Proarrhythmic effects induced by benzethonium chloride and domiphen bromide in vitro and in vivo.

Benzethonium chloride (BZT) and domiphen bromide (DMP) are widely used as antimicrobials in drugs, vaccines and industry. However, no cardiac safety data has been developed on both compounds. Previously we reported BZT and DMP as high-affinity human ether-a-go-go related gene (HERG) channel inhibitors with unknown proarrhythmic risk. Here, we investigate the cardiotoxicity of BZT and DMP in vitro and in vivo, aiming to improve the safety-in-use of both antimicrobials. In the present study, human iPSC derived cardiomyocytes (hiPSC-CMs) were generated and rabbit models were used to examine the proarrhythmic potential of BZT and DMP. Our results found that BZT and DMP induced time- and dose-dependent decrease in the contractile parameters of hiPSC-CMs, prolonged FPDc (≥ 0.1 µM), caused tachycardia/fibrillation-like oscillation (0.3-1 µM), ultimately progressing to irreversible arrest of beating (≥ 1 µM). The IC50 values of BZT and DMP derived from normalized beat rate were 0.13 µM and 0.10 µM on hiPSC-CMs at 76 days. Moreover, in vivo rabbit ECG data demonstrated that 12.85 mg/kg BZT and 3.85 mg/kg DMP evoked QTc prolongation, noncomplex arrhythmias and ventricular tachycardias. Our findings support the cardiac safety of 0.01 µM BZT/DMP in vitro and the intravenous infusion of 3.85 mg/kg BZT and 1.28 mg/kg DMP in vivo, whereas higher concentrations of both compounds cause mild to moderate cardiotoxicity that should not be neglected during medical and industrial applications.

Effects of Consumer Antimicrobials Benzalkonium Chloride, Benzethonium Chloride, and Chloroxylenol on Colonic Inflammation and Colitis-Associated Colon Tumorigenesis in Mice.

Benzalkonium chloride (BAC), benzethonium chloride (BET), and chloroxylenol (PCMX) are antimicrobial ingredients used in many consumer products and are frequently detected in the environment. In 2016, the U.S. Food and Drug Administration removed 19 antimicrobial ingredients from consumer antiseptic wash products, but deferred rulemaking for BAC, BET, and PCMX to allow additional time to develop new safety and efficacy data for these 3 antimicrobials. Therefore, it is important and timely to better understand the effects of these 3 compounds on human health. Here, we report that exposure to low doses of these antimicrobial compounds, in particular BAC, increases dextran sodium sulfate (DSS)-induced colonic inflammation and azoxymethane/DSS-induced colon tumorigenesis in mice. In addition, we find that exposure to BAC increases activation of Toll-like receptor 4 signaling in the systemic circulation, by disrupting intestinal barrier function and thus enhancing circulating levels of bacterial products. Together, our results suggest that these widely used antimicrobial compounds could exaggerate disease development of inflammatory bowel disease and associated colon cancer. Further studies are urgently needed to better characterize the impacts of these compounds on gut diseases.

Benzalkonium chloride, benzethonium chloride, and chloroxylenol - Three replacement antimicrobials are more toxic than triclosan and triclocarban in two model organisms.

With the recent ban of triclosan (TCS) and triclocarban (TCC) from some personal care products, many replacement antimicrobial compounds have been used. Yet the potential health risk and environmental impact of these replacement compounds are largely unknown. Here we investigated the toxicological effects of three commonly used replacement antimicrobials, benzalkonium chloride (BAC), benzethonium chloride (BEC), and chloroxylenol (CX) to two model organisms, the nematode C. elegans and zebrafish (Danio rerio), and compared them to the banned TCS and TCC. We found that these replacement compounds are not any safer than the banned antimicrobials. In the worm, at least one of the three, BAC, showed comparable toxicity to TCS from organismal to molecular levels, with toxic effects occurring at lower hundred µg/L to lower mg/L levels. In the fish, all three compounds at the tested concentration ranges (0.05-5 mg/L) showed toxicity effects to zebrafish embryos, indicated by hatching delay or inhibition, embryonic mortality, morphological malformations, and neurotoxicity. BAC was the most toxic among the three, with acute lethal toxicity occurring at environmentally relevant concentrations (hundreds of µg/L), which is comparable to the banned TCC. However, the toxicity effects of BAC and TCC occurred within different time windows, potentially suggesting different mechanisms of toxicity. CX was the only compound that induced a "body curvature" phenotype among the five compounds examined, suggesting a unique mode of toxic action for this compound. Furthermore, all five compounds except TCS induced neurotoxicity in fish larvae, indicated by alterations in secondary motoneuron axonal projections. Such neurotoxicity has been largely understudied for these antimicrobials in the past years and calls for further investigations in terms of its underlying mechanisms and ecological significance. These findings strongly indicate that scrutiny should be put on these replacement compounds before their introduction into massive use in personal care products.

