Diantimony trioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Cf. Scientific opinion on genotoxicity in Section 13 for complete weight of evidence and read-across assessments.

 

Prokaryotic Test Systems

 

Tests using prokaryotic systems generally provide negative responses for mutagenicity, but interpretation of this negative finding must be qualified by recognition that uptake of ions for metalloids such as Sb by prokaryotic organisms has traditionally been considered to be limited. However, as evidenced by cytotoxicity, the negative Ames test data do not appear to be false negatives related to compound uptake failure.  

Although gene mutations were not observed in bacterial mutation test, positive response were observed for Sb compounds in the B. subtilis rec assay for DNA damage.  This “indicator assay” assesses increases in recombination events that are most likely the result of DNA damage induced by chemical treatment.  Although positive, the rec assay results must be regarded with caution since recombination/repair systems are complex and are subject to modulation by metal ions. In a comparison between the results from the two test systems, negative Ames test mutation data are assigned priority over data from the recombination indicator assay (Boreiko and Rossman, 2020).

 

In Vitro Tests with Mammalian Cells

 

One study evaluated Sb trioxide compounds for forward mutation at the thymidine kinase (TK) locus of cultured L5178Y mouse lymphoma cells (Elliot et al., 1998.;Table 2).  Sb trioxide, tested in the presence and absence of S9 for metabolic activation, failed to induce mutation after 4 h exposure.  Tested concentrations were nominal (i.e. not measured in the cell culture medium) and may have exceeded the aqueous solubility of the test compound.  Little cytotoxicity was observed, further suggesting limited release of Sb(III) ions.  Finally, the 4 h treatment time employed was shorter than the 24 h exposure duration currently recommended by international guidelines (Moore et al., 2002).  Thus, while Sb trioxide was not mutagenic, not all assay conditions were conducive to a positive response.

Elliot et al. (1998) examined the induction of chromosomal aberrations in cultured human lymphocytes at nominal Sb trioxide concentrations that ranged from 10 to 100 µg/ml.  Setting aside concerns over possible exceedance of solubility limits, a dose-dependent increase in chromosome aberrations was observed in the absence of cytotoxicity.  The nature of the aberrations was not explicitly described except to note that chromosome gaps had been excluded. Asakura et al. (2009) also reported that Sb metal powder induced chromosome aberrations.  However, no data on toxicity, doses used or aberrations observed was provided to permit evaluation of this claim.

Given the observation of chromosome aberrations, it is not surprising that studies have reported that treatment with Sb compounds (usually SbCl₃) is associated with micronucleus (MN) induction in a variety of different cell types.  Huang et al. (1998) observed MN induction in a series of studies using Chinese hamster ovary cells, human bronchial epithelial cells and human fibroblasts.  MN induction was concentration-dependent and, at higher concentrations, associated with significant cytotoxicity that significanltly exceeded that permitted under current GLP guidelines.  The authors further observed an influx of calcium into cells after SbCl₃ treatment followed by time-delayed apoptosis and DNA fragmentation.  Calcium influx was noted to potentially be an indication of oxidative stress and to provide a mechanistic pathway for DNA damage via indirect pathways.  Induction of apoptosis was similarly noted to provide an additional pathway for DNA damage to occur independent of direct Sb ion interaction with DNA.  Both mechanisms of actions would be expected to exhibit non-linear dose-response functions (i.e. thresholds). Similar dose-dependent increases in MN induction were observed in V79 cells (Gebel et al., 1998) and cultured human lymphocytes (Schaumloffel and Gebel, 1998). Recently completed study of antimony metal powder and antimony(III) compounds (Simar, 2021) similarly have found weak Mn induction in studies that adequately controlled cytotoxicity to appropriate limits.

The study of Sb compounds in indicator assays yields positive results. Sister chromatid exchange induction and Comet assay results have been generated most frequently but the quality of most studies is low.  Both assays require careful monitoring of, and control for, cytotoxicity, terminal differentiation and/or apoptosis to permit meaningful interpretation of results.  Most studies have failed to implement proper controls for these sources of experimental artifact and have been excluded from consideration here.  Moreover, given the preponderance of positive micronucleus data, indicator assay data adds little to a weight of evidence evaluation.  Indicator assay data considered but excluded from evaluation here are summarized in the CSRs.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

 

Cf. Scientific opinion on genotoxicity in Section 13 for complete weight of evidence and read-across assessments.

