An in vitro toxicological assessment of two electronic cigarettes: E-liquid to aerosolisation.

Interest in the toxicological assessment of iterations of e-cigarette devices, e-liquid formulations and flavour use is increasing. Here, we describe a multiple test matrix and in vitro approach to assess the biological impact of differing e-cigarette activation mechanism (button vs. puff-activated) and heating technology (cotton vs. ceramic wick). The e-liquids selected for each device contained the same nicotine concentration and flavourings. We tested both e-liquid and aqueous extract of e-liquid aerosol using a high throughput cytotoxicity and genotoxicity screen. We also conducted whole aerosol assessment both in a reconstituted human airway lung tissue (MucilAir) with associated endpoint assessment (cytotoxicity, TEER, cilia beat frequency and active area) and an Ames whole aerosol assay with up to 900 consecutive undiluted puffs. Following this testing it is shown that the biological impact of these devices is similar, taking into consideration the limitations and capturing efficiencies of the different testing matrices. We have contextualised these responses against previous published reference cigarette data to establish the comparative reduction in response consistent with reduced risk potential of the e-cigarette products tested in this study as compared to conventional cigarettes.

A contextualised e-cigarette testing strategy shows flavourings do not impact lung toxicity in vitro.

Vaping has the potential to reduce the individual health risks associated with smoking and e-cigarette flavours have been reported to help smokers' transition from cigarettes. In this manuscript, we provide evidence to support the reduced risk potential of e-cigarette aerosols and flavours by assessing commercially available e-liquids (Vuse ePod - Manufactured by British American Tobacco) in a 2D in vitro screening approach. We also analysed selected flavours using a more physiologically relevant 3D (MucilAir) whole aerosol exposure model, measuring toxicity and functional endpoints such as Trans Epithelial Electrical Resistance, Cilia Beat Frequency and Active Area. To contextualise responses, we have compared e-cigarette aerosol to cigarette smoke (1R6F research cigarette) and calculated the percentage reduction using a point of departure approach. We show that aerosolised flavoured e-liquids, (appropriately stewarded) do not increase the overall measured aerosol toxicity when compared to cigarette smoke. In fact, we demonstrate that the measured in vitro cellular toxicity of flavoured e-cigarette products remains > 95% reduced when compared to cigarette smoke toxicity, using point of departure (IC80) approach. These data indicate that the overall product toxicity is not increased in a flavour dependent manner and that flavoured e-cigarette products can potentially play a role in tobacco harm reduction.

A 3D in vitro comparison of two undiluted e-cigarette aerosol generating systems.

In vitro studies play an important role in supporting the toxicological assessment of e-cigarettes, with many current methods reliant on sophisticated in vitro exposure systems designed for conventional cigarette testing. In this study, we have compared two distinct systems; the modified Vitrocell VC10 and Borgwaldt LM4E designed to deliver undiluted e-cigarette aerosol. We assessed the cytotoxicity response of 3D reconstituted lung tissue (MucilAir) exposed to undiluted aerosol from ePen3 (closed modular e-cigarette) using these two exposure systems. As the induced cytotoxicity profiles were comparable, we then compared these responses against historical eBox (open modular e-cigarette) and 3R4F reference cigarette data to show evolution of product technology. This latter approach was deemed possible by monitoring intrinsic donor-to-donor control variability over a three-year period, bridging between exposure systems and observed biological responses. Despite the differences in the technology, on a puff-by-puff basis these machines gave remarkably similar cytotoxicity profiles for ePen3, as determined by MTT, and consistency of pre-cytotoxicity markers: transepithelial electrical resistance (TEER), cilia beat frequency and cilia active area. When responses are compared as a function of exposed nicotine concentration, we see differences due to the dynamics of the exposure systems. The parity of responses between the systems in generated undiluted aerosol has allowed us to compare back to previously published eBox data, irrespective of aerosol generating system and MucilAir donor, showing how evolution from open systems to podmod e-cigarette design can make a step change in the cytotoxicity profile of the product.

A screening approach for the evaluation of tobacco-free 'modern oral' nicotine products using Real Time Cell Analysis.

