Introduction

Tooth pulp is protected from injurious elements by enamel, dentin, and periodontium; however, when these barriers are breached, microorganisms and substances may adversely affect the stability, causing inflammation (pulpitis) and even tissue death (pulp necrosis). To overcome this, the recommended procedures are pulpectomy, which involves surgical removal of all the material in the pulp chamber and root canal, or pulpotomy, which refers only to removal of the coronal portion of the pulp [1]. The root canal system is sealed with a filling material to prevent reinfection and growth of surviving microorganisms. The deposition of cementum is considered a desired healing response and a prerequisite for restoring a functional periodontal attachment. The effectiveness of the filling materials is critically dependent on its physical and chemical properties, however being biological safety a prerequisite for its clinical use [2].

Genotoxicity is a critical issue in determining the safety of agents that might contact biological structures and should be considered within a biological risk assessment process [3]. Mutagens or genotoxic substances induce DNA damage directly or indirectly through inactivation of enzymes involved in the maintenance of genome integrity. Mutagen-target interactions may result in different types of DNA damage (DNA adducts, alkali labile sites, strand breaks) that can be pre-mutagenic. Cellular mechanisms to overcome these harmful effects include protective antioxidant activity (mediating elimination/neutralization processes) and the removal of induced lesions by the DNA repair machinery [4]. Nevertheless, insufficient cell-protecting mechanisms and/or high DNA-inflicted damage result in the disruption of the replication and/or transcription processes hindering the cell self-repairing potential leading, ultimately, to cell apoptosis [5].

Several methodologies able of detecting genetic damage and/or mutations have been established and are approved by international regulatory agencies for validation of chemical agents commercially available. The potential health risks are thus elucidated as it has been established that genetic damage is intimately linked to diseases such as cancer [6]. Understanding the impact of exposure scenarios (dose, chronic, acute) on cancer risk is important but remains a scientific challenge. Dental materials persist in the oral cavity for long periods which imply that risk assessment is required to ensure the safety profile of such materials [7].

Due to the current demand of enhanced clinical performance of dental materials, the number of commercial products is continuously increasing. Physical properties, biocompatibility, sealing ability, adhesion, solubility, and antibacterial efficacy results are abundant for root canal sealers, and some reviews on those issues have been written [2, 8,9,10,11,12,13]. However, genotoxic stress as a reaction to endodontic sealers is also an important parameter to be assessed to validate the safety of biomaterials in clinical practice [14]. So far, limited reviews have reported about the genotoxicity of endodontic sealers [15].

The present review intends to provide detailed information on the genotoxicity of root canal sealers (RCS), displaying the reported results considering the sealer’s composition. A comprehensive literature search on “genotoxicity,” “mutagenicity,” “endodontic sealers,” and “root canal sealers” was performed on studies conducted between 1998 and 2020. In brief, a search of PubMed, MEDLINE, Embase, and Google Scholar for a plethora of articles was carried out using the aforementioned keywords. Case reports and articles not written in English were excluded from this review.

Genotoxicity as a biocompatibility requirement

ISO 10993-1 lists two components for biological response evaluation of biomaterials [16]. The first normative component requires a number of aspects, such as physicochemical information, cytotoxicity, sensitization, irritation or intracutaneous reactivity, material-mediated pyrogenicity, toxicity, implantation, hemocompatibility, genotoxicity, carcinogenicity, reproductive/developmental toxicity, and biodegradation. The second one provides suggestions and considerations. These aspects are combined together with the nature of the tissues and contact time to assist in the selection of an appropriate evaluation technique [14]. However, the clinical relevance of these assays is limited because they do not take into account the complexity of a living organism, as well as the clinical presentation of the apical region. Despite that assessment, it is also mandatory to determine the biocompatibility of a material within in vivo setting [16]. Of the above-mentioned aspects, cytotoxicity and genotoxicity are most commonly reported in the literature [10, 11].

