Antimony pentachloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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 antimony by prokaryotic organisms is generally considered to be limited (Kuroda et al., 1991). Genetic resistance to antibiotics, often carried by DNA plasmids transmissible from one bacterial strain to another, can also impart properties of a “metalloid pump” that actively reduces intracellular concentration of antimony ions (Xu et al., 1998). The presence of such a gene in a bacterial test strain would predispose to false negative test results. Given these caveats, assays for reverse gene mutation in (e.g. the Ames test) have generally produced negative results (Table 1) for Sb(III) and Sb(V) compounds.

 

Although gene mutations were not observed in bacterial mutation test, positive response were observed for antimony 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. Sb₂O₅ did not produce a response but also seemed to lack toxicity as evidenced by lack of a zone of inhibition resulting from Sb₂O₅ treatment. The authors attributed this to limited solubility of the pentoxide but data to substantiate this are not presented. Independent of the reasons, the rec assay results for Sb₂O₅ do not appear to have resulted in significant exposure to antimony ions. The authors further hypothesized that the difference in response in the two bacterial test systems might have been produced by differences in compound uptake or toxicity in the two bacterial strains. False negatives would result in the Ames test if inadequate antimony uptake occurred, whereas false positives can occur in the rec assay if cytotoxicity results in lysosomal nuclease release. In the absence of information that discriminates between these alternate hypotheses, response inconsistency between the bacterial test systems, and between compounds in the rec assay, make it difficult to derive definitive conclusions regarding mutagenicity or genotoxicity from studies using bacteria.

 

In Vitro Tests with Mammalian Cells

 

Two studies have evaluated antimony compounds for forward mutation at the thymidine kinase (TK) locus of cultured L5178Y mouse lymphoma cells (Elliot et al., 1998; Stone 2010). Sb₂O₃, tested in the presence and absence of S9 for metabolic activation, failed to induce mutation after 4 h exposures. 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 Sb₂O₃ was not mutagenic, positive responses might have been induced by longer duration of chemical exposure or the study of more soluble antimony compounds that would yield higher antimony concentrations. Similarly, negative results were obtained in the testing of NaSb(OH)₆ in the presence and absence of S9 using the microtiter fluctuation technique for the assay.

 

Elliot et al. (1998) also examined the induction of chromosomal aberrations in cultured human lymphocytes. at nominal Sb₂O₃ 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.

 

Given the finding of chromosome aberrations, it is not surprising that studies have reported that treatment with antimony 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. 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 antimony 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). Finally, Migliore et al. (1999) observed strong dose dependent induction of micronuclei in cultured lymphocytes from two human volunteers following in vitro treatment with KSbO₃ (potassium antimonate). Fluorescence in situ hybridization was used to examine micronuclei for the presence of centromeres – micronuclei in antimony treated cells generally lacked centromeres suggesting the occurrence of clastogenic events as opposed to aneuploidy. The concentrations tested (240 – 600 µM) are within the range expected for a moderately soluble compound but higher than others have reported as being possible in cell culture medium.

 

The absence of centromeres in antimony induced MN, although consistent with chromosome breakage, also raises technical concerns with respect to the majority of the micronucleus studies conducted of antimony compounds. Studies conducted to date have primarily relied upon Geimsa staining for micronucleus detection, a staining method that lacks specificity for DNA (Nersesyan[C1] et al., 2006). Metalloids such as arsenic have recently been reported (Wedel et al, 2013; Cohen et al., 2013), presumably due to electrophilic interaction with thiol groups on proteins and other macromolecules, to produce cytoplasmic inclusions bodies that can be mistaken for micronuclei if non-DNA specific stains (e.g. Geimsa) are used. There thus remains the possibility that inclusion body formation by antimony may have produced staining artifacts misinterpreted as micronuclei. Further research would be required to determine if this potential source of experimental artifact is applicable to antimony.

 

The study of antimony 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.