In many regulated industries there is an increasing pressure to provide timely and robust risk assessment data to support product launches. Real-time cell analysis (RTCA) is a tool that allows for the fast and relatively labour-free cytotoxic assessment of test compounds, compared to traditional methods. Here, we propose an application for the RTCA platform to provide a screening approach, to evaluate the cytotoxic potential of tobacco-free nicotine pouches, also termed modern oral product (MOP), to determine the contribution of differing nicotine strengths (4-11 mg) and a range of available flavour types from multiple markets, on overall product toxicity. Aqueous extracts were prepared for all products using 1 pouch in 20 mL cell culture media and applied to the cell system for 24 h. Test extract nicotine concentrations reflected the increases in product nicotine strength; however, these changes were not present in the same magnitude in the cytotoxicity data obtained from both primary human gingival fibroblasts (HGF) and an NCI-H292 human bronchial epithelial continuous cell line. Furthermore, across the range of flavours and product nicotine strengths tested, H292 cells whilst not the target organ for oral product use, accurately predicted the results seen in HGFs and could be considered a useful surrogate for fast screening studies. H292 cells are more easily cultured and for longer periods, offering a more compatible test system. In conclusion, the data demonstrate the utility of the RTCA platform for the quick assessment of a large range of product variants. Furthermore, for a cytotoxicity measure with this test product, the simple H292 cell line can predict outcomes in the more complex HGF and provide useful pre-clinical cytotoxicity screening data to inform the risk assessment of MOPs and the relative contribution of flavourings, nicotine and other components.

The in vitro assessment of a novel vaping technology.

We have developed a novel vaping product (NVP) IS1.0(TT), which utilises a stainless-steel mesh to transfer and vaporise the e-liquid, mitigating some of the potential sources of toxicants that can be generated using the more traditional 'wick and coil' approach. The emissions from IS1.0(TT) have previously been found to have lower levels of toxicants overall when directly compared with a commercial wick and coil e-cig. This current study assessed the toxicological responses to aerosols from this NVP. Responses induced by IS1.0(TT)were compared to those from a 3R4F reference cigarette, using in vitro test methods which included regulatory genetic toxicological assays as well as some more contemporary screening approaches. The experimental conditions were designed to facilitate the testing of aerosol from this vaping product at doses that in most cases greatly exceeded those of the 3R4F comparator showed little to no toxicological responses and demonstrated significantly reduced effects in these in vitro assays when compared to 3R4F. Furthermore, the extreme doses tested in the present study indicate that the toxicant profile of this NVP translates to lower biological activity in vitro, and suggests that the absolute risk hazard level associated with electronic cigarettes can be reduced through continuous improvement as the technology evolves.

An approach to testing undiluted e-cigarette aerosol in vitro using 3D reconstituted human airway epithelium.

The data presented here show that to provide an estimate of the relative cytotoxicity and therefore potency of e-cigarettes, undiluted aerosol techniques can be used. With the emergence of electronic nicotine delivery systems, fit-for-purpose in vitro screening methods are required. Reconstituted 3D human airway epithelium, was exposed to undiluted aerosols at the air-liquid interface, using a Vitrocell VC 10. TEER, cilia beat frequency and cytotoxic responses were assessed. Using two smoking regimes (ISO and HCI) a 3R4F reference cigarette, produced IC50s of 5.2 and 2.1 min, 1458 ng/mL and 1640 ng/mL nicotine respectively. Using an open tank e-cigarette device, a full cytotoxicity dose-response curve was obtained giving an IC50 of 30 min with corresponding nicotine of 10,957 ng/mL, 6-14 times less cytotoxic than cigarette smoke. A commonly used e-liquid flavourant cinnamaldehyde and known skin sensitizer was added to the standard e-liquid formulation and used as an aerosolised positive control, at 0.1, 0.025, 0.01 and 0%, demonstrating a full dose response. The delivery of undiluted aerosols in vitro has resulted in increased method sensitivity, throughput and quantitative e-cigarette comparisons. A positive control aerosol generated from a 'safe' e-liquid benchmark can inform risk assessments on supportable levels of flavour ingredients.

Extreme testing of undiluted e-cigarette aerosol in vitro using an Ames air-agar-interface technique.

