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

Read-across: The read-across approach based on dissolved vanadium is based on the assumption that once inorganic vanadium compounds dissolve or become bioavailable, this will be in tetra- or pentavalent vanadium forms. In bioaccessibility tests of tri-, tetra- and pentavalent vanadium substances, tetra- and pentavalent forms dissolved 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). Tri-, tetra- and pentavalent vanadium substances are 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.

The registrant is of the opinion that the toxicity of vanadium, oxalate complexes is driven by the vanadium moiety for the following reasons: Oxalic acid is a dicarboxylic acid occurring in many plants and vegetables and as such part of the daily diet. It is produced in the body by metabolism of glyoxylic acid or ascorbic acid and is not metabolized but excreted in the urine. Based on the lack of any identified systemic human health effect at relevant exposure levels, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) does not specify an ADI for oxalic acid. Further, oxalic acid is unlikely to raise public health concerns because any use in food-producing animals is generally regarded as safe by many national authorities (JECFA).

Based on the above information, one can therefore safely assume that the oxalate anion in vanadium, oxalate complexes is not the driver of genetic toxicity of vanadium, oxalate complexes. It is concluded that only “vanadium” is further considered in the assessment of genetic effects of vanadium, oxalate complexes. Thus, read-across of genetic toxicity data from soluble tri-, tetra- and pentavalent vanadium substances is justified.

In vitro gene mutation assays

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

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

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

 

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

In vivogene mutation assays

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

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

In vitro clastogenicity assays

Trivalent vanadium substance:

In the key study by Lloyd (2010):

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

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

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

 

Tetravalent vanadium substance

In the key study by Lloyd (2010):

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

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

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

 

Pentavalent vanadium substance

In the key study by Lloyd (2010):

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

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

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

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

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

 

In vivo induction of micronuclei

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

In vivo genotoxicity test

Route - exposure

Substance - Valency

Dose

(mg V / kg bw d)

Result - Reliability

Reference

Micronuclei in bone marrow - rats

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

Divanadium pentaoxide - V

i) 16.8

ii) 33.6

iii) 50.4

iV) 67.2

Negative – RL = 1

Beevers, 2011

Micronuclei in peripheral blood - mice

Inhalation – 3 mo

Divanadium pentaoxide - V

0.5 – 9 mg/m3

Negative – RL = 1

NTP, 2002

DNA damage in BAL or pulmonary cells (Comet assay)

Inhalation – 16 d

Divanadium pentaoxide - V

0.1 – 2.2 mg/m3

Negative – RL = 1

Schuler, 2010

Schuler et al, 2011

Micronuclei in peripheral blood lymphocytes & bone marrow - mice

 p.o. via drinking water - 5 wk

Vanadyl sulphate - IV

0.4 – 30

0.1 – 0.4

Negative – RL = 2

Villani et al, 2007

BrdU-incorporation assay

i) Aneuploidy in sperm

ii) Micronuclei in bone marrow – mice

i.p. – once

i) examined after 22 d

ii) examined after 24h

 

Sodium orthovanadate - V

i) 1.4 - 7

ii) 0.3 - 7

i) Positive –RL = 2

ii) Negative – RL = 2

Attia et al, 2005

Micronuclei in peripheral blood lymphocytes & bone marrow - mice

 p.o. via drinking water - 5 wk

Sodium orthovanadate - V

0.1 – 5.5

20.8– 33

Negative – RL = 3

Positive – RL = 3

Leopardi et al, 2005*

Micronuclei in bone marrow - mice

i.p. – once, examined after 30h

Ammonium metavanadate - V

10.2

Negative – RL = 3

Wronska-Nofer et al, 1999*

Sister chromatid exchange

i.p. – once, examined after 24h

Divanadium pentaoxide - V

3.2 – 12.9

Negative – RL = 3

Altamirano-Lozano et al, 1993*

Micronuclei in bone marrow - mice

i.p. – once, examined after 18h

Sodium orthovanadate - V

1.4 – 6.9

Positive – RL = 3

Mailhes et al, 2003*

Micronuclei in bone marrow - mice

p.o. – once, examined after 4-48 h

i) Ammonium metavanadate – V

ii) Sodium orthovanadate – V

iii) Vanadyl sulphate - IV

i) 21.8

ii) 20.8

iii) 31.2

Positive – RL = 3

Ciranni et al, 1995*

 mice

p.o. – once, examined after 24-36 h

i) Ammonium metavanadate – V

ii) Sodium orthovanadate – V

iii) Vanadyl sulphate - IV

i) 21.8

ii) 20.8

iii) 31.2

Positive – RL = 3

Ciranni et al, 1995*

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

 

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

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

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

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

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

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

 

References:

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


Justification for selection of genetic toxicity endpoint
Data of the genetic toxicity are available for tri,- tetra,- and pentavalent substances (including V2O3, VOSO4, V2O5).

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

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

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

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

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

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