Slags, steelmaking, vanadium

Endpoint summary

Additional information

Based on available water solubility data, "Slags, steelmaking, vanadium" is rather inert with respect to the dissolution of slag components in water with the exception of slightly soluble vanadium. Aluminum, magnesium, manganese, molybdenum and zinc dissolved at detectable concentrations but these concentrations are neglectable in any physiological meaning. Therefore, only the potential of the respective vanadium component is adressed. In this dossier, the endpoint genetic toxicity is not addressed by substance-specific information, but instead by read-across approach based on dissolved vanadium. This is related to the assumption that once inorganic vanadium compounds become bioavailable, this will be in tetra- or pentavalent vanadium forms. Thus, read-across of toxicity data from soluble tri-, tetra-, and pentavalent vanadium substances is justified.

 

In vitrogene mutation assays

Testing in bacteria reverse mutation assays (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 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 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 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, 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 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 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 peripheral blood - mice	Inhalation – 3 mo	Divanadium pentoxide - 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 pentoxide - 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 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, vanadium pentaoxide, administered to male and female mice, did not increase the frequency of micronucleated normochromatic erythrocytes in peripheral blood. Thus, for pentavalent V, reliable (RL=1) in vivodata 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]

Short description of key information:
Slags, steelmaking, vanadium is considered inert with the exception of slightly soluble vanadium. Representative soluble 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). 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. 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 a corresponding in vivo (RL=1) assay. Extrapolation of the effects of soluble vanadium substances to "slags, steelmaking, vanadium" is further complicated by its sparingly soluble nature. Slags, steelmaking, vanadium is poorly water soluble and rather inert. In conclusion, based upon a weight of evidence evaluation, genotoxicity is not an endpoint appropriate to be carried forward to risk characterisation. Similarly, classification of slags, steelmaking, vanadium with respect to mutagenic potential does not appear to be supported. Thus, according to Directive EEC 67/548 and to EC Regulation No. 1272/2008, slags, steelmaking, vanadium should not be considered to have a mutagenic potential, and hence no classification or labelling is required.

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, these in vitro studies of effects upon eukaryotic cells in vitro employed high concentrations of soluble vanadium producing significant levels of cytotoxicity and only weak genotoxic responses. A central issue that requires resolution is whether such in vitro results have physiological relevance by virtue of the mechanisms involved or the concentrations required to produce effects. For example, induction of genotoxic effects in cultured cells at dissolved vanadium concentrations in the µM or mM range would have limited relevance to in vivo exposures wherein the concentration of vanadium available for transfer to the soft tissues is in the nM range or lower.

 

In vitro assays for cytogenetic changes conducted with metals are quite often positive, even with some indications that aneuploidy induction may be common. Conversely, upon in vivo testing, most metals with very few exceptions yield negative results, leading to the conclusion that the in vitro results should not be over-interpreted since unphysiologically high concentrations as obtainable in in vitro test systems do not mirror in vivo physiological conditions. This is verified by 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.

 

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 a corresponding in vivo (RL=1) assay. Extrapolation of the effects of soluble vanadium substances to "slags, steelmaking, vanadium" is further complicated by its sparingly soluble nature. In conclusion, based upon a weight of evidence evaluation, genotoxicity is not an endpoint appropriate to be carried forward to risk characterisation. Similarly, classification of "slags, steelmaking, vanadium" with respect to mutagenic potential does not appear to be supported.Thus, according to Directive EEC 67/548 and to EC Regulation No. 1272/2008, "slags, steelmaking, vanadium" should not be considered to have a mutagenic potential, and hence no classification or labelling is required.

 

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