There is a growing consensus that e-cigarettes hold the potential for reducing the harm associated with cigarette smoking. Recently published studies have reported in vitro testing of e-cigarettes, demonstrating reduced toxicological and biological effects. Few studies however have reported the use of e-cigarettes under extreme testing conditions. To assess the full mutagenic potential of a commercially available electronic-cigarette (Vype ePen), this study investigated the delivery of aerosol under extreme conditions, using a scaled-down 35 mm plate Ames bacterial reverse mutagenicity assay. S. typhimurium strains TA98, TA100, TA97, TA104 and E. coli WP2 uvrA pKM101 with or without metabolic activation (S9), were employed. Using a modified Vitrocell VC 10 exposure system 0, 180, 360, 540, 720 or 900 puffs of undiluted e-cigarette aerosol was generated and delivered to bacterial cultures aligned to reported human consumption data. The results demonstrate that no mutagenic activity was observed in any strain under any test condition even when exposed to 900 puffs of undiluted e-cigarette aerosols +/- S9. Positive control responses were observed in all strains +/- S9. Nicotine assessments demonstrated an increased and consistent aerosol delivery, with calculated maximum doses of ∼1 mg/mL delivery of nicotine. These data demonstrate the validity of this unique testing approach and adds further information to the growing weight of evidence that e-cigarettes offer substantially reduced exposure when compared to conventional cigarette smoke. For future in vitro assessments of next generation tobacco and nicotine products, the generation, delivery and testing of undiluted aerosols can now be considered.

The mutagenic assessment of an electronic-cigarette and reference cigarette smoke using the Ames assay in strains TA98 and TA100.

Salmonella typhimurium strains TA98 and TA100 were used to assess the mutagenic potential of the aerosol from a commercially available, rechargeable, closed system electronic-cigarette. Results obtained were compared to those for the mainstream smoke from a Kentucky reference (3R4F) cigarette. Two different test matrices were assessed. Aerosol generated from the e-cigarette was trapped on a Cambridge filter pad, eluted in DMSO and compared to cigarette smoke total particulate matter (TPM), which was generated in the same manner for mutagenicity assessment in the Salmonella assay. Fresh e-cigarette and cigarette smoke aerosols were generated on the Vitrocell® VC 10 smoking robot and compared using a modified scaled-down 35mm air agar interface (AAI) methodology. E-cigarette aerosol collected matter (ACM) was found to be non-mutagenic in the 85mm plate incorporation Ames assay in strains TA98 and TA100 conducted in accordance with OECD 471, when tested up to 2400µg/plate. Freshly generated e-cigarette aerosol was also found to be negative in both strains after an AAI aerosol exposure, when tested up to a 1L/min dilution for up to 3h. Positive control responses were observed in both strains, using benzo[a]pyrene, 2-nitrofluorene, sodium azide and 2-aminoanthracene in TA98 and TA100 in the presence and absence of metabolic activation respectively. In contrast, cigarette smoke TPM and aerosol from 3R4F reference cigarettes were found to be mutagenic in both tester strains, under comparable test conditions to that of e-cigarette exposure. Limited information exists on the mutagenic activity of captured e-cigarette particulates and whole aerosol AAI approaches. With the lower toxicant burden of e-cigarette aerosols compared to cigarette smoke, it is clear that a more comprehensive Ames package of data should be generated when assessing e-cigarettes, consisting of the standard OECD-five, TA98, TA100, TA1535, TA1537 (or TA97) and E. coli (or TA102). In addition, TA104 which is more sensitive to the carbonyl based compounds found in e-cigarette aerosols under dry-wicking conditions may also prove a useful addition in a testing battery. Regulatory standard product testing approaches as used in this study will become important when determining whether e-cigarette aerosols are in fact less biologically active than cigarette smoke, as this study suggests. Future studies should be supported by in vitro dosimetry approaches to draw more accurate comparisons between cigarette smoke, e-cigarette aerosol exposure and human use.

Bilateral androblastoma (Sertoli-stromal cell tumor) of the ovary: a rare cause of virilization in a teenager.

A case of a 17-year-old patient diagnosed with bilateral androblastoma of the ovary is presented. The patient was admitted because of secondary amenorrhea, hirsutism and acne. After clinical, ultrasonographic and hormonal examinations an androgen-producing ovarian tumor was suspected and consequently laparotomy with right ovarian tumor excision and left ovary exploration was carried out. During surgery the right ovarian tumor was excised and exploration of the left ovary revealed an ovarian tumor with a diameter of 10 mm, which was then also excised. The pathologic diagnosis was a bilateral androblastoma of the ovary measuring 40 mm x 30 mm x 20 mm in the right ovary and 10 mm in diameter in the left ovary. We concluded that androblastomas, in spite of their low incidence, are a possibility that should always be considered in women of all ages presenting with signs of virilization.