Comparative study on toxic effects induced by oral or intravascular administration of commonly used disinfectants and surfactants in rats.

Accidental ingestion or injection of household products sometimes occurs due to their accessibility, but the toxic manifestations have not been well characterized when they are internally administered. The aim of this study was to investigate the toxic effects induced by ingestion or injection of different ionic surfactants and disinfectants in rats. The test drugs involved benzalkonium and benzethonium (BZK and BZT, both cationic surfactants used as disinfectants), alkyldiaminoethylglycine (AEG, an amphoteric surfactant used as a disinfectant), linear alkylbenzenesulfonate (LAS, an anionic surfactant), polyoxyethylene cetylether (PEC, a nonionic surfactant), chlorhexidine (CHX, not a surfactant but a disinfectant) and saline (control). Male Sprague-Dawley rats were administered one of the test drugs orally (p.o.), intravenously (i.v.) or intraarterially (i.a.). The fatal effects appeared rapidly (<30 min) in i.v.-administered rats, while taking hours (>5 h) in i.a./p.o.-administered rats after a dose of around LD(50) , although the progress and degree of toxic effects varied among the drugs tested. In intravascular administration, BZK and BZT were fatal at doses of 15-20 mg kg(-1) . Higher concentrations in lung and kidney than in blood were determined. CHX showed a high toxic effect compared with cationic surfactants. The rats administered anionic (LAS) or amphoteric (AEG) surfactant died in less than 24 h at doses over 100 mg kg(-1) . In p.o. administration, the toxic effects were concentration/dose-dependent, and all rats administered high doses of surfactants except for PEC died at 5-20 h. The overall toxic ranks could be: cationic surfactant/CHX> anionic/amphoteric surfactant > nonionic surfactant.

Benzethonium increases the cytotoxicity of S(+)-ketamine in lymphoma, neuronal, and glial cells.

INTRODUCTION: Ketamine has been demonstrated to be neurotoxic in animals as well as in patients. Preservatives added to ketamine have been accused to induce this neurotoxicity. Therefore, we investigated whether the most widely used preservative of ketamine-benzethonium chloride-enhances the toxicity of S(+)-ketamine in vitro in lymphoma, neuroblastoma cells and primary astrocytes. METHODS: Human Jurkat T-lymphoma- and neuroblastoma cells (SHEP) were incubated for 24 hours with commercially available S-ketamine containing benzethonium, pure S-ketamine and pure benzethonium chloride. The rate of early- and late-apoptotic cells was evaluated by flowcytometry. In a second step the combined toxicity of benzethonium and ketamine was investigated in neuroblastoma cells and primary rat astrocytes in a mitochondrial activity assay (XTT). The additivity of the toxicities was evaluated by employing isobolographic analysis. RESULTS: In Jurkat T-lymphoma and neuroblastoma cells benzethonium increased the toxicity of ketamine from 32% to 80% and from 64% to 84% cell deaths, respectively. In neuroblastoma cells as well as in primary rat astrocytes the measured combined toxicity was within the confidence interval of the calculated pure additive toxicity as seen in the isobolograms. CONCLUSIONS: We conclude that benzethonium increases the local toxicity of ketamine in cells of hematopoietic, neuronal and glial origin in an additive manner. Therefore, caution is recommended especially when using preservative containing S-ketamine as an additive for long-term neuraxial analgesia.

