrently. Mice from the positive control group received a single intraperitoneal injection of 80 mg/kg bw methyl methanesulfonate 24 hours before euthanasia. The micronucleus assay using bone marrow erythrocytes was composed of main experiment (concentration levels: 7.5, 75, 750, and 1500 mg/L) and a repeat experiment (concentration levels: 0.75, 7.5, and 75 mg/L) performed to investigate on effects in low dose groups. Peripheral blood reticulocytes used for the second micronucleus assay were obtained, from the same test animals from experiment 1 and 2 of the bone marrow micronucleus test, at treatment start as well as on days 7, 21 and 35 of exposure. Additionally, the elemental vanadium concentration was measured in the femur, tibia, spleen, liver, kidney, and testis using ICP-OES. In the peripheral blood micronucleus assay, the micronucleus frequency was statistically significantly increased at the third week in test animals dosed at 75 and 750 mg/L sodium orthovanadate, and at the fifth week in test animals dosed at 0.75, 7.5, and 750 mg/L. However, no such effect was observed in the highest dose group and the dose appears to be not dose related. Cytotoxicity was not observed under the conditions of the test. However, increased general toxicity was observed in the two highest dose groups as discussed below. The biological relevance of the findings presented herein is questionable. In the first micronucleus assay experiment using bone marrow erythrocytes, the frequency of micronuclei was statistically significantly increased after exposure to sodium orthovanadate concentration levels of 7.5, 750, and 1500 mg/L, when compared to untreated controls. However, the no such effect was observed at a sodium orthovanadate concentration level of 75 mg/L. Moreover, in the repeat experiment testing concentration of 0.75 to 75 mg/L, the micronucleus frequency was not statistically significantly altered in any dose group. Thus, the positive finding observed in the first experiment, at a concentration of 7.5 mg/L, is considered to be of an incidental nature. The proportion of immature among total erythrocytes was not statistically significantly under any condition tested. Notably, the positive findings observed in the first experiment at sodium orthovanadate concentration levels of 750 and 1500 mg/L and in the peripheral blood micronucleus assay at a concentration level of 750 mg/L were associated with increased general toxicity as indicated by observations, which are discussed in the following. I) The water consumption of animals treated with 750 and 1500 mg/L sodium orthovanadate was markedly and statistically significantly decreased to 47.1 and 37.1% of the daily intake of control animals, respectively. The reduced water consumption was most probably due to effects on palatability. II) Moreover, the body weight gain was decreased by 40% and 41.1% in animals dosed at 750 and 1500 mg/L, respectively. III) Both dose groups were lethargic during the last two weeks of the treatment. IV) One animal of the high dose groups died prematurely during the third week of the treatment. The cause of the death was not specified. Premature deaths, however, clearly indicate that the maximum tolerable dose was exceeded. In conclusion, the statistically significant increases in the micronucleus frequency observed in the two highest dose groups have to be interpreted with caution. The effects are not clearly assignable to be genotoxic effects but could have been potentially confounded by general toxicity. The analytical investigation on the vanadium levels showed concentration related increases in all tissue investigated. The MTD was exceeded at the highest concentration tested, which is not in accordance with in vivo genotoxicity test guidelines. The number of animals scored for the presence of micronuclei was, in the high dose group, lower than recommended (4 vs. 5) due to the premature death of one test animal. The description of the test animals is insufficient, since details on housing conditions, food consumption, and the body weights at study initiation and termination are missing. The test material was insufficiently characterised, since information on the physical appearance and manufacturer are missing. The evaluation and scoring criteria were not specified. Historical control data is not included. The response in the micronucleus frequency in bone marrow cells the control group showed some variation. One of the control test animals showed 27 micronuclei, while highest frequencies observed in the high dose were only slightly higher (24, 25, 27, 29). However, the combined micronucleus frequency was 1.9-fold above control values. The number of cells analysed was lower than requested by the test guideline effective at that time (1000 vs. 2000 cells per animal). The methodology of the cytotoxicity test used in the peripheral blood micronucleus assay is only poorly described. Moreover, the results of the cytotoxicity are neither shown nor sufficiently discussed. The results of the peripheral blood micronucleus experiments are presented only in a single diagram without any information on standard deviations. In the diagram, it is not indicated whether the results on the 7.5 and 75 mg/L dose groups stem from experiment 1 or experiment 2.