Efficiency of postoperative pain management after gynecologic oncological surgeries with the use of morphine + acetaminophen + ketoprofen versus morphine + metamizol + ketoprofen.

Surgical treatment used in gynecological oncology involves acute postoperative pain which requires efficient treatment. This study covered a group of 128 patients who were randomly divided into two groups. In the postoperative period patients in group I were administered morphine subcutaneously, acetaminophen intravenously and naproxen per rectum. The pain intensity level was checked by means of the pain intensity numeric rating scale (NRS). In the instances of pain rated at 5 or more, patients were additionally administered ketoprofen intravenously. Patients in group II were administered morphine, naproxen, and metamizole instead of acetaminophen and ketoprofen additionally. In group I after the administration of morphine and acetaminophen 22 patients (34.37%) needed additional doses of ketoprofen. In group II 33 women (51.56%) required ketoprofen after the administration of morphine and metamizole (N1 = 22 vs N2 = 33, p < 0.05). The use of metamizol with morphine (without ketoprofen) gave worse analgesic results than acetaminophen with morphine, but the combination of morphine, acetaminophen and ketoprofen or morphine, metamizol and ketoprofen gave satisfactory analgesic results.

The activation of synovial mast cells: modulation of histamine release by tryptase and chymase and their inhibitors.

Mast cells have been implicated as having pivotal roles in arthritis, but little is known of the processes leading to the activation of synovial mast cells or their potential for pharmacological control. We have investigated the ability of tryptase and chymase, and inhibitors of these major mast cell proteases to modulate the activation of mast cells from human synovial tissue. The tryptase inhibitor drug N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride (APC366) inhibited immunoglobulin E (IgE)-dependent histamine release in a dose-dependent manner, with about 70% inhibition being achieved at a dose of 300 microM. Histamine release stimulated by calcium ionophore A23187 was also inhibited by this compound. The chymase inhibitor chymostatin inhibited IgE-dependent histamine release by approximately 60% at 1 microg/ml. Tryptase at concentrations of 3.0 microg/ml and greater stimulated histamine release from synovial cells, which was dependent on catalytic activity, whereas chymase had little effect on these cells. The activation of mast cells by tryptase may represent an amplification process in the synovium. The mast cell stabilising properties of inhibitors of tryptase and chymase could be of therapeutic value in arthritis.

Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells.

BACKGROUND/AIMS: Connective tissue growth factor is a recently described mitogenic protein implicated in a variety of fibrotic disorders. Connective tissue growth factor may be a downstream mediator of the pro-fibrotic and mitogenic actions of transforming growth factor-beta, promoting extracellular matrix deposition and fibrogenesis. As transforming growth factor-beta is considered important to the pathogenesis of hepatic fibrosis, we examined the possible contribution of connective tissue growth factor to this process. METHODS: Connective tissue growth factor expression was examined in normal and fibrotic human and rat livers using RT-PCR and ribonuclease protection assays, and in primary cultures of rat hepatic stellate cells by Northern and Western blotting. RESULTS: Ribonuclease protection assays demonstrated connective tissue growth factor mRNA was increased 3-5-fold in human fibrotic liver compared with normal. RT-PCR showed this mRNA was increased in carbon-tetrachloride-treated rat liver. Northern analysis showed connective tissue growth factor mRNA was increasingly expressed during progressive activation of cultured rat hepatic stellate cells. Western analysis confirmed that freshly isolated hepatic stellate cells secreted relatively little connective tissue growth factor compared with hepatic stellate cells activated in culture. Hepatic stellate cells stimulated with transforming growth factor-beta showed increased expression of connective tissue growth factor mRNA and protein. CONCLUSIONS: Connective tissue growth factor mRNA is consistently upregulated in human liver cirrhosis of various aetiologies, supporting a role for this growth factor in hepatic fibrogenesis. Our studies suggest that hepatic stellate cells may be an important source of hepatic connective tissue growth factor in vivo, particularly following stimulation with transforming growth factor-beta.