 

Gurnani et al. (1992) evaluated the effects of single and repeated doses of Sb trioxide chromosome aberrations in mouse bone marrow.  Oral gavage of 400 -1000 mg/kg in a single dose, followed by analysis of chromosome aberrations after dosing did not detect an increase in aberration frequency.  In a repeated dosing protocol, mice were exposed to 400, 667 and1000 mg/kg Sb trioxide by oral gavage for up to 21 days and animals sacrifice at 7, 14 and 21 days for evaluation of chromosome aberrations.  Day 21 evaluations were restricted to the 400 and 667 mg/kg dosing group since lethality occurred on day 20 in the 1000 mg/kg treatment group.  The authors reported a variety of chromosome alterations including chromatid gaps and breaks, polyploid cells and “centric fusions” that increased as a function of dose through day 7 and 14 and then declined at day 21.  Presentation of the data is less than straightforward and statistical evaluations were conducted after pooling of data for aberration types that should have been evaluated independently (e.g. chromatid breaks and polyploid cells should have been evaluated separately).  Kirkland et al. (2007) have noted a number of deviations from GLP protocols in the conduct of the study of Gurnani et al. (1992), questioned the purity of the test substance used and noted irregularities in the nature of the chromosomal changes observed (i.e. breaks and centric fusions should have been associated with chromosome fragments but were not).  The study deficiencies are significant and indicate a need for validation from other studies.  A later publication by Gurnani et al. (1993) would at first seem to provide confirmation of Gurnani et al. (1992) but, as also noted by Kirkland et al. (2007), is merely republication of the data originally published in 1992.  Gurnani et al. (1993) has thus been excluded from Table 4 since it is not a new study.

Kirkland et al. (2007) mirrored the protocols of Gurnani et al. (1992) in a study of male and female rats administered 250, 500 and 1000 mg/kg Sb trioxide by oral gavage for 21 days. Six male and six female rats were included in each treatment group and the protocol included a positive control treatment group (lacking in the Gurnani et al., 2002 study). Treatment with Sb trioxide produced few signs of clinical toxicity other than a modest reduction in weight gain in the highest dosing group.  Additional toxicokinetic studies confirmed both the uptake of Sb into the blood and the presence of Sb in bone marrow.  Animals were then evaluated for the induction of both bone marrow chromosome aberrations and micronuclei in polychromatic erythrocytes on day 22. No treatment-related increases in chromosome aberrations or micronuclei were observed. This study strongly adhered to GLP guidelines and possesses technical rigor superior other in vivo studies evaluating clastogenic effects of Sb compounds.

Other studies evaluating the genotoxic impacts of Sb in vivo followed protocols limited in scope.  Elliot et al. (1998) examined the impacts of a single 5000 mg/kg oral gavage Sb trioxide dose upon micronucleus induction.  No evidence was obtained for micronucleus induction but the use of only a single treatment and one dose limits the significance of this negative finding.  The same authors also examined the induction of unscheduled DNA synthesis in rat liver after a single dose of Sb trioxide administered by oral gavage at doses of 3200 and 5000 mg/kg.  No treatment-related impacts upon unscheduled DNA synthesis were observed.

The National Toxicology Program of the United States (US NTP) recently conducted inhalation cancer bioassays upon rats and mice, exposing animals to 3, 10 and 30 mg/m³ Sb trioxide for two years (NTP, 2017).  The NTP also conducted studies to evaluate the genotoxic effects of exposure to Sb trioxide after one year of inhalation exposure.  Flow cytometric procedures were also applied to enumerate induction of micronuclei in the erythrocytes and white blood cells from rats and mice. Increased micronuclei were not observed in cells from rats but a low level of micronucleus induction was observed in mouse erythrocytes. The incidence of micronuclei increased in both male and female mice generally increased in a dose-dependent fashion but the response magnitude was small.  For example, normochromatic erythrocytes exhibited an average of 1.04 micronuclei per 1000 cells in controls, increasing to a maximum of 1.38 per 1000 cells in female mice exposed to 30 mg/m³ of Sb trioxide.  This level of response is statistically significant by virtue of 1,000,000 cells having been scored but would not have been detectable or significant without the application of flow cytometry to screen large numbers of cells.  While the response observed may be statistically significant, the biological significance of the response is unclear.

Other laboratories have observed that conditions which accelerate or perturb erythropoiesis produce small increases in erythrocyte micronuclei.  Thus, induction of anemia by blood loss or dietary iron restriction causes modest increases in micronucleus incidence - generally accompanied by the appearance of immature reticulocytes in the blood (Tweats et al., 2007; Molloy et al, 2012).  The pulmonary toxicity of Sb trioxide produced hypoxia and bone marrow hyperplasia that perturbed erythropoiesis as evidenced by increased prevalence of immature reticulocytes in the blood of mice.  Although NTP (2017) interprets the induction of micronuclei in mice as evidence of genotoxicity, the small magnitude of the response and evidence of disturbed red blood cell production indicates that designation of this as a positive response is not inappropriate.