 

Villani, P. et al. (2007) tested vanadyl sulphate pentahydrate (VOSO4 5H2O) for its potential to induce clastogenic and aneugenic effects in bone marrow and peripheral blood cells of CD-1 mice treated for five weeks via drinking water. The potential aneugenicity and clastogenicity were evaluated in two independent experiments for both of tissues examined. In the first experiment, groups of eight to ten male mice were exposed to vanadyl sulphate at concentration levels of 10, 100, 500, and 1000 mg/L. In a repeat experiment, eight male mice per group were treated with 2 and 10 mg/L vanadyl sulphate. Untreated and positive (methyl methanesulfonate) control groups were run concurrently. For bone marrow micronucleus assay, the cells were obtained from femurs subsequent to euthanasia and stained with Giemsa. A total of 2000 polychromatic erythrocytes per animas were scoored for the presence of micronuclei. Moreover, cytotoxicity was evaluated by the determination of the proportion of polychromatic among total erythrocytes. For the peripheral blood micronucleus assay, blood was obtained at treatment initiation, and 7, 14, 21, 28, and 35 days thereafter. A total of 1000 reticulocytes were scored for the micronucleus formation frequency. The micronucleus frequencies were compared within each group before and during treatment. In order to examine possible effects of the treatment on erythropoiesis, reticulocyte types were characterised and scored among the 1000 cells analysed. Additionally, the elemental vanadium concentration was determined via ICP-OES. The ICP-OES analyses revealed that elemental vanadium was found in all tissues examined, i.e. in the femur, tibia, liver, kidney, spleen, and testis. The vanadium concentration increased with the vanadyl sulphate concentration. In the second experiment at vanadyl concentration levels of 2 and 10 mg/L, elemental vanadium could be detected only in the femur and tibia. The values results observed for the spleen at testis in the low dose groups were considered to be of an artificial nature. Moreover, vanadyl sulphate could not be detected in the intestine and stomach. The vanadyl sulphate exposed mice did not show any effects on food consumption, body weight gain, and absolute organ weights. However, the daily water consumption per mouse was statistically significantly to 65.1% and 60.0% of the concurrent control level in mice exposed to 500 and 1000 mg/L vanadyl sulphate, respectively. Moreover, one high dose mice died prematurely during the fourth week of the treatment. In the bone marrow erythrocyte micronucleus test, vanadyl sulphate did not induce at statistically significant increase in the frequency of micronucleated polychromatic erythrocytes under any condition tested. In contrast, the positive control group showed a marked and statistically significant increase in the micronucleus frequency. Thus, the sensitivity of the test system was demonstrated. Moreover, no biologically relevant alteration in the proportion of polychromatic among total erythrocytes was observed. In the first experiment of the peripheral blood micronucleus assay, mice exposed to vanadyl sulphate at a concentration level of 10 mg/L showed statistically significantly increased micronucleus frequencies at weeks 4 and 5, when compared to the value of the same group at treatment start. Similarly, in the repeat experiment, mice treated with 2 mg/L vanadyl sulphate showed statistically significantly increased levels of micronucleated reticulocytes at week 3 and 4, while mice treated with 10 mg/L showed increased micronucleus frequencies at weeks 4 and 5. However, no such effects were observed in the higher dose groups. Moreover, the results show in general no concentration and time related response. Furthermore, the sporadic findings observed in the repeat experiment were within negative control ranges (approx. 4-5‰) and t0-values (approx. 3-7.5‰) observed in a study performed by the same research group (Leopardi et al., 2005). Thus, the findings are considered to be of an incidental nature. Methyl methanesulfonate induced a marked and statistically significant increase the frequency of micronucleated reticulocytes. The cytotoxicity evaluation revealed no significant effects in vanadyl sulphate exposure groups. The publication shows some deficiencies with regard to reporting. In the high dose group, one mouse died prematurely. However, the cause of the prematurely died test animal was not discussed. Moreover, information on clinical signs are not provided. The use of a pre-treatment control is considered acceptable only for short-term treatments. Blood withdrawal during longer treatments could potentially increase the micronucleus background frequency. The description of the test animals is insufficient, since details on housing conditions, and the body weights at study initiation and termination are missing. Detail on the positive control group are missing. The evaluation and scoring criteria were not specified. Historical control data is not included. In the peripheral blood micronucleus assay, the number of cells analysed was lower than requested by the test guideline effective at that time (1000 vs. 2000 cells per animal). The methodology of the cytotoxicity test used in the peripheral blood micronucleus assay is only poorly described. Moreover, the results of the cytotoxicity are neither shown nor sufficiently discussed. The results of the second bone marrow micronucleus experiment are not shown.