Inhibitors of chymase as mast cell-stabilizing agents: contribution of chymase in the activation of human mast cells.

There has long been evidence that inhibitors of chymotryptic proteinases can inhibit the degranulation of rodent mast cells, but their actions on human mast cells and the contribution of mast cell chymase itself have received little attention. We investigated the ability of the selective chymase inhibitor Z-Ile-Glu-Pro-Phe-CO(2)Me and other proteinase inhibitors to inhibit chymase and cathepsin G activity, and we examined their potential to modulate the responsiveness of mast cells dispersed from human skin, lung, and tonsil tissues. IgE-dependent histamine release from skin mast cells was inhibited by up to about 80% after preincubation with Z-Ile-Glu-Pro-Phe- CO(2)Me (up to 0.1 microM), 70% with chymostatin (17 microM), and 60% with soybean trypsin inhibitor (0.5 microM). The mast cell-stabilizing properties of chymase inhibitors appeared to be greater for skin mast cells than for those from lung, whereas tonsil mast cells were relatively unresponsive. There were marked differences in the time course of responses to inhibitors, and the effect was dependent on the stimulus, with calcium ionophore-induced histamine release being unaffected. Incubation of dispersed skin, lung, or tonsil cells for up to 45 min with purified chymase failed to induce histamine release, although preincubation of cells with chymase was able to suppress IgE-dependent activation. Chymase could thus contribute to mast cell degranulation and after secretion could provide a feedback mechanism to limit this process. Nevertheless, inhibitors of chymase can be potent mast cell stabilizers, particularly in the skin.

Progelatinase A is produced and activated by rat hepatic stellate cells and promotes their proliferation.

Activated hepatic stellate cells (HSCs) are a potential source of gelatinase A, which accumulates in fibrotic livers. Progelatinase A activation requires its binding to a complex of membrane-type matrix metalloproteinase (MT-MMP) and tissue inhibitor of metalloproteinases (TIMP)-2. These studies examine gelatinase A, MT1-MMP, and TIMP-2 synthesis by HSCs during activation in vitro and the potential role of gelatinase A in promoting HSC proliferation. Gelatinase A, MT1-MMP, and TIMP-2 messenger RNA (mRNA) were all upregulated in HSCs activated on plastic over 5 to 14 days. Gelatinase A expression was maximal at 7 days of culture, coinciding with the peak of HSC proliferation and the onset of procollagen I and alpha-smooth muscle actin (alpha-SMA) mRNA expression. Active forms of gelatinase A of 62 kd and 66 kd were secreted by activated HSCs and reached a maximum of 12.1% of total enzyme in 14-day culture supernatants. Treatment of HSCs with concanavalin A (con A) induced activation of MT1-MMP and enhanced secretion of activated gelatinase A, which reached a maximum of 44.4% of the total enzyme secreted into culture supernatants using 30 microgram/mL con A. [(14)C]-gelatin degradation assays confirmed the presence of gelatinolytic activity in activated HSC supernatants, which reached a maximum level at 7 days of culture. Antisense oligonucleotide inhibition of endogenous progelatinase A production, or the MMP inhibitor 1,10-phenanthroline inhibited (3)H-thymidine incorporation into HSC DNA by greater than 50%. We conclude that HSCs produce progelatinase A during activation in vitro and activate this enzyme coincident with MT1-MMP and TIMP-2 synthesis. Gelatinase A activity is required for maximal proliferation of HSCs in vitro suggesting this metalloproteinase is an autocrine proliferation factor for HSCs.

Human and rat hepatic stellate cells produce stem cell factor: a possible mechanism for mast cell recruitment in liver fibrosis.