Genotoxicity is defined as toxicity that affects DNA structure, i.e., the ability of a substance (genotoxin) to modify the chemical structure of DNA, causing DNA lesions [14]. However, not all genotoxins act directly on the DNA molecule. Some genotoxins interact with DNA repair proteins, increasing mutation rate, or mitotic spindle proteins, leading to chromosomal misaggregation or even with proteins involved in the cell cycle, increasing the proliferation rate [17]. Several assays are addressed to detect DNA damage, i.e., comet assay, sister chromatid exchange, detection of γH2AX or 32P-postlabeling assay, and its transition to mass spectrometry [18]. Once damaged DNA can mispair during replication, an alteration in nucleotide sequence can arise, characterizing the mutation, which can involve a single (point mutation), few base pairs or a whole chromosome. Mutagenicity can be assessed using the Ames test, cytogenetics, or micronucleus (MN) assay [19]. The aim of genotoxicity assays is the identification of probable mutagens.

Regulatory agencies require testing for biomaterials to be available in the market. However, multiple tests are needed to monitor all potential endpoints related to DNA damage or mutations [14]. Registration, Evaluation, Authorisation, and restriction of Chemicals (REACH) is a European Union (EU) regulation adopted to improve protection of human health and the environment from the risks posed by chemicals. REACH Annexures describe the requirements on genotoxicity, specifying the information that must be submitted for purposes of registration and evaluation [20]. A set of specifications for testing chemicals and also medical devices is established by the Organization for Economic Co-operation and Development (OECD) with appropriate model systems, methodologies, reference standards, and recommendations for statistical analysis [21]. For medical devices, ISO10993-33 provides guidance on tests to evaluate the potential genotoxicity, carcinogenicity, or reproductive toxicity [22]. Table 1 summarizes the methodologies used to assess materials genotoxicity according to EU/OECD/ISO10993-33 guidelines.

Table 1 Methodologies used to assess materials genotoxicity according to EU/OECD/ISO guidelines
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Results

Commonly used sealers are based on zinc oxide-eugenol (ZOE sealers), silicone (S sealers), glass ionomer cements (GIC sealers), methacrylate resin (MR sealers), epoxy resin (ER sealers), polyvinyl resin (PR sealers), calcium silicate (CS sealers), calcium hydroxide (CH sealers), and novel sealers (Table 2). Detailed information of the commercial sealers with reported results on genotoxicity is presented in the Supplementary material (Table S1).

Table 2 Endodontic sealers classified by composition
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The present survey addresses the information on the genotoxicity of available root canal sealers reported from 1998 to the present date. A total of 39 articles were included. Figure 1 shows the publication periodicity grouped over a 4-year time span. On average, 1.9 articles were published per year, with two peak periods (2006 to 2009, 9 articles; 2014 to 2017, 11 articles). The two more recent articles were available in 2018 and 2020. Table 3 presents an overview of the included studies in terms of the experimental protocol (cell line/type, exposure time, concentration range, genotoxicity assay) and the relevant results, considering the sealers’ groups. Most of the studies were performed in vitro; the comet assay and the micronucleus test were widely used and broadly applied to assess genotoxic and mutagenic effects, respectively. Relevant results are given below.

Fig. 1
figure 1

Number of articles that performed genotoxicity studies with root canal sealers grouped over a 4-year time span, from 1998 to the present

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Table 3 Overview of genotoxicity/mutagenicity studies addressing endodontic sealers
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Zinc oxide-eugenol-based sealers

Six articles addressed the genotoxicity of eight ZOE sealers (five of them published from 1999 to 2009, and the last one in 2016). One or two studies were performed for each sealer (Table 3). All sealers were considered genotoxic in the tested cell lines through different methods, i.e., comet assay [36, 40], DNA precipitation assay [37], proto-oncogenes expression [38], and micronuclei assay [39]. The Ames test was performed for Tubli-Seal and Endométhasone N, and the results were considered negative [35].

Silicone-based sealers

RoekoSeal demonstrated time-dependent positive genotoxicity results on V79 cells using comet assay [41], and GuttaFlow showed negative results performing chromosomal alteration analysis and comet assay in hPB lymphocytes [40].