Lung tissues from a separate cohort of rats and mice exposed to Sb trioxide for 12 months were analyzed for DNA damage by the Comet assay.  No DNA damage was observed in exposed rats while positive assay responses are reported for cells within mouse lung tissue.  Although the NTP report does not attribute great significance to the positive Comet assay results, it must be noted that the protocols employed for conduct of the Comet assay do not meet current minimal quality standards (Speit et al., 2015).  Application of the Comet assay to intact tissues must carefully control for natural process that can produce DNA fragmentation and false positive assay outcomes.  Cytotoxicity, apoptosis and terminal differentiation must all be carefully assessed for their impact upon assay outcomes.  The study controlled for none of these sources of artifact, casting doubt upon the significance of the modest positive response observed in mice.  Lack of genotoxicity in rats remains a significant observation since the uncontrolled sources of experimental artifact would create false positive assay response and would not mask genotoxicity to create a false negative response.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

 

Cf. Scientific opinion on genotoxicity in Section 13 for complete weight of evidence and read-across assessments.

 

The mechanism(s) by which Sb compounds exert genotoxic effects in vitro remain(s) to be determined.  There is no evidence that Sb ions undergo covalent interaction with DNA.   Genotoxicity is thus believed to involve indirect mechanisms.  De Boeck et al. (2003) suggest that the generation of oxygen radicals constitute an indirect pathway for inducing genotoxic responses.

Not all evidence supports oxidative stress as a mechanism for Sb genotoxicity.  Shaumloffel and Gebel (1998) did not observe attenuation of Sb induced Comet assay responses by the exogenous addition of superoxide dismutase or catalase, but it is not clear whether the positive Comet assay results reported were artifacts of cytotoxicity or apoptosis. 

Additional information

 

Cf. Scientific opinion on genotoxicity in Section 13 for complete weight of evidence and read-across assessments.

 

In order to assess the potential genotoxicity of Sb substances, it is important to initiate definition of the mechanism(s) by which Sb compounds produce positive response in some in vitro test systems, the speciation of the Sb(III) moiety responsible for the induction of genotoxicity and the ease with which genotoxic Sb forms can be generated in aqueous environments. The following hypotheses have been put forward based upon the available genotoxicity information and knowledge of the behavior of Sb in aqueous environments:

Most genotoxicity studies have been evaluated using soluble Sb in the form of trivalent Sb trichloride and the assumption made that any genotoxic activity observed could be attributed to the release of the electrophilic Sb ion via hydrolysis to yield Sb(OH)₃ or the oxyanion Sb(OH)₄ .The behavior of trivalent Sb compounds in solution is complex and, depending upon pH or the presence of other ions, can entail the formation of Sb oxide chloride (SbOCl), Sb oxide hydroxide (SbO(OH) and ultimately the formation of Sb trioxide (Sb₂O₃) (Hashimoto et al., 2003). A range of different oxyanions may thus be formed in solution by Sb metal or trivalent Sb compounds, with speciation being dictated by the properties of the surrounding aqueous environment and not the composition of the original parent compound. From this simplified perspective, all trivalent antimony compounds that release Sb(III) will have a similar potential for genotoxicity that can be indexed to the impacts of Sb release by from trivalent compounds. For purposes of read across, Sb metal and trivalent Sb compounds can be regarded as insoluble and functionally inert unless the release of Sb ions occurs. Released Sb(III) rapidly hydrolizes to form Sb(OH)₃, which then polymerizes to yield ring-like (SbOH₃)_3, the form of Sb taken up or excreted by cells by specific binding to the ArsB or GlpF proteins, highly conserved aquaglyceroporin channel analogs ubiquitous in eukaryotic and prokaryotic cells (Meng et al., 2004). The stereospecificity of the aquaglyceroporin transporters is highly selective, making SbOH₃/(SbOH₃)₃ the predominant form of Sb taken up into cells and most likely to mediate manifestations of toxicity by most (if not all) Sb(III) compounds.