 

The objective of the study by Attia, S.M. et al. (2005) was to investigate the ability of sodium orthovanadate to induce micronuclei in bone marrow cells of (102/E1 x C3H/EI)F1 mice. Groups of five male mice received i.p. orthovanadate doses of 1, 5, 15 or 25 mg/kg bw, followed by bone marrow sampling 24 hours after treatment. A total of 2000 polychromatic erythrocytes were scored for the formation rate of micronuclei. The proportion of polychromatic erythrocytes among all erythrocytes was determined in order to evaluate potential cytotoxicity. None of the orthovanadate doses caused a significant increase micronucleus formation frequency. Moreover, cytotoxicity was not observed as evidenced by the ratio of polychromatic to total erythrocytes. However, the publication by Attia, S.M. et al. shows major deficiencies with regard to reporting and the methodology applied. The test material was administered via intraperitoneal injection, which is considered to be a non-physiological exposure route, and thus, considered not relevant for the hazard assessment. Moreover, target organ exposure was not demonstrated. The dose selection is not consistent with the criteria of comparable test guidelines, since the top dose did not result in toxicity and was far below the recommended limit dose. Furthermore, a positive control group was not included, thus, the sensitivity of the test system was not demonstrated. Further, the bone marrow cells were only sample once after 24 hours, whereas the test guideline (OECD TG 474, 1997) foresees a least two different sampling times. The test material was only poorly characterised and information on the test material preparation are insufficient. The description of the methodology lacks details, including information on the bone marrow cell harvest, preparation, and stain. Evaluation and scoring criteria are missing. Historical control data are not included. Based on the above-mentioned shortcomings the reference is considered not reliable and therefore disregarded for the hazard assessment.

 

Attia, S.M. et al. (2005) evaluated orthovanadate for its aneugenic potential in epididymal sperm cells of mice after a single intraperitoneal injection. The (102/EI x C3H/EI)F1 mice received a single intraperitoneal injection of orthovanadate at doses of 5, 15, and 25 mg/kg bw. A solvent control group was run concurrently. After 22 days, the epididymal sperms were obtained from euthanised mice. The sperm cells were stained with three different probes specific for each of the gonosomes as well as for the chromosome 8. The distribution of the signals was scored in approximately 10,000 sperm cells per animal using fluorescence microscopy. In a pre-test, a potential delay in meiosis was examined via BrdU staining. Moreover, the animals were examined for potential effects on the body weight, testes weight, and sperm counts. The treatment with orthovanadate at a dose of 25 mg/kg bw did not induce a meiotic delay as evidenced by the BrdU proliferation assay. Based on the results obtained in the BrdU assay, a post-exposure period of 22 days was selected for the analysis of potential aneugenic effects. The exposure to 15 and 25 mg orthovanadate/kg bw resulted in a slight but statistically significantly increased frequency of hyperhaploid epididymal sperm cells, when compared to solvent control mice. The response showed a positive dose-response correlation. According to the authors, of the individual classes of aneuploid sperm, only the frequency of sperm with two X-chromosomes was statistically significantly higher in the high dose groups when compared to controls. Body and testes weight were not statistically significantly altered after the orthovanadate injection. Moreover, the sperm counts were comparable between groups and showed no statistically significant differences. Due to the unsuitable study design with the following major restrictions this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information. The experiments performed followed no currently available test guideline. The test material was administered via intraperitoneal injection, which is considered to be a non-physiological exposure route, and thus, considered not relevant for the hazard assessment. The test material was only poorly characterised and information on the test material preparation are insufficient. Further, information of the test animal’s housing conditions are largely missing. The description of the methodology lacks details, including information on the cell preparation and staining procedure. Evaluation and scoring criteria are insufficiently described. Two different tests were performed to analyse statistically significant differences on the same data set showing divergent results. Historical control data are not included.