Glass-ionomer cement-based sealers

Ketac Endo was not mutagenic on S. typhimurium strains [35]. Vitrebond was tested on CHO cells exhibiting a clear genotoxic effect in the hprt test and umu chromotest [42]. Additionally, the genotoxic compounds, being hydrophilic, leached out rapidly when in contact with saliva [42], and when tested on V79 cells using the MN test, results were also considered positive [43].

Resin-based sealers

The prototype of R sealers was developed as a bis-phenol resin using methenamine for polymerization; however, the product released formaldehyde during setting [74]. Alternatives were sought and variants include phenol-formaldehyde, resorcin-formaldehyde, and methylmethacrylate, which were strongly antibacterial however presenting shrinkage and discoloration and poor biocompatibility during setting. Genotoxicity results for resin-based sealers were considered for methacrylate resin (MR)-, epoxy resin (ER)-, and polyvinyl resin (PR)-based sealers and are summarized in Table 3.

MR sealers were involved in a variety of studies performed from 2009 to 2018, and a higher number revealed absence of genotoxicity. Several studies that compared single MR sealer (EndoRez) with multi-MR sealer (RealSeal) found no toxic effects on FMM1 cells and transfected COS-7 cells [47] and also hGF [44]; EndoRez caused low double-strand break (DSB) formation in PDL cells while RealSeal did not [46]. EndoRez also showed positive results on V79 cells [41, 45] and on PDL cells [46]. Epiphany, using the comet assay, showed negative effects on hPB lymphocytes [40] and hPB leucocytes [49]; however, results were positive with the micronucleus test on V79 cells [48]. Coming to RealSeal, only one study (out of six) reported dose- and time-dependent DNA damage, namely on BHK-21 cells using the comet assay [50], while others reported negative genotoxicity on several cell types [45, 49]. Hybrid Root Seal, a self-etching hydrophilic material as a result of the inclusion of 4-META, showed significant DSB formation in PDL cells [46].

ER-based sealers have also been extensively tested, with published studies from 1998 to 2020, prevailing the results reporting genotoxicity. Results are available for Topseal, AH26, AH Plus/AH Plus Jet, and Acroseal (Table 3). Most concern AH Plus. AH26 and AH Plus showed positive genotoxicity [36, 37, 51] but negative effects on the Ames test [35, 52,53,54]. AH Plus was also genotoxic in V79 cells [41, 48, 55], FMM1 cells [56], UO2S cells [38], hDPS cells [57], and, recently, in hGF cells [58]. However, controversial results were found with the same cell lines, i.e., results were negative with V79 cells [45], FMM1 cells [47], hGF cells [44], and PDL cells [46] perhaps due to different methodologies. Other ER sealers have also been investigated. Topseal showed some DNA damage in OC2 cells using the comet assay [36], and Acroseal increased the production of ROS and MN and delayed the cell cycle in G2 phase on V79 cells [48]; however, it did not alter DSB formation on PDL cells [46].

Diaket, a PR sealer, sets by chelation, but contains polyvinyl chloride as polymer and showed positive genotoxicity on hPB lymphocytes [40].

Calcium silicate-based sealers

CS sealers were the most tested, from 2005 to 2020, and only few studies demonstrated genotoxicity in mammalian cells (Table 3). Portland cement (PC) did not cause genotoxic effects in four cell lines, using the comet assay [59,60,61,62]. ProRoot MTA was also safe in most studies [43, 63, 65, 66], except for one exhibiting some genotoxicity on L929 cells using comet assay [64], perhaps due to the high concentrations used. Endocem MTA [65] and MTA Plus [50] were not genotoxic using the comet assay on CHO cells and BHK-21 cells, respectively. MTA Fillapex was tested in five cell lines being genotoxic in four of them [46, 50, 55, 57], except with hGF cells [58]. MTA Angelus, the most tested CS sealer, showed absence of genotoxicity, with the comet assay, on L5178Y cells [67], CHO-K1 cells [60, 68], hPB lymphocytes [61, 69], and 3T3-L1 cells [62]. MTA Angelus also showed negative results using the MN test with V79 cells [55] and the bone marrow of Swiss mice [39]. Genotoxicity was observed on hDPS cells, evaluated by gene expression analyses [57]. Other CS-based sealers not displaying genotoxicity were a CS cement [63] and TheraCal LC [66], a light-cure single-component material for direct and indirect pulp capping.

