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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 is generally considered to be limited. Negative findings in the Ames test must be qualified by recognition that uptake of ions for metals such as Sb by prokaryotic organisms is seldom measured and is generally considered to be quite limited (Kuroda et al., 1991, Zangi et al., 2012). Genetic resistance to antibiotics, often carried by DNA plasmids transmissible from one bacterial strain to another, can impart properties of a “metalloid pump” that actively reduces intracellular concentration of Sb ions (Xu et al., 1998). The presence of such a gene in a bacterial test strain would predispose to (potentially false) negative test results. Five structural proteins that can function as efflux transporters were initially identified and the metalloid binding domains of the transporter proteins established (Ruan et al., 2008). A sixth family of novel efflux transporters that will be activated by, and reduce intracellular concentrations of, Sb, has recently been identified (Shi et al., 2018). The multiplicity and prevalence of metal-resistance genes in bacteria is such that negative Ames tests in bacteria have limited significance unless it is demonstrated that Sb is taken up and retained by the test organisms.

 

Although gene mutations were not observed in bacterial mutation test, positive response were observed for Sb compounds in theB. subtilisrec 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. Sb2O5did not produce a response but also seemed to lack toxicity as evidenced by lack of a zone of inhibition resulting from Sb2O5treatment. 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 Sb2O5do not appear to have resulted in significant exposure to Sb 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 Sb 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 Sb compounds for forward mutation at the thymidine kinase (TK) locus of cultured L5178Y mouse lymphoma cells (Elliotet al., 1998; Stone 2010). 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 3+ ions. Finally, the 4 h treatment time employed was shorter than the 24 h exposure duration currently recommended by international guidelines (Mooreet al.,2002). Thus, while Sb trioxidewas not mutagenic, positive responses might have been induced by longer duration of chemical exposure or the study of more soluble Sb compounds that would yield higher Sb concentrations. Similarly, negative results were obtained in the testing of Sodium hexahydroxoantimonat (NaSb(OH)6) in the presence and absence of S9 using the microtiter fluctuation technique for the assay.

 

Elliotet al. (1998) also 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 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. Huanget 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 SbCl3treatment 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). 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 Sb treated cells generally lacked centromeres suggesting the occurrence of clastogenic events as opposed to aneuploidy. The concentrations tested (240 – 600 M) 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 Sb induced MN, although consistent with chromosome breakage, also raises technical concerns with respect to the majority of the micronucleus studies conducted of Sb compounds. Studies conducted to date have primarily relied upon Giemsa staining for micronucleus detection, a staining method that lacks specificity for DNA (Nersesyan 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. Giemsa) are used. There thus remains the possibility that inclusion body formation by Sb 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 Sb.

 

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.

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 trioxidechromosome 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 trioxideby 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 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 trioxideby 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 otherin vivostudies evaluating clastogenic effects of Sb compounds.

 

Other studies evaluating the genotoxic impacts of Sbin vivofollowed 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 trioxideadministered 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/m3Sb trioxide for two years (NTP, 2017). The NTP also conducted studies to evaluate the genotoxic effects of exposure to Sb trioxideafter 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/m3of 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. 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 Sb trioxidefor 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.

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 effectsin vitroremain(s) to be determined. There is no evidence that Sb ions undergo covalent interaction with DNA but the nature of interactions is influenced by valence state (Li et el., 2011). No interaction or binding of Sb 5+ with DNA was detected by Li et al. (2011). An apparent binding of Sb 3+ to DNA was observed, with initial binding of Sb 3+ to the ribose group of guanosine. This binding action is generally slow to take place and may have a covalent basis. In binding to ribose, Sb 3+ does not alter the base pair specificity of guanine and primarily poses an impediment to the efficacy of DNSA polymerase function during replication or DNA repair. 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. The NTP inhalation cancer bioassays of Sb trioxide (NTP, 2017) observed activation of the EGFR oncogene in a number of mouse lung tumors and “fingerprinted” the DNA sequence changes presumed to be responsible for activation. The observed changes were not characteristic of DNA sequence changes associated with oxygen radicals. A high frequency of G to T transversions was not observed in activated oncogenes, the DNA sequence alteration that is most commonly associated with interaction of active oxygen species with DNA to form 8-hydroxyguanine and a subsequent G to T transversion (Tchouet al., 1991; Hong et al., 2016). Oncogene activation in mouse lung tumors may thus result from events unrelated to oxidative stress and/or may not be the critical event by which Sb trioxideinduces mouse lung tumors.

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 understand the mechanism(s) by which Sb compounds produce positive response in some in vitro test systems, and the chemical species involved in such a response. The following hypothesis have been put forward based on the available genotoxicity information: 

 

Most genotoxicity studies have been conducted using soluble Sb in the form of trivalent Sb trichloride and the assumption made that any activity observed in various test systems could be attributed to the release of the electrophilic Sb ion via hydrolysis to yield Sb(OH)3. The behavior of trivalent Sb compounds in solution is likely to be complex and involve the sequential formation of Sb oxide chloride (SbOCl), Sb oxide hydroxide (SbO(OH) and ultimately the formation of Sb trioxide (Sb2O3) (Hashimoto et al., 2003). The pentavalent Sb pentachloride is similarly an electrophilic oxidizing agent which, as a function of pH, will also undergo a series of hydrolytic transformations to oxychlorides and oxide hydroxides that result in the formation of Sb2O5(Zheng, Zhi and Chen, 2006). Although the chemical moiety that might be responsible for producing a possible genotoxic response is uncertain, hydrolysis products are the likely mediator for positive test responses.

