Maternal exposure to cetylpyridinium chloride impairs oogenesis by causing mitochondria disorder in neonates.

Cetylpyridinium Chloride (CPC) is a common disinfectant with potential mitochondrial toxicity. However, the effects of CPC on female reproduction remains unclear. In the present study, pregnant mice were exposed to environmentally relevant doses of CPC for 3 days, the effects were evaluated in the female offspring. Maternal exposure to CPC caused loss of oocytes in neonatal ovaries. TEM analysis of neonatal ovaries showed CPC caused aberrant mitochondrial morphology including vacuolated and disorganized structure, reduced functional cristae. In addition, CPC decreased mitochondrial membrane potential in neonatal oocytes. Seahorse analysis showed that CPC hampered mitochondrial reserve, manifested as reduced spare respiratory capacity. Furthermore, CPC damaged mitochondrial function and impaired developmental competence of MII oocytes, suggesting a persisting impact into adulthood. In summary, this is the first known demonstration that maternal exposure to CPC caused mitochondrial disorders in neonatal ovaries and had long-term effects on fertility of the female offspring.

Plasma membrane damage triggered by benzalkonium chloride and cetylpyridinium chloride induces G0/G1 cell cycle arrest via Cdc6 reduction in human lung epithelial cells.

Quaternary ammonium compounds, including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC), are widely used as disinfectants. Increased use of inhalable products containing BAC or CPC has raised concerns for lung toxicity. This study sought to elucidate the microstructure of plasma membrane damage caused by BAC and CPC and the subsequent mechanism by which the damage is mediated, as assessed using two human pulmonary epithelial cell lines (A549 and BEAS-2B). Scanning electron microscopic observation showed that exposure to BAC or CPC for 3 hr reduced the length and density of microvilli on the plasma membrane in A549 cells. Analysis of cell cycle distribution following plasma membrane damage revealed that BAC and CPC promote G0/G1 cell cycle arrest in both cell lines. The protein levels of Cdc6, an essential regulator of DNA replication during G1/S transition, are decreased significantly and dose dependently by BAC or CPC exposure. CPC and BAC decreased the Cdc6 levels that had been increased by a PI3K agonist in A549 cells, and levels of phosphorylated AKT were reduced in response to BAC or CPC. Conversely, exposure to equivalent concentrations of pyridinium chloride (lacking a hydrocarbon tail) induce no changes. These results suggest that plasma membrane damage triggered by BAC or CPC causes Cdc6-dependent G0/G1 cell cycle arrest in pulmonary cells. These effects are attributable to the long alkyl chains of BAC and CPC. The reduction of Cdc6 following plasma membrane damage may be caused, at least in part, by diminished signaling via the PI3K/AKT pathway.

Controlled release, antimicrobial activity, and oral mucosa irritation of cetylpyridinium chloride-montmorillonite incorporated in a tissue conditioner.

This study examined the controlled release of cetylpyridinium chloride (CPC) from a tissue conditioner (TC) containing CPC-montmorillonite (CPC-Mont), the associated antimicrobial activity, and oral mucosa irritation. The CPC release test was performed daily for 28 days in three test solutions: distilled water, 0.2 M NaCl, and 0.2 M HCl. The antimicrobial activities for 7, 14, 21, and 28 days against Candida albicans, Staphylococcus aureus, and Streptococcus mutans were assessed according to the JIS Z 2801/ISO 22196 standard. An oral mucosa irritation test was conducted using cheek pouches in five male hamsters according to the ISO 10993-10:2010 standard. The amount of CPC released each day and the cumulative amount released over 28 days (6.12 mg) were less than the daily safe maximum of sore throat medicines (8 mg). Additionally, TC with CPC-Mont could sustain antimicrobial activity against adherent bacteria for 14 days and has no oral mucosa irritation potential.

Comprehensive pulmonary toxicity assessment of cetylpyridinium chloride using A549 cells and Sprague-Dawley rats.