Effects of high-dose major components in oral disinfectants on the cell cycle and apoptosis in primary human gingival fibroblasts in vitro.

We evaluated the effects of high-dose major components in oral disinfectants on oral cells from the standpoints of the cell cycle and apoptosis. We examined the viability and cell cycle of human gingival fibroblasts (HGFs) treated with the components of dental disinfectants, benzethonium chloride (BEC), benzalkonium chloride (BAC), and povidone iodine (PVD-I) using a cell counting kit and flow cytometry. The IC(50) inhibitory concentration value in HGF cultures at 24 hours was 1.3x10(-2) mM BEC, 6.0x10(-3) mM BAC, and 2.6x10(-1) mM PVD-I. In the cell cycle analysis, propidium iodide-stained HGFs were arrested in G(0)/G(1) of the cell cycle by all three disinfectants, and in the apoptosis assay, annexin V-FITC/PI-stained HGFs that became apoptotic at 5.0x10(-2) and 1.0x10(-1) mM BEC and 5.0x10(-2) and 1.0x10(-1) mM BAC, but not in PVD-I at concentrations as high as 5.0x10(-1) mM. Our findings describe the effects of high-dose oral disinfectants, rather than clinical concentrations. Nevertheless, appreciating the effects of high-dose disinfectants absorbed into the human body is important, where they may accumulate in specific tissues and cells.

Effects of nonionic and ionic surfactants on survival, oxidative stress, and cholinesterase activity of planarian.

Eight widely used surfactants (cetyltrimethylammonium bromide; CTAB, benzethonium chloride; Hyamine 1622, 4-nonylphenol; NP, octylphenol ethoxylate; Triton X-100, dodecylbenzene sulfonate; LAS, lauryl sulfate; SDS, pentadecafluorooctanoic acid; PFOA, and perfluorooctane sulfonate; PFOS) were selected to examine their acute toxicities and effects on oxidative stress and cholinesterase (ChE) activities in Dugesia japonica. The differences in acute toxicity among eight surfactants to planarians were at least in the range of three orders of magnitudes. The toxicity rank of surfactants according to estimated 48-h LC(50) was SDS>NP>LAS>Hyamine 1622>CTAB>Triton X-100>PFOS>PFOA. The toxicity rank of surfactants according to 96-h LC(50) was as follows: SDS>CTAB>NP>LAS>Hyamine 1622>Triton X-100>PFOS>PFOA. There were significant increases in catalase activities in planarians exposed to LAS at nominal concentrations of 0.5 or 1 mgl(-1) and to PFOS at nominal concentrations of 5 or 10 mgl(-1) after 48-h exposure. Inhibitions of ChE activities were found in planarians exposed to Hyamine 1622 at all concentrations tested, to PFOS at nominal concentration of 10 mgl(-1), to PFOA at nominal concentrations of 50 or 100 mgl(-1) and to NP at nominal concentration of 0.5 mgl(-1). A significant increase in ChE activities was also observed in planarian exposed to Triton X-100 at nominal concentration of 5 mgl(-1). The implication of ChE inhibition by NP, PFOS and PFOA on neurological and behavioral effects in aquatic animals requires further investigation.

Cationic surfactants induce apoptosis in normal and cancer cells.

Cationic surfactants, such as benzalkonium chloride and benzethonium chloride, possess quaternary ammonium salt. These surfactants have antimicrobial action and are used as a preservative and an antiseptic. The positively charged polar head of cationic surfactants seems to play a role in the antimicrobial action of these compounds. Recently, benzalkonium chloride in eye drops has been reported to induce apoptosis in conjunctival cells. Here, we examined whether various types of surfactants including anionic and amphoteric surfactants induce apoptosis or not in mammalian cells. Antimicrobial cationic surfactants induced apoptosis at lower concentration than its critical micelle concentration (CMC) in rat thymocytes. Other quaternary ammonium surfactants, such as cetyltrimethylammonium bromide, similarly increased biochemical and morphological features of apoptosis, whereas both anionic and amphoteric surfactants had no significant effect on these apoptotic features. These results suggest that the positive charge of quaternary ammonium surfactants is involved with onset of the apoptotic process. The treatment of benzethonium chloride also led to apoptotic cell death in Jurkat cells. These results indicate that cationic surfactants induce apoptosis in the normal and cancer cells.