 

Rojas-Lemus et al. (2014) concluded that vanadium pentoxide (V2O5) induced a sex-dependent increase in the MN frequency. In this study, males were more susceptible to genotoxic damage than females. The study shows deficiencies in reporting and methodology. Thus, no conclusion on the genotoxicity can be drawn based on the results presented in this study. Major: Only one dose group was included, which precludes evaluation of the dose-response relationship. Historical control data is missing. A concurrent positive control group was not included. The results of the concurrent negative control groups are missing. The body weights at study initiation as well as bodyweights and food consumption during the study were not recorded. Moreover, information on clinical signs are not provided. Description of animal housing is insufficient. The information on the exposure scheme is contradictory (abstract: 2 h/twice a week; main text: 1 h/twice a week). Furthermore, information provided on the data set are contradictory (Methods (section: peripheral blood micronucleus test in vivo): 5 animals per group vs. (section: statistical analysis) study was performed with six mice per group). The MN rate observed (ca. 0.05-0.25%) was well-within historical control data range of other laboratories (e.g. Krishna, G. et al., 2000 (reference was also cited in this study; but no reference to historical control data of the study): 0.09-0.31%). The results on MN frequency and proportion of reticulocytes are only presented in a diagram and not summarised in a tabular format with means and standard deviations. Moreover, individual data is not reported. Information on scoring and acceptability criteria are missing. The GSD is not specified. The statements on reticulocyte proportion in females are contradictory. Main text: “In both sexes, a significant decrease in reticulocyte counts during the experiments was noticed.” Vs. diagram (Fig. 3): No significant difference between pre-treatment and treatment animals was observed. The preparation procedure of the inhalation formulation is not specified. The percentage of reticulocytes was always below 50%. After 24 hours of the first treatment, the proportion was less than 20%, which could potentially have confounded the MN frequencies. Week 1-2: ca. 35%. Week 3-4: ca. 50%. The first sampling (after a single exposure) was performed after 24 hours. However, according to OECD 474, sampling should be done not before 36 h post-treatment. The dosing scheme (2 treatment per week; sampling 24 h after treatment and afterwards weekly sampling) was not in accordance with OECD 474 (1. Singe treatment; sampling 36-72 h after treatment, 2. 2 daily treatments; sampling: once 36-48h, 3. >2 daily treatments (e.g. 3 ore more treatments at approx. 24 h intervals; sampling not later than 40 h after last treatment). Minor: The quantification method of test substance levels is not specified (only referenced). The MMAD was specified but its determination was not described. The MMAD (0.5-5 µM) was not in the range recommended by validated test guidelines (OECD 403): “aerosols with mass median aerodynamic diameters ranging from 1 to 4 μm with a geometric standard deviation (σg) in the range of 1.5 to 3.0 are recommended”. The number of metaphases scored for chromosomal damage is not compliant with the requirements of the test guideline in force at that time.

 