Calcium hydroxide-based sealers

CH sealers have a complex and inhomogeneous setting reaction, producing a hard surface with the deeper part retaining a dough-like structure. They perform remarkably well in laboratory leakage tests and also on biological, animal, and human tests [12]. Lack of physical sturdiness, poor clinical and radiographic outcomes when compared with other sealers are causes for concern [75]. CH is also added to other cements such as resins and ZOE sealers [76,77,78,79].

As a group, CH sealers appeared safe, as suggested from the studies performed through 1999 to 2018 (Table 3). CRCS [35], Sealapex [36, 38], Hydro C [43], Calcicur [44], P.A. CH [39], and Apexit [47] showed negative results. Positive genotoxicity was only noted for Calcipex II and Vitapex with CHO-K1 cells, observed by a higher incidence of micronuclei and higher tail moment values when compared with MTA [65].

Other novel sealers

Bioceramics exhibit excellent biocompatibility properties due to compositional similarity with biological hydroxyapatite, producing mineral hydroxyapatite with an osteoconductive effect, leading to bone formation at the interface between material and bone. Even though these advantages have contributed to their rapid spread in the dental field, they are not widely used, and products on the market are not yet known or used by many dentists [9]. Other novel sealers include the use of medicinal plants with studies showing the effect of natural products in pulpal and dentin repair with variable effectiveness such as alkaloids, coumarins, saponins, and flavonoids [80].

Genotoxicity results of novel sealers are summarized in Table 3. Studies were reported through 2008 to 2020. EndoSequence BC and iRoot SP are the same product, a hydrophilic sealer where the moisture inside the tubules initiates setting and shows genotoxicity [46], however lower than ZOE with L929 cells [70] and AH Plus with FMM1 cells [56]. Biodentine, a Ca3SiO5-based material indicated as a dentine substitute, was not mutagenic or genotoxic [66, 71]. However, using the MN test, an increase in the frequency of micronuclei was observed [72] which may have occurred because the high concentrated test solution BioRoot RCS demonstrated negligible DSB formation [46]. Two novel natural resin-based sealers have been recently created, namely Polifill [43, 45] (based on a polymer from R. communis—castor oil) and Bioseal [45] (based on Copaifera multijuga oil-resin—copaiba—with CH and zinc oxide) which tested negative for genotoxicity with V79 cells. Other experimental sealers tested with the comet assay include calcium silicate-hydroxyapatite (CS+HA) which was not genotoxic with hPB lymphocytes [73] and calcium-enriched mixture (CEM) which was genotoxic with L929 cells [64]. Novel sealers with favorable biocompatibility results that require genotoxicity studies include polymer nanocomposite resins [81], PC-based partial stabilized cement with zinc [82], and new resin cement [83].

Discussion

In 1981, Grossman listed requirements for RCS, as they should (1) be tacky when mixed to provide good adhesion to the canal wall when set; (2) make a hermetic seal; (3) be radiopaque to be visualized on radiographs; (4) have fine particles of powder to mix easily with liquid; (5) not shrink upon setting; (6) not discolor tooth structure; (7) be bacteriostatic or at least not encourage bacterial growth; (8) set slowly; (9) be insoluble in tissue fluids; (10) be well tolerated by the periapical tissue; and (11) be soluble in common solvents if it is necessary to remove it [84]. In 2007, de Ingle et al added two more requirements: (1) should not provoke an immune response and (2) should not be mutagenic or carcinogenic [85]. Thus, a RCS should have an acceptable level of biocompatibility and according to regulations, successfully pass a clinical risk assessment before commercialization, fulfilling the technical, biological, handling, and biocompatibility requirements [86].

