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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data on genetic toxicity are available for tetra- and pentavalent vanadium substances (including V2O5). The GLP conform, in vitro mammalian cell gene mutation tests by Loyd (2010) according to OECD 476 are considered key studies and will be used for classification. Representative vanadium candidates from the +4 and +5 valency groups, including divanadium pentaoxide, did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells (Loyd 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. Divanadium pentaoxide appears to express clastogenicity in vitro, but only at high concentrations.

Currently, Divanadium pentoxid has a harmonized classification as Muta. Cat 2 (H341) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 Annex VI.

Genetic toxicity in vivo

Description of key information

Data on genetic toxicity are available for tetra- and pentavalent vanadium substances (including V2O5).

Divanadium pentaoxide appears to express clastogenicity in vitro, but only at high concentrations, and is not mirrored in corresponding in vivo (RL=1) GLP-conform assays.

Currently, Divanadium pentoxid has a harmonized classification as Muta. Cat 2 (H341) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 Annex VI.

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

Additional information

The test substance Divanadyl pyrophosphate was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium, in a reverse mutation assay. In an AMES-test (Life Science Research, 1991; Validity 2) performed according to OECD guideline 471 and under GLP, the test substance was tested from 25 – 2500 µg/plate in the strains TA 1535, TA 100, TA 1537, and TA 98. An increase in the number of revertants was not observed either without S-9 mix or after the addition of a metabolizing system. A weakly bacteriotoxic effect was observed at doses > 2500 µg/plate.

In an in vitro cytogenetics assay using human lymphocyte cultures (Life Science Research, 1991; Validity 2), the test substance was tested both in the absence and presence of metabolic activation (S9 mix), according to the OECD Guideline 473 and in compliance with the principles of GLP. Tests were conducted with or without the inclusion of a rat liver-derived metabolic activation system (S-9 mix): without S-9 mix cells were exposed for 24 h to solvent vehicle or test substance at 0, 5, 10, 20, 40 µg/ml. With S-9 mix exposure was limited to three hours and cells were harvested 21 h later. Treatment with produced reductions in mean mitotic index of 46 and 54% at concentrations of 20 and 40 µg/ml, respectively in the absence of S-9 mix and 30% at 40 µg/ml in its presence. Some of the metaphases showed wide separation of the chromatids with no apparent link at the centromere. Metaphases affected in this way could not be scored for chromosomal aberrations. No statistical significant increases in the frequency of metaphases with chromosomal aberrations, compared to corresponding untreated control values, were observed for any group treated, whether gaps were included or excluded.

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 as well as in vivo, vanadium substances, including sodium metavanadate, should be considered void of genotoxicity.

Read-across: The read-across approach based on dissolved vanadium is based on the assumption that once inorganic vanadium compounds dissolve or become bioavailable, this will be in tetra- or pentavalent vanadium forms.In bioaccessibility tests of tetra- and pentavalent vanadium substances, tetra- and pentavalent forms dissolved completely within 2h in various media selected to simulate relevant human-chemical interactions (i.e. PBSmimickingthe 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.

In vitro gene mutation assays

Testing in bacteria reverse mutation assays (NTP 2002, Wolf 2006), although of limited relevance for metals (HERAG, 2007), yielded negative results:

- 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).

- 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.

Guideline-conform in vitro mammalian cell gene mutation tests according to OECD 476 conducted by Loyd (2010a,b) under GLP are considered as key studies and will be used for classification. In these studies, soluble tetra- and pentavalent vanadium substances, including divanadium pentaoxide, 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 vivo gene 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

Tetravalent vanadium substance

In the key study by Loyd (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 Loyd (2010),

- divanadium pentaoxide showed 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%).

- Divanadium pentaoxide showed 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).

- Vanadium pentaoxide was 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 Loyd (2010) are true effects based on VOSO4or V2O5induced 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 vitrowith soluble vanadium substances. 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.

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 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

Micronuclei in peripheral blood lymphocytes & bone marrow - mice

 

p.o. via drinking water - 5 wk

Vanadyl sulphate - IV

0.39 – 30

0.10 – 0.40

Negative –RL = 2

Villani et al, 2007

Micronuclei in peripheral blood lymphocytes & bone marrow - mice

 

p.o. via drinking water - 5 wk

Sodium orthovanadate - V

0.06 – 5.49

20.8– 33

Negative–RL = 2

Positive –RL = 2

Leopardi et al, 2005

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 bone marrow - mice

i.p. – once, examined after 30h

Ammonium metavanadate - V

10.19

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*

chromosome aberration in bone marrow, 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 reliable in vivo data indicate a negative potential for tetravalent V, unreliable in vivo data appear to report the contrary.The in vivo genetic toxicity of V2O5was 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. Furthermore, the genetic toxicity of divanadium pentaoxide was assessed by testing the ability of V2O5to 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, 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).

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.

References:

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

Justification for classification or non-classification

Currently there is limited substance specific data on Divanadyl pyrophosphate for mutagnicity available. However data from tetra- and pentavalent vanadium source compounds (e.g. Divanadium pentoxide (CAS 1314-62 -1)) can be taken into account for hazard assessment.

Based on the available data and according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 Divanadyl pyrophosphate

has to be classified as Muta. Cat 2 (H341).