García-Rodríguez et al. (2016) concluded that the MN frequency in vanadium pentoxide (V2O5) treated mice was significantly but only marginally increased above concurrent control values and should be considered with caution. The study shows deficiencies in reporting and methodology. Thus, no conclusion on the genotoxicity can be drawn based on the results presented in this study. Major: Only one dose was tested, and thus, a dose-response relationship cannot be derived. The exposure via intraperitoneal injection is a non-physiological route of exposure. The evaluated cell number was too low due to the fact that only 2000 cells were scored for MN-PCE, whereas scoring of at least 4000 cells is required by OECD 474 (2016 and 2014). Only 1000 cells were scored for PCE/NCE ratio, whereas scoring of at least 2000 cells is required by OECD 474 (2016 and 2014). The test item was only poorly characterised, since information on e.g. purity and physical appearance not described. Information on scoring criteria are missing. All samples of V2O5 treated animals showed a significantly increased proportion of MN-PCEs (1.4, 2.2, 3.0, and 3.9‰ at 0, 24, 48, and 72 h, respectively), when compared to the concurrent vehicle control group. Historical control data are not provided. However, comparing the obtained data with historical control data from other laboratories, it becomes evident that only the MN-PCEs number at 72 h was slightly above the historical control data range (Krishna, G. et al. 2000: CD-1: 0.09-0.31%). Moreover, according to the publication: “…the EPA Gene-Tox Program and the Collaborative Study Group for the Micronucleus Test have proposed a threshold of 4/1000 MN-PCE increase to define a compound as genotoxic agent…”. Thus, if the value is credible it is still a significant increase without toxicological relevance. Apoptosis and cell viability were determined only before and 48 h after treatment. The viability was decreased to 73% after 48 h. Moreover, number of apoptotic and necrotic cells were significantly increased. However, these parameters were not determined at 72 h after treatment. There could have been increased cytotoxicity after 72 h potentially confounding the MN-PCE results observed. Both the concurrent water control group and the V2O5 treatment group showed a time-dependent increase in the MN-PCE frequency. A high variability of MN-PCE frequencies in controls (0.05-0.14‰) was observed. The concurrent negative control blood samples showed MN-PCEs at 0 and 24 h below historical control data ((Krishna, G. et al. 2000: CD-1: 0.09-0.31%). The data on MN-PCE net induction frequency was contradictory and implausible. The difference in depicted and anticipated values are either due to i) wrong means MN-PCEs (Tab. 1), ii) a wrong formula, iii) error in calculation of NIFs. A positive control group was not included. Distinction between apoptotic and necrotic cells remains unclear. In the example (Fig. 2), apoptotic cells are not clearly distinguishable from necrotic cells. The PCE-MN NIF and cell viability are only depicted in diagram but not presented in a tabulated format. Information on toxicity are missing. Minor: Information on animal housing are missing (temperature variation, humidity, group size in cages, details on food and water quality). Details on preparation of test substance are missing.

 

The publication by Altamirano-Lozano et al. (1996) reports on results obtained in an in vivo comet assay as well as a dominant lethal assay – both are reported separately in the CLH report. Since both studies show similar shortcomings in study design, reporting and interpretation, they are discussed together here. The CLH report rates both studies as reliable with restriction (RL=2), which in the light of the shortcomings appears unjustified.

-            In both studies, the (poorly characterised) test substance divanadium pentaoxide was administered via intraperitoneal injection. Intraperitoneal injection is a non-physiological route of exposure (see also comment on administration routes further below) and should not be considered relevant for hazard assessment of industrial chemicals.

-            The health status of the male CD-1 mice may be questioned, since authors state a weight of 26-29 g for animals aged 45 days – information from a commercial breeder suggests a mean body weight of 34-36 g for a 6-7 weeks CD-1 mouse. No further information on the health status, housing conditions and clinical observations of the animals was provided.

Comments specifically on the comet assay:

-            The cell isolation and processing as well as the electrophoresis and scoring is insufficiently described, since information on: time elapsed between cell harvest and preparation, neutralization step after alkalic treatment, scoring system, scoring criteria and “hedgehog” occurrence is missing. The degree of DNA damage was assessed by migration length, which is not the recommended parameter for DNA damage (OECD 489 recommends %DNA in tail). Cytotoxicity as a potential confounder of the read-out was not determined and is not discussed. However, in the repeated exposure experiment of the same study, sperm counts decreased by 75% compared to controls.

-            As generic comment on the suitability of the comet assay for germ cells, the OECD guideline 489 (2016) states: “Whilst there may be interest in genotoxic effects in germ cells, it should be noted that the standard alkaline comet assay as described in this guideline is not considered appropriate to measure DNA strand breaks in mature germ cells. Since high and variable background levels in DNA damage were reported in a literature review on the use of the comet assay for germ cell genotoxicity (Speit et al. 2009), protocol modifications together with improved standardization and validation studies are deemed necessary before the comet assay on mature germ cells (e.g. sperm) can be included in the test guideline.”