Genetic toxicity in vivo

Description of key information

Gurnani et al. (1992) evaluated the effects of single and repeated doses of Sb₂O₃ 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 doing protocol, mice were exposed to 400, 667 and1000 mg/kg Sb₂O₃ 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.

 

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 Sb₂O₃ 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., 1992 study). Treatment with Sb2O3 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 antimony into the blood and the presence of antimony 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 antimony compounds.

 

Other studies evaluating the genotoxic impacts of antimony in vivo followed protocols limited in scope. Elliot et al. (1998) examined the impacts of a single 5000 mg/kg oral gavage Sb₂O₃ 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 Sb₂O₃ 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 recently conducted inhalation cancer bioassays upon rats and mice, exposing animals to 3, 10 and 30 mg/m³ Sb₂O₃ for two years (NTP, 2017).   The NTP also conducted studies to evaluate the genotoxic effects of exposure to antimony trioxide after one year of inhalation exposure. Sensitive 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/m3antimony 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 antimony 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. Indeed, as acknowledged by NTP (2017) an independent Peer Review Panel had evaluated the genotoxicity study results and indicated that evidence of genotoxicity was lacking in the NTP studies.

 

Lung tissues from a separate cohort of rats and mice exposed to antimony 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.

 

Considering the available genotoxicity data, antimony trioxide does not induce gene mutations in vitro, but has been shown to induce structural chromosome aberrations in cultured mammalian cells in vitro. However, negative in vivo results on induction of chromosome aberrations and/or micronuclei were obtained in two different species – mouse (Elliot et al., 1998) and rat (Whitwell, 2006), (Kirkland et al., 2007). An in vivo UDS assay in rats was also negative (Elliot et al., 1998). These tests were performed according to GLP and using OECD test guideline protocols and oral administration. According to a recent guideline-conform toxicokinetic study (De Bie, 2005) conducted under GLP at a dose of 1000 mg/kg as used in these mutagenicity studies, tissue distribution data confirmed that the bone marrow was exposed. It can therefore be concluded that diantimony trioxide (and via read-across also antimony pentachloride) does not cause systemic mutagenicity in vivo after oral administration.

 

Human data

 

In the human study the genotoxicity of antimony trioxide in lymphocytes from occupationally exposed workers was assessed. No induction of micronuclei or sister chromatid exchanges could be seen between the two exposed groups and the unexposed control. In an enzyme-modified comet assay a significantly higher proportion of the workers in the “high exposure group” showed oxidative DNA damage in their lymphocytes compared to control. However, the workers were exposed to diverse chemicals and no monitoring was performed on the control group, therefore a correlation between the oxidative DNA damage and air concentration of antimony trioxide (and via read-across also antimony pentachloride) is uncertain.

Additional information

Antimony pentachloride hydrates in the presence of water/moisture, and then decomposes to either antimony trichloride and chlorine, antimony pentoxide and chlorine, or H₃SbO₄ and hydrochloric acid. Conditions under which these various reactions occur are not clear, but require each different pressure and moisture. Assuming that antimony pentachloride can yield Sb³⁺ ions under some sort of extreme conditions, and that Sb³⁺ ions are reported to be more toxic than Sb5+ ions, it is proposed to refer to mutagenicity information available from both trivalent and pentavalent antimony forms. This enables a conservative assessment of antimony pentachloride, which is extremely difficult to test due to its chemical instability.

Inorganic antimony compounds and antimony metal powder, have been evaluated for mutagenic and/or genotoxic potential in in vitro and in vivo test systems. Most studies have been conducted using soluble antimony in the form of trivalent antimony trichloride and the assumption made that any activity observed in various test systems could be attributed to the release of the antimony ion via hydrolysis to yield Sb(OH)₃. The behavior of antimony compounds in solution is likely to be complex (Hashimoto et al., 2003) and involve the sequential formation of antimony oxide chloride (SbOCl), antimony oxide hydroxide (SbO(OH) and ultimately the formation of antimony trioxide (Sb₂O₃). The chemical moiety responsible for producing a genotoxic response is thus uncertain. 