Cetylpyridinium chloride (CPC), a quaternary ammonium compound and cationic surfactant, is used in personal hygiene products such as toothpaste, mouthwash, and nasal spray. Although public exposure to CPC is frequent, its pulmonary toxicity has yet to be fully characterized. Due to high risks of CPC inhalation, we aimed to comprehensively elucidate the in vitro and in vivo toxicity of CPC. The results demonstrated that CPC is highly cytotoxic against the A549 cells with a half-maximal inhibitory concentration (IC50 ) of 5.79 µg/ml. Following CPC exposure, via intratracheal instillation (ITI), leakage of lactate dehydrogenase, a biomarker of cell injury, was significantly increased in all exposure groups. Further, repeated exposure of rats to CPC for 28 days caused a decrease in body weight of the high-exposure group and the relative weights of the lungs and kidneys of the high recovery group, but no changes were evident in the histological and serum chemical analyses. The bronchoalveolar lavage fluid (BALF) analysis showed a significant increase in proinflammatory cytokines interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α levels. ITI of CPC induced focal inflammation of the pulmonary parenchyma in rats' lungs. Our study demonstrated that TNF-α was the most commonly secreted proinflammatory cytokine during CPC exposure in both in vitro and in vivo models. Polymorphonuclear leukocytes in the BALF, which are indicators of pulmonary inflammation, significantly increased in a concentration-dependent manner in all in vivo studies including the ITI, acute, and subacute inhalation assays, demonstrating that PMNs are the most sensitive parameters of pulmonary toxicity.

In Vitro Evaluation of Mitochondrial Function and Estrogen Signaling in Cell Lines Exposed to the Antiseptic Cetylpyridinium Chloride.

BACKGROUND: Quaternary ammonium salts (QUATS), such as cetylpyridinium chloride (CPC) and benzalkonium chloride (BAK), are frequently used in antiseptic formulations, including toothpastes, mouthwashes, lozenges, throat and nasal sprays, and as biocides. Although in a recent ruling, the U.S. Food and Drug Administration (FDA) banned CPC from certain products and requested more data on BAK's efficacy and safety profile, QUATS, in general, and CPC and BAK, in particular, continue to be used in personal health care, food, and pharmaceutical and cleaning industries. OBJECTIVES: We aimed to assess CPC's effects on mitochondrial toxicity and endocrine disruption in vitro. METHOD: Mitochondrial O2 consumption and adenosine triphosphate (ATP) synthesis rates of osteosarcoma cybrid cells were measured before and after CPC and BAK treatment. Antiestrogenic effects of the compounds were measured by a luciferase-based assay using recombinant human breast carcinoma cells (VM7Luc4E2, ERalpha-positive). RESULTS: CPC inhibited both mitochondrial O2 consumption [half maximal inhibitory concentration (IC50): 3.8µM] and ATP synthesis (IC50: 0.9µM), and additional findings supported inhibition of mitochondrial complex 1 as the underlying mechanism for these effects. In addition, CPC showed concentration-dependent antiestrogenic activity half maximal effective concentration [(EC50): 4.5µM)]. BAK, another antimicrobial QUATS that is structurally similar to CPC, and the pesticide rotenone, a known complex 1 inhibitor, also showed mitochondrial inhibitory and antiestrogenic effects. In all three cases, there was overlap of the antiestrogenic activity with the mitochondrial inhibitory activity. CONCLUSIONS: Mitochondrial inhibition in vitro occurred at a CPC concentration that may be relevant to human exposures. The antiestrogenic activity of CPC, BAK, rotenone, and triclosan may be related to their mitochondrial inhibitory activity. Our findings support the need for additional research on the mitochondrial inhibitory and antiestrogenic effects of QUATS, including CPC and BAK. https://doi.org/10.1289/EHP1404.

Influence of surfactants and humic acids on Artemia Franciscana's embryonic phospho-metabolite profile as measured by 31P NMR.