Comments specifically on the dominant lethal assay:

-            For the fertility assessment, data on general toxicity are missing. Mortality, clinical signs, and body weights are not specified. However, when interpolating the data from the sperm assessment, it can be seen that in this study, 60 days of treatment lead to reduced body weights in the male mice. The body weights were reduced by 10.2% compared to the initial body weight and were 21% lower, when compared to the vehicle control group. Similarly, in the sperm assessment, clinical signs are not specified, and the body weight data is only partially discussed. The body weights are only compared within the test groups (initial vs terminal), however, testis weight and sperm count/motility/morphological abnormalities are compared between groups. Comparison of the terminal body weights revealed that all treatment groups show distinctly lower terminal body weights (66.8-87% of the control group). Moreover, the absolute testis weights are found to be reduced. However, the relative testis weight is similar compared to the control animals. Only limited information was presented on the female animals.

Based on the above described deficiencies, the Registrant does not agree with the rating of the study by Altamirano-Lozano et al. as given in the CLH report as “reliable with restriction” (RL=2) but proposes to reduce the reliability to “not reliable” (RL=3) for both experiments reported.

 

Altamirano and Alvarez-Barrera (1996) investigated on the potential of vanadium pentoxide (V2O5) to induce chromosomal aberrations in male CD-1 mice treated via intraperitoneal injection. Moreover, the authors analysed the mitotic index (MI) and average generation time (AGT). The authors summarised that vanadium pentoxide (V2O5) induced no chromosomal aberration in bone marrow cells of CD-1 treated male mice. However, the publication presented herein shows major deficiencies in reporting and methodology. Thus, no conclusion on the genotoxicity can be drawn based on the results presented in this study.

 

Thus, for pentavalent V, reliable (RL=1) and GLP-conform in vivo data indicate a negative potential. However, reliable (RL=2) in vivo data also indicate positive threshold effects, but in these studies, the toxicological significance could not be properly assessed due to reporting and experimental deficiencies.

 

In vivo DNA damage studies

The GLP compliant study by Schuler (2010 and 2011) 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. 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 (0, 0.25, 1, 4 mg/m³). 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, receiving methylmethanesulfonate via oral route. 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 a 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, showing a structure typical for dead cells, complicating the macroscopic differentiation. Nevertheless, the validity of the study was not jeopardised, since the comet results showed a clear and statistically significant increase in DNA damage in cells treated with the positive control, after exclusion of the severely damaged cells (% tail intensity above 80%).

 

In the study by Leopardi et al (2005), the DNA damaging potential of sodium orthovanadate was evaluated in different tissues of CD-1 mice after short-term exposure via drinking water. The male CD-1 mice were exposed for 5 weeks to sodium orthovanadate at concentration levels of 7.5, 75, 750, and 1500 mg/L via drinking water. An untreated control group was run concurrently. Mice dosed with methyl methanesulfonate 24 hours before euthanasia served as positive control group. After euthanasia, splenocytes, bone marrow cells, testis cells, and epididymal spermatozoa were obtained and evaluated for DNA damage via the alkaline comet assay. A total of 100 cells per animal (n=3) and tissue were scored for the mean tail moment. Additionally, the authors investigated on DNA denaturation in epididymal sperm cells and the distribution of testis cell types. Furthermore, the elemental vanadium concentration was measured in the femur, tibia, spleen, liver, kidney, and testis using ICP-OES.

According to the authors, cells obtained from the bone marrow and testis of sodium orthovanadate-exposed mice did not show a statistically significant increase in the mean tail moment, when compared to untreated control mice. Moreover, the epididymal spermatozoa did neither show statistically significantly increased mean tail moment values nor increased DNA denaturation, as evidenced by the comet and sperm chromatin structure assay. In contrast, the high dose group showed a statistically significantly increased mean tail moment in splenocytes. The increase, however, is considered to be not dose related and associated with increased general toxicity. The increased general toxicity was evidenced by following observations: I) The water consumption of animals treated with 750 and 1500 mg/L sodium orthovanadate was markedly and statistically significantly decreased to 47.1 and 37.1% of the daily intake of control animals, respectively. The reduced water consumption was most probably due to effects on palatability. II) Moreover, the body weight gain was decreased by 40% and 41.1% in animals dosed at 750 and 1500 mg/L, respectively. III) Both dose groups were lethargic during the last two weeks of the treatment. IV) One animal of the high dose groups died prematurely during the third week of the treatment. The cause of the death was not specified. Premature deaths, however, clearly indicate that the maximum tolerable dose was exceeded.