The assumption that Sb(OH)₃ (or its polymer) is the toxophore that mediates Sb toxicity is supported by the aquaglyceroporin transport mechanisms responsible for the uptake of antimony (III) into cells. Aquaglyceroporins are ubiquitous transporter systems highly conserved in bacteria and animals and provide the main transport pathway for the entry of Sb into a cell in vivo or in vitro. (Boreiko and Rossman, 2020). As is true for most metal/metalloid transporters, aquaglyceroporins exhibit strong specificity for the chemical composition and stereochemistry of the molecular moieties that they will bind and subsequently transport in or out of a cell. Sb(OH)₃ displays the strong Sb binding for the metalloid transporter system and is the probable “toxophore” taken up by cells to yield toxic effects from different Sb(III) compounds. Indirect evidence for the identity of this common toxophore is further derived from the cytotoxicity dose response functions for Sb derived from Sb metal or trivalent antimony compounds. For example, the LC50’s, indexed to solubilized Sb(III), for Sb derived from Sb metal powder, diantimony trioxide, antimony trisulphide, antimony trichloride and antimony tris(ethylene glycolate were found to be 0.54, 0.72, 1.0 and 0.51 mg/ml, respectively. Such tight clustering of LC50s for antimony released from chemically diverse substances supports the role of a common toxophore (e.g. Sb(OH)_3- in the induction of antimony toxicity.

Direct covalent interaction of Sb with DNA has not been detected although ionic interactions that interfere with the fidelity of DNA replication or repair are plausible. Consistent with this are impacts of Sb upon the repair of DNA strand breaks (Beyersmann and Hartwig, 2008; Koch et al., 2017) and excision repair (Grosskopf et al., 2010). Genotoxicity responses might thus be mediated by indirect impacts upon DNA repair that permit the persistence of promutagenic lesions that will result in mutations following cell division. However, the relevance of these in vitro DNA repair impacts to in vivo exposure frameworks is uncertain since the Sb concentrations required to produce effects in vitro are generally significantly higher than plausible systemic levels of Sb in vivo. However, such concentrations may be within the range of feasibility for tissues experiencing direct or local exposure to Sb compounds (e.g. inhaled material deposited in the lung). 

De Boeck et al. (2003) emphasize that there may be more than one pathway through which a toxicant can result in the generation of oxygen radicals. Supportive evidence for this is derived from the calcium influx studies of Elliot et al. (1998). The cytotoxic effects of potassium antimony tartrate upon cardiomyocytes also appears to be associated with the generation of oxygen radicals (Tirmenstein et al. 1995). Finally, Jiang et al. (2016) have observed that apoptosis induced by Sb appears to be a response to the generation of reactive oxygen species. Multiple pathways thus exist whereby Sb might promote ROS induction or interfere with the fidelity of DNA replication or repair.

If reactive oxygen species mediate most in vitro observations of genotoxicity, this could explain why the majority of in vivo studies have not observed genotoxicity. Antioxidant system in an intact animal are robust and would mitigate against oxidative damage. The expression of genotoxicity would be absent in vivo or exhibit a threshold with genotoxicity only resulting when the protective capacity of anti-oxidant systems is exceeded (Kirkland et al., 2015). Consistent with this is the lack of correlation between urinary Sb and oxidative biomarkers in humans (Domingo-Relloso, 2019) although others have suggested oxidative damage detected by the Fpg modified Comet assay is induced by occupational exposures other than to antimony (Cavallo et al., 2002).

In summary, the available data suggest that Sb hydrolysis products do not induce point mutations but that clastogenic events can result from in vitro exposures. In vivo evaluations of Sb genotoxicity have generally produced negative or, at best equivocal, results that vary as a function of study quality. Negative studies tend to be those possessing the highest technical rigor. Studies with positive or equivocal findings have significant technical deficiencies that raise uncertainty as to the reliability of the study results. Thus, whereas in vitro studies suggest genotoxic properties, there is little compelling evidence that this is expressed in vivo.

Justification for classification or non-classification

 

Cf. Scientific opinion on genotoxicity in Section 13 for complete weight of evidence and read-across assessments.

 

The available evidence currently indicates that:

• Sb(III)compounds are clastogenic in vitro but do not induce point mutations; indirect mechanisms of action are probable.


• Sb(III) substances are not genotoxic in vivo. Any genotoxicity observed (in vitro) is the result of indirect, thresholded, mechanisms most likely entailing changes in reactive oxygen species, valence state and ROS generation. Methylation of Sb occurs and may play a role in genotoxicity.


• The available data do not support classification for mutagenicity

Further Research Options: The ongoing combined study evaluating Comet assay and micronucleus responses in cells of the respiratory tract and liver of mice exposed to antimony trioxide by nose-only inhalation