Surfactants, such as triton X-100 (Tx-100), cetylpyridinium chloride (CPC), and sodium dodecyl sulfate (SDS) are known to be toxic to Artemia Franciscana (Artemia) - an organism, frequently used to monitor the health of the aquatic environment. The phospho-metabolite profile of a living organism is often indicative of imbalances that may have been caused by environmental stressors, such as surfactants. This study utilizes in vivo31P NMR to monitor temporal changes in the phospho-metabolite profile of Artemia caused by Tx-100, CPC, and SDS and the ability of humic acid (HA) to mitigate the toxicity of these surfactants. It was found that, while Tx-100 does not have any effect on the phospho-metabolite profile, both CPC and SDS cause a complete retardation in growth of the phosphodiester (PDE) peak in the 31P NMR spectrum, which is indicative of the inhibited cell replication. This growth inhibition was independently verified by the decreased guanosine triphosphate (GTP) concentration in the CPC and SDS-exposed Artemia. In addition, upon introduction of HA to the CPC and SDS-exposed Artemia, an increase of PDE peak over time is indicative of HA mitigating toxicity.

Developmental and acute toxicity of cetylpyridinium chloride in Bombina orientalis (Amphibia: Anura).

In an effort to evaluate the toxicity of cetylpyridinium chloride (CPC), a cationic surfactant in amphibians, we examined the developmental and acute toxicity of CPC in Bombina orientalis embryos and tadpoles. Embryonic exposure to 2.0µM (0.72mg/l) CPC for 7 days significantly decreased the survival rates and increased DNA damage in the intestine of developed tadpoles. Exposure to 1.5µM (0.54mg/l) CPC significantly decreased the growth of embryos and increased developmental abnormalities. The 168-h LC50 and EC50 values of CPC were 1.95µM (0.697mg/l) and 1.48µM (0.531mg/l) in embryos, respectively. In an extended acute toxicity test using tadpoles, the 168-h LC50 value of CPC was 5.07µM (1.82mg/l). In terms of survival and growth rates, the lowest observed effective concentration of CPC was 1.5µM. At sub-lethal concentrations (1.0 and 2.0µM) CPC treatment to embryos increased lipid peroxidation in the intestine and gills of developed tadpoles, indicating that CPC can impose oxidative stress. At 2.0µM CPC, pro-apoptotic Bax and Bak mRNA levels were significantly increased together with DNA fragmentation, indicative of apoptotic cell death. CPC in freshwater system may threaten the normal development of amphibian embryos.

Cetylpyridinium chloride and miramistin as antiseptic substances in chronic wound management - prospects and limitations.

The antimicrobial activity of cetylpyridinium chloride (CPC) and miramistin (MST) solutions at different concentrations (5×10(-5) to 0.4%) and a dressing, containing 0.15% CPC, were tested against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli after 30 (solutions) and 60 min (fleece) incubation, respectively. Furthermore, the cytotoxic effects of CPC and MST were examined in human keratinocyte (HaCaT) and murine fibroblast (L929) cell lines. A dose of 3×10(-3)% CPC or MST was sufficient to entirely eradicate S. aureus after 30 min incubation. To achieve the same effect, higher concentrations were required against E. coli (0.025% CPC; 0.0125% MST) and P. aeruginosa (0.5% CPC; 0.05% MST). The CPC-fleece showed a high antiseptic effect against all three bacterial strains, although it did not completely eliminate P. aeruginosa. Both substances showed a high cytotoxic impact at higher tested concentrations (CPC >3×10(-3)%; MST >8×10(-4)%). CPC showed high antimicrobial potency at low concentrations against S. aureus, accompanied by low cytotoxic (side) effects at these concentrations, whilst the required minimal concentration to eradicate E. coli and P. aeruginosa was shown to be cytotoxic for keratinocytes and fibroblasts. The necessary antibacterial amounts of MST were lower, but also cytotoxic in direct contact with typical human wound cells. With regard to demographic changes and increasing bacterial resistance, new effective antiseptics, such as CPC and MST, incorporated in wound dressings without releasing an active substance could help to improve the treatment and healing rates of chronic wounds.

Survival, mobility, and membrane-bound enzyme activities of freshwater planarian, Dugesia japonica, exposed to synthetic and natural surfactants.