 

Antimony pentachloride is a strong oxidizing agent which, as a function of pH, will similarly undergo a series of hydrolytic transformations to oxychlorides and oxide hydroxides that result in the formation of Sb₂O₅. Once again, hydrolysis products are the likely mediator for positive test responses but the chemical moiety responsible for effects remains unknown.

 

The available data suggest that antimony hydrolysis products do not induce point mutations but that clastogenic events result from in vitro exposures. In vivo assessments of genotoxicity have generally produced negative or, at best, equivocal results. Several negative studies possess the highest technical rigor – those with equivocal findings have significant technical deficiencies. Thus, whereas in vitro studies suggest genotoxic properties, there is little evidence that this is expressed in vivo.

 

The mechanism(s) by which antimony compounds produce positive response in some in vitro test systems is not clear. Direct covalent interaction of antimony with DNA has not been detected, leading to suggestions that genotoxicity responses may be mediated by indirect mechanisms. De Boeck et al. (2003) suggest that the generation of oxygen radicals constitute an indirect pathway for inducing genotoxic responses. If reactive oxygen species mediate most in vitro observations of genotoxicity, this could explain why most in vivo studies have not observed genotoxicity. Anti-oxidant system in an intact animal are robust and would mitigate against oxidative damage. Expression of genotoxicity would with 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).

 

Antimony has also been suggested to interfere with DNA repair processes (Beyersmann and Hartwig, 2008) and this may facilitate genotoxic responses. Inhibition of repair of double strand DNA breaks (Takahashi et al., 2002) and excision repair (Grosskopf et al., (2010) have both been reported. However, the relevance of these observations to in vivo exposure scenarios is uncertain since the concentrations required to produce effects in vitro is significantly higher than plausible systemic levels of antimony in vivo.

 

Antimony hydrolysis products would also be expected to undergo electrophilic interactions with cellular constituents such as thiol rich proteins. Such interactions provide the mechanistic basis for intracellular inclusion body formation. Documented to form after exposure to metals and metalloids, such inclusion bodies can be mistaken for micronuclei unless staining procedures are employed that are specific for the presence of DNA (Wedel et al, 2013; Cohen et al., 2013). Studies of micronucleus induction after treatment with antimony compounds have not routinely employed such high affinity staining procedures. There is thus an element of uncertainty now associated with the interpretation of existing micronucleus studies.

Justification for classification or non-classification

Based upon the preceding, the following conclusions can be drawn:

1. Most antimony (III) compounds produce positive results when tested for genotoxicity in vitro. Clastogenic events, usually the formation of micronuclei, appear to be most commonly observed endpoint. Antimony (V) compounds have been tested with less frequency and available data are inconsistent.
2. Reliance upon genotoxicity data that is heavily weight towards the observation of micronuclei is potentially problematic. Recent studies have observed that some metals and metalloids (see Technical Annex) can form cytoplasmic inclusion bodies that can be mistaken for micronuclei if stains used for conduct of the assays are not highly specific for DNA (Wedel et al, 2013; Cohen et al., 2013).  It cannot be precluded that some reports of micronucleus induction by antimony compounds may reflect staining artefacts.
3. In vitro genotoxicity, assuming it occurs, is likely mediated by indirect mechanisms (e.g. induction of oxidative stress or interference with DNA repair processes). The available data do not permit discrimination between alternative mechanisms – nor do the mechanisms need to be mutually exclusive.
4. There is no technically sound evidence to suggest expression of genotoxic potential in vivo. This difference may reflect factors such as defense mechanisms against oxidative stress that block genotoxic insults. Alternatively, or in addition, the poor uptake efficiency and rapid excretion of antimony may prevent the attainment of systemic antimony concentrations capable of producing a genotoxic impact.
5. Based upon the negative in vivo data, there is no need to apply classification for mutagenicity.