Ammonium trioxovanadate

Endpoint summary

Additional information

Ammonia/ammonium (i) is ubiquitous in the environment (air, soil and water) and as part of the natural nitrogen cycle intrinsically present in the human body due to a natural background in all dietary sources, and (ii) ammonia/ammonium is a normal constituent of all body fluids. The sources of endogenous ammonium include bacterial hydrolysis of urea and other nitrogenous compounds in the intestine, the purine-nucleotide cycle, amino acid transamination in skeletal muscle, and other metabolic processes in kidneys and liver. At physiological pH, it exists mainly as ammonium ion with reference serum levels just below 35 µmol/L. Any excess ammonia is excreted as urea, which is synthesised in the liver through the urea cycle. In consequence, because of its intrinsic nature as an endogenous physiological compound, any genotoxic effect of ammonium is not to be expected.Therefore, only the potential of the respective vanadium anion is addressed. 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 ammonium trioxovanadate, 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 vitrogene 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 Loyd (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 locusof 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 Loyd (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 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):

- 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 Loyd (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. 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 vivoinduction 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/m^3	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.

 

While reliablein vivodata indicate a negative potential for tetravalent V, unreliablein vivodata appear to report the contrary.Thein vivogenetic 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, 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).

Thus, for pentavalent V, reliable (RL=1) and GLP-conformin vivodata indicate a negative potential. However, reliable (RL=2)in vivodata 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 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 Loyd (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 (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. 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 yield 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 ammonium trioxovanadate with respect to mutagenic potential does not appear to be supported. Thus, according to EC Regulation No. 1272/2008, ammonium trioxovanadate should not be considered to have a mutagenic potential, and hence no classification or labelling is required.