Surfactants are a major class of emerging pollutants widely used in large quantities in everyday life and commonly found in surface waters worldwide. Freshwater planarian was selected to examine the effects of different surfactants by measuring mortality, mobility, and membrane-bound enzyme activities. Among the 10 surfactants tested, the acute toxicities of betaine and polyethylene glycol (PEG-200) to planarians were relatively low, with a median lethal concentration (LC50) greater than 10,000 mg/L. The toxicity to planarians of the other eight surfactants based on 48-h LC50 could be arranged in the descending order of cetylpyridinum chloride (CPC) > 4-tert-octylphenol (4-tert-OP) > ammonium lauryl sulfate > benzalkonium chloride > saponin > sodium lauroylsarcosinate > dioctyl sulfosuccinate > dodecyl trimethyl ammonium bromide (DTAB). Both CPC and 4-tert-OP were very toxic to planarians, with 48-h LC50 values <1 mg/L. The median effective concentrations (EC50s) of planarian mobility were in the 0.1 to 50 mg/L range and were in the same range as the 24-h LC50 of planarians exposed to different surfactants, except for DTAB. In addition, significant inhibition of cholinesterase activity activities was found in planarians exposed to 4-tert-OP at 2.5 and 5 mg/L and to saponin at 10 mg/L after 2-h treatments. This result suggests that planarian mobility responses can be used as an alternative indicator for acute toxicity of surfactants after a very short exposure period.

Cytogenetic damage induced by mouthrinses formulations in vivo and in vitro.

The aim of the present study was to comparatively evaluate DNA damage and cellular death in cells exposed to various commercially available mouthrinses: Listerine Cepacol, Plax alcohol free, Periogard, and Plax Whitening. A total of 75 volunteers were included in the search distributed into five groups containing 15 people each for in vivo study. Exfoliated buccal mucosa cells were collected immediately before mouthrinse exposure and after 2 weeks. Furthermore, blood samples were obtained from three healthy donors for in vitro study. The micronucleus test was used to evaluate mutagenicity and cytotoxicity in vivo. The single-cell gel (comet) assay was used to determine DNA damage in vitro. After 2 weeks exposure, Periogard showed 1.8% of micronucleated cells with significant statistical differences (p < 0.05) compared to before exposure (0.27%). Plax Whitening presented high tail moment value (4.5) when compared to negative control (0.6). The addition of all mouthrinses to cells incubated with methyl methanesulfonate did not alter the number of strand breaks in the genetic material. Listerine was able to reduce genetic damage induced by hydrogen peroxide because a decrease of tail moment was noticed. The results of the present study suggest that Periogard and Plax Whitening can induce genetic damage, whereas Listerine is an antioxidant agent. Since DNA damage is considered to be prime mechanism during chemical carcinogenesis, these data may be relevant in risk assessment for protecting human health and preventing carcinogenesis.

Airway effects of inhaled quaternary ammonium compounds in mice.

Quaternary ammonium compounds (QAC) constitute a family of widely used chemical substances. The QAC benzalkonium chloride (BAC) has caused bronchoconstriction in human beings by poorly understood mechanisms and lung damage at high concentration as shown in a single rat study. This study evaluates acute airway effects in mice after inhalation of aerosols of the QACs, BAC, hexadecyl trimethyl ammonium bromide (HTA), cetyl pyridinium chloride (CPC) and dimethyl dioctadecyl ammonium bromide (DDA). The QACs gave rise to concentration-dependent decreases in the tidal volume (VT) and a concomitant increase in respiratory rate indicating pulmonary irritation. The potencies of the QAC to induce these effects were in the order: BAC > HTA = CPC > DDA. Furthermore, inhalation of BAC and CPC aerosols gave rise to pulmonary inflammation as apparent from bronchoalveolar lavage. Stimulation of nasal trigeminal nerve endings by QAC, which may serve as a warning signal, was absent.

A new strategy to probe the genotoxicity of silver nanoparticles combined with cetylpyridine bromide.

The environmental genotoxic behavior of silver nanoparticles (nanoAg) combined with the detergent cetylpyridine bromide (CPB) was examined in vitro. The experimental results showed that the genotoxicity of nanoAg itself is weak, but nanoAg shows obvious genotoxicity after combined with CPB. The combined materials have a strong coeffect on calf thymus DNA (ctDNA) at a concentration of 3.3 x 10(-6 )gmL(-1) nanoAg and 6.0 x 10(-6) molL(-1) CPB. After the addition of ctDNA to the nanoAg-CPB system, the particles are scattered and the diameter decreases, which indirectly reveal that nanoAg-CPB has genotoxicity.