 

As oxyanions,Sb hydrolysis products would also be expected to undergo electrophilic interactions with cellular constituents such as thiol rich proteins(Verdugo et al., 2017). 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 Sb compounds have not routinely employed such high affinity staining procedures. There is an element of uncertainty associated with the interpretation of existing micronucleus studies.

 

Direct covalent interaction of Sb 5+ with DNA has not been detected (Li et al., 2011) althoughbinding of Sb 3+ to the ribose of guanosine appears to occur, leading to suggestions that interference with the function and/or fidelity of DNA polymerases may be impacted. 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 may thus be mediated by indirect impacts upon DNA repair. The relevance of these in vitro observations to in vivo exposure scenarios is uncertain, since the 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 exposure directly to Sb compounds (e.g. inhaled material in the lung).

 

De Boeck et al. (2003) suggest that thegeneration of oxygen radicals constitute another indirect pathway for inducing genotoxic responses. 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 (Tirmensteinet al. 1995). Finally, Jianget al.(2016) have observed that apoptosis induced by Sb appears to be a response to the generation of active oxygen species.

 

Mechanistic aspects of Sb genotoxicity have recently been evaluated in the “ToxTracker” assay, a novel test system that employs mammalian stem cell lines containing fluorescent reporters that respond to the expression of genes expected to be induced following the direct induction of DNA damage, oxidative stress and protein damage (Hendriks, 2017). None of the Sb compounds tested yielded responses indicative of direct DNA damage. However, all but two compounds (Sb pentoxide and sodium hexahydroxoantimonate)were found to be strong inducers of oxidative stress. All but three (sodium antimonate, Sb pentoxide and sodium hexahydroxoantimonate) exerted protein damage that could reflect interference with DNA repair functions. Although still in validation, the ToxTracker assay has generated a response profile for all Sb substances registered under REACH consistent with theinduction of genotoxicity though indirect mechanisms that entail oxidative stress or protein damage. 

 

If reactive oxygen species mediate most in vitro observations of genotoxicity, this could explain why most in vivostudies have not observed genotoxicity. Anti-oxidant system in an intact animal are robust and would mitigate against oxidative damage. 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 exposure (Cavallo et al., 2002).

 

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. The NTP inhalation cancer bioassays of Sb trioxide (NTP, 2017) observedactivation of the EGFR oncogenein a number of mouse lung tumors and “fingerprinted” the DNA sequence changes presumed to be responsible for activation. The observed changes were not characteristic of DNA sequence changes associated with oxygen radicals. A high frequency of G to T transversions was not observed in activated oncogenes, the DNA sequence alteration that is most commonly associated with interaction of active oxygen species with DNA to form 8-hydroxyguanine and a subsequent G to T transversion (Tchouet al., 1991; Hong et al., 2016). Sb 3+ binding to the ribose of guanosine also does not appear to impact the nature of mutations observed. Oncogene activation in mouse lung tumors may thus result from events unrelated to oxidative stress and/or may not be the critical event by which Sb trioxide induces mouse lung tumors.

 

In summary, the available data suggest that Sb 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.

 

More information on the weight of evidence and read-across approach for the assessment of the genotoxicity of Sb substances is attached to Section 13.

Justification for classification or non-classification

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

 

The assessment of the genotoxicity dataset on Sb substances concludes the following:

·       Sb 3+ in vitro genotoxicity: Sb 3+ compounds are clastogens but do not induce point mutations in vitro

·       Sb 3+ in vivo genotoxicity: Sb trioxide is not genotoxic in vivo

·       Sb 3+ overall genotoxicity: Sb 3+ compounds are not genotoxic

·       Sb 5+ in vitro genotoxicity: Sodium hexahydroxoantimonate is not genotoxic in vitro, there is no binding with DNA

·       Sb 5+ in vivo genotoxicity: Sb 3+ evidence used to infer that Sb 5+ are not genotoxic in vivo

·       Sb 5+ overall genotoxicity: Sb 5+ compounds are not genotoxic.

 

There is some indication of consistency that neither Sb 3+ compounds, nor Sb 5+ compounds (including Sb pentoxide) are genotoxic. The consistency will be confirmed upon assessment and consideration of the evidence which will be completed with the following research options:

·       In vitro micronucleus study to verify the possible micronucleus staining artifacts caused by non-DNA-specific stains

·       In vitro assay on respiratory cells to understand the mechanism of action yielding positive genotoxicity results in vitro but negative genotoxicity results in vivo, and to inform on subsequent in vivo research needs

·       One or more in vivo studies to validate the hypothesis developed on the basis of the in vitro evidence, and understand the extent and significance of the transformation (and methylation) and Sb compounds following absorption

 

Recently generated data from the ToxTracker assay has suggested compound-specific differences in the ability to induce oxidative stress and/or protein damage. It remains to be seen if the in vitro genotoxic properties of these compounds exhibit differences concordant with their ToxTracker response profiles. Effects that correlate with physical chemical properties such as solubility in aqueous cell culture medium would validate read-across strategies for in vitro genotoxicity.

 

The research strategy developed by the International Antimony Association, which supports REACH registrants with their Registration and Evaluation obligations, already foresees the above research options.