Cytotoxic effects of dental desensitizers on human gingival fibroblasts.

The purpose of this study was to evaluate the effects of three different desensitizers on the cell viability and morphology of human gingival fibroblasts (HGF). Human gingival tissues were obtained from individuals who have clinically, healthy periodontium. HGF were grown at 37 degrees C in humidified atmosphere of 5% CO2 in Dulbecco's modified eagle's medium, supplemented with glutamine, penicillin, streptomycin, and 10% fetal bovine serum. The cells were treated with different concentrations (0.1, 0.3, and 0.5 microL/mL) of desensitizers (Gluma Desensitizer, Seal&Protect, and MicroPrime). After 24- and 48-h exposure to the desensitizer solutions, the viable cells were examined using a hemocytometer. To monitor HGF viability, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used and cell morphology was also observed at 48 h. Following exposure to concentrations of 0.1 microL/mL of test materials for 24 h, cell survival rates for Gluma Desensitizer (106%) and Micro Prime (62%) were not significantly different from the control, while it was significant for Seal&Protect (50%). Growing cells were significantly inhibited by all tested materials for 48 h (p < 0.05) in survival rates of 51, 47, and 31%, respectively. On the basis of the MTT assay, the cytotoxic effect of MicroPrime was more prominent, especially at high concentrations, than does Gluma Desensitizer and Seal&Protect. After exposure to Seal&Protect and MicroPrime, HGF became retracted, rounded in appearance and had loss of normal organization, leading to enlargement of intercellular space when compared with Gluma Desensitizer. As a conclusion, taking the limitations of an in vitro experiment into consideration, the cytotoxic effects were varied, depending on the chemical composition and exposure periods of the tested desensitizers.

Peptidergic innervation of the crop and the effects of an ingested nonpeptidal agonist on longevity in female Musca domestica (Diptera: Muscidae).

Dromyosuppressin (DMS) immunoreactive neurons were discovered running along the crop duct and on the surface of the crop in the house fly, Musca domestica L. DMS is a myoinhibitory neuropeptide that has been shown to inhibit crop contractions, in vitro, in the blow fly, Phormia regina (Meigen), and in Drosophila melanogaster Meigen. Various concentrations of benzethonium chloride (Bztc), an agonist of DMS with shown inhibitory effects on blow fly crop contractions, were fed to adult female M. domestica. Flies exhibited a dose-dependent mortality; avoidance and subsequent dehydration are probably the cause of the low survivorship at higher Bztc concentrations.

Comparison of cytotoxicity of various surfactants tested on normal human fibroblast cultures using the neutral red test, MTT assay and LDH release.

We used the neutral red test, MTT assay and lactate dehydrogenase (LDH) release to compare the potential cytotoxicity of six surfactants belonging to different classes--three non-ionic surfactants (Triton x100, octylphenoxypolyethoxy alcohol, from Orion; Tween 60, polyoxyethylene (20) sorbitan monostearate, from ICI Speciality Chemicals; Tween 80, polyoxyethylene (20) sorbitan monolaurate, from Labosi), two anionic surfactants (Texapon K1298, sodium lauryl sulphate, from Henkel; Texapon N40, sodium laurylether sulphate, from Henkel) and one cationic surfactant (benzethonium chloride, from Siber Hegner)--on human fibroblast cultures. According to the LC50 (microg ml(-1)), the tested surfactants can be classified in the following order of increasing cytotoxicity: Tween 80 < Texapon N40 < Tween 60 < Texapon K1298 < Triton x100 < benzethonium chloride.

Subarachnoid ketamine in swine--pathological findings after repeated doses: acute toxicity study.