Due to the unsuitable study design with the following major restrictions this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information. The MTD was exceeded at the highest concentration tested, which is not in accordance with in vivo genotoxicity test guidelines. The description of the test animals is insufficient, since details on housing conditions, food consumption, and the body weights at study initiation and termination are missing. The test material was insufficiently characterised, since information on the physical appearance and manufacturer are missing. Only three mice per group were evaluated for DNA damage, which is considerably low for robust statistical analysis. Moreover, potential cytotoxic effects were not evaluated for any of the tissues investigated except for the epididymal sperm cells. However, the cytotoxicity in sperm cells was not evaluated according to generally accepted scientific standards. The design of the comet assay deviated from standard recommendations. The electrophoresis duration was significantly low as recommended (7 vs. ≥20 minutes). Moreover, the alkaline solution had a considerably lower pH (12.1 vs. ≥13) which eliminates the expression of alkaline labile sites as single strand breaks. The time point of cell sampling and testing is not explicitly stated. Information on the occurrence and frequency of hedgehogs are not provided. Historical control data is not included. Evaluation and scoring criteria are not specified. The results are only depicted in diagrams, but not presented in a tabular format with means and standard deviations.

 

Villani, P. et al. (2007) Investigated on DNA damage induced by vanadyl sulphate pentahydrate in bone marrow and testis cells of CD-1 mice using the alkaline comet assay. Groups of eight to ten male mice were exposed for five weeks to vanadyl sulphate via drinking water. In the main experiment, the mice were exposed to concentration levels of 10, 100, 500, and 1000 mg/L. A confirmatory experiment was performed at concentration levels of 2 and 10 mg/L in order to exclude potential effects on low dose groups. Untreated and positive (methyl methanesulfonate) control groups were run concurrently. After euthanasia, the femoral bone marrow and testis cells were obtained and stained using ethidium bromide. A total of 100 nucleoids per tissue and animal was scored for the tail moment. Additionally, the elemental vanadium concentration was determined via ICP-OES.

The ICP-OES analyses revealed that elemental vanadium was found in all tissues examined, i.e. in the femur, tibia, liver, kidney, spleen, and testis. The vanadium concentration increased with the vanadyl sulphate concentration. In the second experiment at vanadyl concentration levels of 2 and 10 mg/L, elemental vanadium could be detected only in the femur and tibia. The values results observed for the spleen at testis in the low dose groups were considered to be of an artificial nature. The vanadyl sulphate exposed mice did not show any effects on food consumption, body weight gain, and absolute organ weights. However, the daily water consumption per mouse was statistically significantly to 65.1% and 60.0% of the concurrent control level in mice exposed to 500 and 1000 mg/L vanadyl sulphate, respectively. Moreover, one high dose mice died prematurely during the fourth week of the treatment. In the comet assay, the testis cells of vanadyl sulphate exposed mice showed neither a concentration related nor statistically significant increase in the tail moment values, when compared to untreated control mice. The femoral bone marrow cells showed a statistically significantly increased tail moment value in mice exposed to 10 mg/L vanadyl sulphate. However, the effect was neither concentration related nor confirmed in a repeat experiment. Thus, the finding observed at 10 mg/L is considered to be a chance finding. The positive control substance, methyl methanesulfonate, induced a statistically significant increase in the tail moment value when compared to the negative control group. Thus, the sensitivity of the test system was demonstrated.

Due to the unsuitable study design with the following major restrictions this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information. The methodology was only poorly described, as information on cell preparation, lysis, and electrophoresis are completely missing. The only information given are two different references dealing with the methodology applied. The description of the test animals is insufficient, since details on housing conditions, food consumption, and the body weights at study initiation and termination are missing. Moreover, information on clinical signs are not provided. Potential cytotoxic effects on testis cells were not evaluated. The evaluation and scoring criteria were not specified. Historical control data is not included. Information on the occurrence and frequency of hedgehogs are not provided. Historical control data is not included. The tail moment values observed in the positive control group are not specified.