This review put together the results concerning the studies addressing the genotoxicity of commercially available endodontic sealers and endodontic agents, included in 39 articles published from 1998 to the present data. The results’ outcome was presented considering the chemical composition of the sealers and are summarized in Table 3. Most studies were performed in vitro, with only three studies being conducted in vivo [39, 72, 87]. A great heterogeneity on the experimental protocols was evident, involving a variety of animal and human cell lines, exposure conditions (sealers’ concentration and exposure time) and the genotoxicity assays, hampering the results’ analyses and the characterization of genotoxicity patterns. Nevertheless, some trends were clearly noted. As a whole, all sealers’ groups presented agents with reported genotoxicity, but differences were observed among the groups, and also within each group. Figure 2 gives a quantitative overview of the reported studies, displaying also the genotoxicity outcome. Resin-based sealers (methacrylate- and epoxy-based) and calcium silicate sealers were addressed in a higher number of studies, followed by the zinc oxide-eugenol sealers and calcium hydroxide sealers.

Fig. 2
figure 2

Root canal sealers’ genotoxicity studies presented by composition: methacrylate-based sealers (MR), epoxy resin-based sealers (ER), polyvinyl resin-based sealers (PR), calcium silicate-based sealers (CS), zinc oxide eugenol-based sealers (ZOE), silicone-based sealers (S), glass ionomer cement-based sealers(GIC), calcium hydroxide-based sealers (CH), and other novel sealers

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As displayed in Fig. 2, resin-based sealers were tested in a great number of studies. Detailing the methacrylate-based sealers, EndoRez, Epiphany and RealSeal presented different outcomes depending on the cell line and experimental protocol; however, absence of genotoxic effects was reported in most studies. RealSeal was the most tested sealer of this group, and genotoxicity was rarely observed [50]. Otherwise, MetaSeal was only addressed in one study, being genotoxic [46]. On the epoxy resin-based sealers, AH 26 but, particularly, AH Plus, were thoroughly tested. This sealer yielded different outcomes although prevailing an absence of toxicity [44, 46, 47]. TopSeal and Acroseal were less addressed. The same was verified with Diaket, a polyvinyl resin-based sealer [40]. In the R sealers, the monomers and co-monomers appear potentially associated with genotoxicity, and a relationship between the structural and biological activities has been reported [88]. Resin monomers enhance the formation of ROS affecting the expression and levels of protective antioxidant enzymes as superoxide dismutase, glutathione peroxidase, and catalase [89], and the redox imbalance triggers DNA damage and apoptosis [90]. In methacrylate resin-based sealers, leached compounds as BisGMA, BisDMA, HEMA, PEGDMA, TEGDMA, UDMA, and the photoinitiator camphorquinone [91], possibly due to their solubility [92], alter tightly regulated metabolic pathways, by inducing cellular stress responses, including oxidative DNA damage and DSB [93,94,95]. In the oral environment, HEMA increases production of ROS and causes oxidative DNA damage through DSB as evidenced by the presence of micronuclei, cell-cycle delay, and apoptosis [96], as well as the transcription of early inflammatory genes [97]. For epoxy resin-based sealers, identified leachable components include bisphenol-A diglycidyl ether and formaldehyde, which are considered carcinogens and probably responsible for cellular toxicity [98] and apoptotic cell death [91].

Calcium silicate-based sealers are mainly composed of mineral trioxide aggregate (MTA) and were described as the most biocompatible material with most predictable outcomes [99], however with some drawbacks [100]. These sealers were tested in a large variety of studies and genotoxicity was observed only occasionally (Fig. 2). The most tested were Portland cement, ProRoot MTA, MTA Fillapex and, particularly, MTA Angelus. Portland cement (and also MTA Plus, Endocem MTA, CS cement) did not reveal genotoxic effects. ProRoot MTA showed toxic effects in only one study, as well as MTA Angelus. Comparatively, MTA Fillapex displayed higher toxicity. As a whole, MTA-based sealers are associated with low genotoxicity potential [65, 69]. However, calcium-enriched mixtures of MTA were genotoxic [64]. Nevertheless, pure CS-based cements, modified CS-based cements, and three resin-based CS cements showed no genotoxicity in human osteoblast cells [101].