BACKGROUND AND OBJECTIVES: The purpose of this study was to investigate whether 5% ketamine with and without preservative, administered intrathecally to swine, produced a clinical anesthetic effect and caused direct subacute neurotoxicity. METHODS: Twenty pigs were used. Under general anesthesia, a subarachnoid catheter was placed at L5-L6 or L6-S1 spinal interspace. Five animals were used for initial clinical evaluation of the anesthetic effects of subarachnoid ketamine (12.5 and 25.0, and 500 mg). Two animals were excluded because of bloody taps, two served as controls (catheterization without drug administration), four received ketamine racemate (25.0 mg/d), four received ketamine racemate preservative free (25.0 mg/d), and three received benzethonium chloride, the ketamine excipient (0.05 mg/d). All drugs were administered for 7 days. The catheters were withdrawn at the end of the treatment period. After 35 days, the pigs were euthanized and the spinal cord removed and preserved for histopathologic study with hematoxilyn-eosin and luxol-fast blue myelin staining. Histopathologic effects were defined as absent/minimal, mild, or severe by a pathologist, unaware of group allocation, by evaluating the presence and intensity of peripheral and/or central chromatolysis, spongiosis, neuronal loss, perivascular neuroglia, neuronolysis, and myelin degeneration. RESULTS: All doses of ketamine produced immediate cutaneous anesthesia and motor block; benzethonium chloride did not. Histopathologic examination showed no neurotoxic effect of ketamine without preservative; ketamine with preservative showed a discrete neurotoxic effect, and the preservative alone produced a moderate neurotoxic effect. CONCLUSIONS: Clinically, in swine, subarachnoid ketamine without preservative is a safe and effective anesthetic and did not show significant neurotoxic effects. However, ketamine with preservative produces minimal changes, and benzethonium chloride alone produces moderate neurotoxic effects.

[Cytotoxicity of benzethonium chloride on cultured mammalian cells].

Cytotoxicity of benzethonium chloride on cultured mammalian cells was studied. Growth of V79 cells, derived from Chinese hamster lung was inhibited by treatment with benzethonium chloride at above 30 micrograms/ml for 24 to 48 hr. Inhibitory effect on survival of V79 cells was not observed in cultures treated with benzethonium chloride at 3 micrograms/ml for 2 to 24 hr. Meanwhile, treatment with benzethonium chloride at 10 micrograms/ml for 2 to 24 hr elicited 11.4% to 75.0% inhibition of cell survival. Exposure of cells to benzethonium chloride at 3 to 30 micrograms/ml for 2 hr resulted in inhibition of DNA-, RNA-, and protein-syntheses of the cells, in a dose-dependent manner.

Surfactants selectively ablate enteric neurons of the rat jejunum.

Surfactants, a group of nonspecific membrane perturbating substances, can cause nerve damage. Various concentrations of the cationic surfactants benzalkonium chloride (BAC) and benzethonium chloride, the anionic surfactants sodium ricinoleate, dioctyl sodium sulfosuccinate and sodium lauryl sulfate and the nonionic surfactant Triton X-100 were applied to the serosal surface of the rat jejunum every 5 min for 0.5 hr and then rinsed off with saline. Thirty days after surfactant application, the treated and an untreated segment of jejunum were removed and examined histologically. All surfactants which were tested significantly reduced the number of ganglion cells in the myenteric plexus. In addition, sodium ricinoleate significantly reduced the number of ganglion cells in the submucosal plexus. Higher concentrations of the cationic agents BAC and benzethonium chloride caused a generalized tissue damage including disruption of the smooth muscle, lymphocytic infiltration, intestinal perforation and death. Using BAC as a prototype surfactant, peptidergic neuron distribution and gut electrical activity were examined. BAC treatment markedly reduced the immunoreactivity of somatostatin, substance P, met-enkephalin and vasoactive intestinal peptide in the myenteric plexus. In addition, the electric properties of the smooth muscle were altered. BAC treatment resulted in an erratic, markedly distorted basic electric rhythm and an alteration in spike potential generation. These studies demonstrate that surfactants in appropriate concentrations selectively ablate the myenteric neurons and alter peptidergic neuron distribution and gut electrical parameters in the rat jejunum.