 

Altamirano-Lozano et al. (1999) performed a comet assay to evaluate DNA damage after intraperitoneal injection of divanadium pentoxide solution in male CD-1 mice. Groups of four male mice received single doses of 5.75, 11.5, and 23 µg divanadium pentoxide/g bw. Twenty-four hours after treatment, the test animals were sacrificed, and several organs were dissected (liver, lung, heart, bone marrow, kidney, and spleen). Afterwards, one-hundred cells per animal were evaluated for DNA damage by analysis of DNA migration distance, proportion of DNA in head vs tail, and tailed cells vs untailed cells. The authors concluded that vanadium pentoxide (V2O5) induced DNA damage in several organs and tissues. The sensitivity to vanadium pentoxide-induced DNA damage varied between the organs. The publication presented herein shows some major reporting and methodological deficiencies as well as deficiencies in the study design. Thus, no conclusion on the genotoxicity can be drawn based on the results presented in this study. The test material was administered via intraperitoneal injection, which is considered as a non-physiological route of exposure. The publication contains some contradictory statements on dosing. Moreover, the laboratory’s proficiency in preparing and analysing comets was not demonstrated. Further, the publication shows some contradictions in the results section. A positive control group was not included. Cell viability not discussed in context of genotoxicity. The authors provided no information on the evaluation and scoring criteria. The methods section and the description of the test animals lacks details. The test substance is insufficiently characterised. The cell types used are not specifically stated. The results are partially unclear. The housing conditions deviated from standard in vivo test guidelines. The historical control data neither shown nor discussed.

 

in vivo studies using a non-physiological route of exposure

In vivo studies using the intraperitoneal route of administration are not to be considered equally relevant and adequate as studies using a physiological route of exposure. In contrast, an evaluation is to be conducted in-line with (i) the provisions laid down in regulation (EC) 1272/2008 (ii) the test guidelines specified in Article 13(3) of the REACH regulation and (iii) ECHA guidance (Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.4):

i.            the CLP Regulation states in Annex I: 3.5.2.3.9 “The relevance of the route of exposure used in the study of the substance compared to the most likely route of human exposure shall also be taken into account.”. For industrial chemicals, the inhalation, dermal and oral route are considered relevant for human exposure. The intraperitoneal injection may only be relevant for substances used in pharmaceutical applications, which is clearly not the case for inorganic vanadium substances. Consequently, only studies via physiological routes should be considered relevant as foreseen in the CLP Regulation.

 ii.           all recent OECD Test Guidelines for in vivo genetic toxicology testing (OECD 474, 475, 483, 488 and 489) state in section “Administration of doses” that “Intraperitoneal injection is generally not recommended since it is not an intended route of human exposure, and should only be used with specific scientific justification.”. As stated above, the intraperitoneal route may be relevant for pharmaceutical applications, but not for industrial chemicals. None of the studies using the intraperitoneal (IP) route described in the CLH report provides a justification for this route of exposure. Consequently, these studies should be considered non-compliant with the validated and accepted test guidelines and not relevant for the purposes of classification.

 iii.          the ECHA Guidance on IR & CSA, Chapter R.4: Evaluation of available information (2011) provides guidance on the reliability rating of studies (Chapter 4.2, page 3). Studies are considered not reliable in case organisms/test systems were used which are not relevant in relation to the exposure (e.g. non-physiological pathways of application).

 In a weight-of-evidence approach, studies using a non-physiological route of administration should therefore be considered to contribute to the overall assessment to a much lesser extent than information obtained respecting the relevant test guidelines and guidance documents.

Justification for classification or non-classification

Based on the available weight-of-evidence and considering guideline-conform studies conducted under GLP both in vitro as well as in vivo, the vanadium category substances should be considered void of genotoxicity.

Divanadium pentaoxide as well as other 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. 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 producing significant levels of cytotoxicity and only weak genotoxic responses. Conversely, in guideline- and GLP- compliant vivo tests, negative results were obtained via oral and inhalation route: (i) a study conducted by NTP (2002) involving in vivo exposure to a V2O5 aerosol for 3 months, which failed to elicit any clastogenic effects in mice, and (ii) a 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. Further negative results were obtained in a transgenic rodent assay, showing no increase in mutations in lung cells and tissue.

Based on the available weight-of-evidence, and considering guideline-conform studies conducted under GLP both in vitro as well as in vivo, the vanadium category substances should be considered void of genotoxicity. Thus, according to EC Regulation 1272/2008 no classification or labelling for heritable gene mutation is applicable.