BACKGROUND/AIMS: Mast cell numbers are markedly increased in advanced liver fibrosis. Stem cell factor may recruit mast cells to the liver following injury as it induces mast cell proliferation, survival and differentiation from resident tissue precursors. This study examines stem cell factor production in human fibrotic liver and by hepatic stellate cells during culture in vitro. METHODS: Stem cell factor production was examined in human fibrotic livers by ELISA and in human and rat hepatic stellate cell cultures using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, Western blotting and immunocytochemistry. Co-culture studies examined adhesion between hepatic stellate cells and purified mast cells. RESULTS: RT-PCR showed stem cell factor mRNA was more consistently expressed in fibrotic human livers relative to normal, and ELISA confirmed this by showing stem cell factor protein was significantly increased 2-3-fold in homogenates of human cirrhotic liver (primary biliary cirrhosis, primary sclerosing cholangitis) relative to normal. RT-PCR detected stem cell factor mRNA in human and rat hepatic stellate cells activated by culture on plastic. This was confirmed by Western blotting, which showed that freshly isolated hepatic stellate cells expressed relatively little 30 kD stem cell factor compared to late primary culture activated hepatic stellate cells (14 day) and passaged hepatic stellate cells. As assessed by fluorescence immunocytochemistry, stem cell factor protein was homogeneously expressed by populations of culture-activated rat hepatic stellate cells. During co-culture, purified human skin mast cells adhered to hepatic stellate cell monolayers on plastic, and this adherence was inhibited >50% by addition of antibodies against stem cell factor. CONCLUSIONS: Hepatic stellate cells activated in vitro produce stem cell factor. These cells may play an important role in recruiting mast cells to liver during injury and fibrosis.

Two distinct forms of human mast cell chymase--differences in affinity for heparin and in distribution in skin, heart, and other tissues.

Chymase, a chymotrypsin-like protease secreted by human mast cells, is generally considered to be a single enzyme. However, by heparin-agarose chromatography of high-salt extracts of human skin, we have consistently resolved three peaks of chymotryptic activity, eluting at 0.4 M NaCl (peak A), 1.0-1.2 M NaCl (peak B) and 1.8-2.0 M NaCl (peak C), with peak B containing 75-90% of the recovered activity. Each peak retained its identity upon rechromatography. The three peaks of activity were similar in substrate specificity and inhibitor profile and distinctly different from other chymotryptic enzymes, including cathepsin G and the stratum corneum chymotryptic enzyme. Examination of different tissues revealed that peak C was virtually absent from synovial tissue, was present as a minor component in skin and heart, but constituted the predominant chymotryptic activity in lung. Peaks B and C from skin tissue were further purified by chromatography on Sephacryl S-200. Both had a molecular mass of 28-29 kDa, yielded the N-terminal sequence reported for chymase, and on western blots reacted with a panel of polyclonal, monoclonal and antipeptide antibodies against chymase. Chymase C required higher concentrations of NaCl to overcome the stimulatory effects of heparin than did chymase B, but had a similar pH profile. Thus, human chymase exists in at least two distinct but similar forms, and the differences in heparin binding and tissue distribution could have important consequences for enzyme function.

A role for tryptase in the activation of human mast cells: modulation of histamine release by tryptase and inhibitors of tryptase.

Tryptase, the most abundant protein product of human mast cells is emerging as an important mediator and target for therapeutic intervention in allergic disease. We have investigated the potential of tryptase and inhibitors of tryptase to modulate histamine release from human mast cells. Addition of purified human tryptase in concentrations ranging from 1 to 100 mU/ml stimulated a concentration-dependent release of histamine from cells dispersed from tonsil, although not from skin tissue. The reaction dependent on an intact catalytic site being inhibited by heat inactivation of the enzyme, or by preincubating with the tryptase inhibitors APC366 or leupeptin or the tryptic substrate N-benzoyl-DL-arginine-p-nitroanilide (BAPNA). Tryptase-induced histamine release took approximately 6 min to reach completion, appeared to require exogenous calcium and magnesium, and on the basis of inhibition by antimycin A and 2-deoxy-D-glucose, seemed to be a noncytotoxic process. Pre-incubation of cells with tryptase at concentrations that were suboptimal for histamine release had little effect on their responsiveness to anti-immunoglobulin (Ig) E or to calcium ionophore A23187, but at higher concentrations their subsequent activation was inhibited. APC366 significantly inhibited histamine release induced by anti-IgE or calcium ionophore from both tonsil and skin cells, with up to 90% inhibition being observed at a concentration of 100 microM with skin. IgE-dependent histamine release was inhibited also by leupeptin, benzamidine and BAPNA. Tryptase may act as an amplification signal for mast cell activation, and this could account at least partly for the potent mast cell stabilizing properties of tryptase inhibitors.