Kinetic analysis on the skin disposition of cytotoxicity as an index of skin irritation produced by cetylpyridinium chloride: comparison of in vitro data using a three-dimensional cultured human skin model with in vivo results in hairless mice.

PURPOSE: The aim of this study was to kinetically and dynamically analyze in vitro cytotoxicity as an index of skin irritation by use of a three-dimensional cultured human skin model and to compare the in vitro assay data with data from living animals. METHODS: A cationic surfactant, cetylpyridinium chloride (CPC), was selected as a model irritant. Living skin equivalent-high (LSE-high) and hairless mice were used for the in vitro and in vivo tests, respectively. Skin irritation dermatodynamics was evaluated by calorimetric thiazoyl blue (MTT) conversion assay both for in vitro and in vivo tests, whereas dermatokinetics of CPC in LSE-high and mouse skin were evaluated using HPLC. RESULTS: The time course of cell viability in the skin after application of CPC to intact skin was distinctly different from that of stratum-corneum-stripped skin in both LSE-high and hairless mice. Biphasic behavior characterized by two first-order rates with an inflection time point was observed in intact skin, whereas cell viability monoexponentially decreased immediately after CPC application in stripped skin. The time courses of cell viability in the skin and dermatodynamics were closely related to that of dermatokinetics of CPC. CONCLUSION: The present study demonstrates that the in vitro cytotoxic profile was similar to the in vivo cytotoxicity test and that dermatodynamics was related to dermatokinetics of CPC.

Adjuvant effect of quaternary ammonium compounds in a murine model.

It has been suggested that occupational exposure to quaternary ammonium compounds (QACs) may promote the development of allergic airway diseases. In this study, hazard identifications of the adjuvant effect of cetylpyridinium chloride (CPC), dimethyldioctadecylammonium bromide (DDA), hexadecyltrimethylammonium bromide (HTA), and tetraethylammonium chloride (TEA) were performed in a screening bioassay. Female BALB/c mice were injected subcutaneously with the model allergen ovalbumin (OVA) alone or together with different quantities of one of the QAC test compounds. After one or two boosters, levels of OVA-specific IgE, IgG1 and IgG2a antibodies were measured in sera. CPC and DDA increased IgE and IgG1 antibody production, respectively, compared to the OVA control group, whereas HTA and TEA showed no adjuvant effect. Nevertheless, when TEA was given in combination with DDA, the adjuvant effect was up to six-fold higher than the adjuvant effect of DDA alone. Only DDA had a statistically significant adjuvant effect on IgG2a antibody levels.

Utility of MTT assay in three-dimensional cultured human skin model as an alternative for draize skin irritation test: approach using diffusion law of irritant in skin and toxicokinetics-toxicodynamics correlation.

PURPOSE: A cytotoxicity assay using a three-dimensional cultured human skin model, Living Skin Equivalent-high (LSE-high) was evaluated as an alternative to the Draize skin irritation tests using animals. A relation between the cytotoxicity and calculated concentration of an irritant in skin was also evaluated. METHODS: Colorimetric thiazoyl blue (MTT) conversion assay and a surfactant, cetylpyridinium chloride (CPC), were selected as a cytotoxicity assay and a model irritant. The fraction of dead cell number in the MTT assay or the Draize irritation score (in vitro and in vivo irritation data, respectively) was treated as a function of CPC concentration in the viable skin of LSE-high and guinea pig. Separately, in vitro permeations of CPC through the LSE-high or excised guinea pig skin were determined to calculate the average concentration of CPC in the viable skin using the Fickian diffusion theory. The obtained relations between the irritation scores and CPC concentration were evaluated by the Emax model (Hill equation). RESULTS: CPC concentration showing 50% irritation (IC50) was similar for the MTT assay (18.9%) and Draize test (12.3%), and a good relationship (r = 0.981) was observed between the fraction of dead cell number and the Draize score. In contrast, IC50, 1.32%, for the MTT assay in LSE-high was much lower than that using guinea pig skin. We then corrected the results for the MTT assay using a ratio of IC50 in guinea pig skin against LSE-high, resulting in a good relation between both MTT results in guinea pig skin and LSE-high. CONCLUSION: The present results suggest that the MTT assay using LSE-high may be utilized as an alternative for the Draize test in animals for evaluating skin irritation.