Zinc oxide-eugenol sealers were also involved in a number of studies. Overall, these sealers appeared to be genotoxic. The eight tested sealers presented deleterious effects in several in vitro [51,52,53,54] and in vivo [39] assays. In the other way, calcium hydroxide-based sealers showed low genotoxicity [35, 36, 38, 39, 43, 44, 47]. Eight sealers were analyzed, and only Calcipex II and Vitapex were genotoxic [65]. Sealapex did not exhibit genotoxicity [36, 38].

The other sealers’ groups were less tested, as shown in Fig. 2. Two silicone-based sealers were tested, i.e., RoekoSeal and Gutta Flow presenting, respectively, positive [41] and negative [40] genotoxicity. For the glass ionomer cement sealers, three genotoxicity studies were reported, namely involving Ketac-Endo [35] and Vitrebond [42, 43]. The presence of the resin component in the sealer composition might play a role in the observed deleterious effect [102, 103]. Miscellaneous commercially available sealers were grouped together, and tested sealers included Endosequence BC [56, 70], iRootSP [56, 70], Biodentine [66, 71, 72], BioRoot RCS, Polifil, CEM, and CS+HA. Although only one or two studies were conducted per sealer, overall, genotoxicity potential was low (Fig. 2).

Considering the three groups of sealers involved in a higher number of studies, calcium silicate-based sealers presented the lower genotoxicity potential, followed by methacrylate resin-based sealers, which appeared slightly lower genotoxic than the epoxy resin-based sealers, whereas zinc oxide based sealers presented the higher genotoxic potential. The other groups of sealers were less addressed. Calcium hydroxide-based sealers appeared to present low genotoxic potential, whereas silicone-based and glass ionomer-based sealers showed variable outcomes. Nevertheless, establishing rankings of genotoxicity is a risky exercise. Major drawbacks are the great heterogeneity of the experimental protocols, namely the use of multiple cell lines, some of them not recommended by OECD guidelines, differences in the exposure conditions and in the performed genotoxicity assays. Also, there is insufficient information to draw firm conclusions concerning the sealers’ safety. Gene expression profiles and epigenetic mechanisms for damage response, antioxidant, or DNA repair genes, despite being indirect methods of analysis and not commonly recommended by standard guidelines, could provide relevant information on the involved genotoxicity mechanisms [104].

In a translational view, the reported information on the sealers’ genotoxicity must be placed in a proper context. Results were collected mostly from in vitro studies, using static cell culture models, and exposure conditions that are far from those anticipated in a clinical setting. Following the endodontic treatment, the levels of degradable/leachable compounds eventually observed are expectably low due to the very small contact area of the sealer potentially yielding leachable components. Also, due to the dynamic in vivo conditions, namely the continuous extracellular fluid flow, eventual leachates are continuously being cleared decreasing the local levels. The exposure features also deserve some observations. In vivo, cells of the periapical tissues are embedded in a collagenous extracellular matrix organized in a three-dimensional structure, thus with lower susceptibility to deleterious effects from the surrounding environment. Due to these unique physiological features and exposure conditions, the genotoxicity trends observed in vitro might not translated to the periapical environment with, eventually, less noticeable differences and/or outcomes among sealers [105]. Nonetheless, efforts to regulate the in vitro and in vivo genotoxicity assays is critical, in order to standardize protocols allowing representative information and translational usefulness.

Conclusions

An overview of the collected studies suggests that most sealers present some degree of genotoxicity and DNA damage, however with a trend toward low or high genotoxic potential. As the available information is mostly from in vitro studies, and involving a great heterogeneity on the experimental protocols, there is insufficient information to draw firm conclusions concerning the sealers’ safety.