Cationic solid-lipid nanoparticles can efficiently bind and transfect plasmid DNA.

The suitability of cationically modified solid-lipid nanoparticles (SLN) as a novel transfection agent was investigated. SLN were produced by hot homogenisation using either Compritol ATO 888 or paraffin as matrix lipid, a mixture of Tween 80 and Span 85 as tenside and either EQ1 (N,N-di-(beta-steaorylethyl)-N,N-dimethylammonium chloride) or cetylpyridinium chloride as charge carrier. The resulting particles were approximately 100 nm in size and showed zeta potentials around +40 mV at pH 7.4. DNA binding was tested by agarose gel electrophoresis. The resulting SLN-DNA complexes were further characterised by AFM and zeta potential measurements. Only the SLN batch SII-13, composed of 4% Compritol, 4% Tween/Span and 1% EQ1, was able to form stable complexes with DNA. Typical complexes were 300 to 800 nm in size. Cytotoxicity and transfection efficiency was tested in vitro on Cos-1 cells. Cationic SLN produced by modification with EQ1 were well tolerated, with LD50 values >3 mg/ml in the LDH release assay and >0.6 mg/ml in the WST-1 assay. Further, SLN-DNA complexes containing between 10 and 200 weight equivalents of SII-13 (matrix lipid) efficiently transfected the galactosidase expression plasmid pCMVbeta in the absence and presence of the endosomolytic agent chloroquine.

Ocular and dermal irritation studies of some quaternary ammonium compounds.

The primary ocular and dermal irritations of four quaternary ammonium compounds, namely cetylpyridinium chloride (CPC), stearylphenylethyldimethylammonium tosylate (SPDAT), dimethyldistearylammonium bisulfate (DDABS) and tri(N-butyl)benzylammonium 4-hydroxynapththalene-1-sulfonate (TBAHNS), were studied. Both CPC and SPDAT were extremely or severely irritating to the eyes of the test animals, whereas DDABS and TBAHNS were mildly or minimally irritating. Both CPC and SPDAT were also severely or extremely irritating to the skin of the test animals, while DDABS and TBAHNS were non-irritating. These quaternary ammonium compounds have little similarity in chemical structure and possess different solubilities. CPC is very soluble in both lipid and water; SPDAT is very soluble in lipid but only slightly soluble in water; and DDABS and TBAHNS are poorly soluble in either lipid or water. The irritancy of these compounds is likely to be related to their solubility, in addition to the cationic characteristics. It appears that not all quaternary ammonium compounds in this study are irritants. Those that are not soluble are not expected to be absorbed in eye/skin tissues and thus irritation reactions will not take place. The use of in vitro alternatives should be considered when assessing the ocular and dermal irritancy potential of water- and lipid-soluble quaternary ammonium compounds.

Acute inhalation toxicity of cetylpyridinium chloride.

Cetylpyridinium chloride (CPC) is a quaternary ammonium salt and cationic surfactant. It has been used as a biocide in personal hygiene products and a charge control additive in some reprographic toners. CPC is orally toxic to rats, mice and rabbits and can cause severe eye irritation. Acute inhalation toxicity studies of CPC and other quaternary ammonium salts have not, however, been reported. Groups of five rats per sex were exposed to aerosols containing 0 (control), 0.05, 0.07, 0.13 and 0.29 mg CPC/litre for 4 hr and observed for toxicity and ocular effects for 14 days thereafter. All animals were subjected to autopsy and the eyes were examined microscopically. The LC50 (sexes combined) of CPC was 0.09 mg/litre with upper and lower 95% confidence limits of 0.13 and 0.07 mg/litre, respectively. Clinical signs of toxicity included weight loss, nasal discharge, chromodacryorrhoea, respiratory difficulty and eye irritation, and all these non-lethal effects were reversible. Acute inflammation of the cornea, iris and/or aqueous humour were found in one, seven and four of 10 rats exposed to 0.07, 0.13 and 0.29 mg CPC/litre, respectively. Corneal epithelial hyalinization, without evidence of ongoing inflammation, was found in three additional rats among the 10 exposed to 0.29 mg CPC/litre.