In vitro evaluation for estrogenic mechanisms of the disinfectant benzalkonium chloride as an emerging contaminant

This study aimed to evaluate in vitro the possible mechanisms underlying the estrogenic potential of benzalkonium chloride (BAC) as a disinfectant emerging contaminant. Effects of BAC at the environmentally-relevant concentrations on estrogen synthesis and estrogen receptor (ER) signaling were assessed using the H295R steroidogenesis assay and the MCF-7 proliferation assay, respectively. Results showed that exposure to BAC at concentrations of 1.0-1.5 mg/L for 48 h significantly increased estradiol production of H295R cells in a concentration-dependent manner. Transcription of steroidogenic genes 3β‐HSD2, 17β‐HSD1, 17β‐HSD4, and CYP19A were significantly enhanced by BAC. In ER-positive MCF-7 cells, exposure to 0.5-1.5 mg/L BAC for 48 h significantly promoted cell proliferation and increased the expressions of ERα and G-protein coupled estrogen receptor 1. Flow cytometry analysis showed that 0.5-1.5 mg/L BAC significantly decreased the percentage of cells in G0/G1 phase, increased the percentage in S phase, and BAC at concentrations of 1.0 and 1.5 mg/L increased the G2/M phase cells. Findings of the study suggested that BAC at environmentally-relevant concentrations might act as a xenoestrogen through its inhibitory effect on steroidogenesis and ER-mediated mechanism.

Analysis of clinical features of seven patients with granular parakeratosis after benzalkonium chloride exposure / 中华皮肤科杂志

Objective:To summarize clinical manifestations and histopathological features of granular parakeratosis (GP) after exposure to benzalkonium chloride.Methods:A retrospective analysis was performed on 7 GP cases with a history of benzalkonium chloride exposure in the Department of Dermatology at Wuhan No.1 Hospital from April to August 2022. Data were collected on the general condition, skin lesion manifestations, pathological examination, treatment, and follow-up of the patients, and retrospectively analyzed.Results:The 7 adult patients with GP typically presented with erythema and brown scales in the intertriginous area, exhibiting an annular distribution pattern. All the 7 patients reported recent exposure to disinfectants containing benzalkonium chloride. A total of 10 skin biopsies were taken from the 7 patients. Histopathological examination showed characteristic hyperkeratosis and fine blue-gray parakeratotic granules in the stratum corneum. All skin lesions improved 1 month after cessation of exposure to benzalkonium chloride.Conclusion:GP has a distinct clinical pattern and histopathological manifestations, and a history of exposure to benzalkonium chloride can be helpful for the diagnosis of GP.

Influence of EDTA and its Association with Benzalkonium Chloride on Enterococcus faecalis Adhesion to Dentin / Influencia del EDTA y su Asociación con Cloruro de Benzalconio en la Adhesión de Enterococcus faecalis a la Dentina

ABSTRACT: The aim of this in vitro study was to investigate the influence of ethylenediaminetetraacetic acid (EDTA) associated with the benzalkonium chloride (BAK) on the adhesion and formation of Enterococcus faecalis biofilms attached to coated dentin. Discs standard bovine dentin blocks were treated with the coating materials evaluated: Saline solution (control), 17 % EDTA, 17 % EDTA associated with 1 % BAK for 5 minutes and subsequently washed with saline solution. Afterwards, biofilms of E. faecalis (ATCC 29212) were grown on the surface of coated dentin blocks for time intervals of 1 hour and 7 days (n = 20) and were subsequently washed with phosphate-buffered saline (PBS). Bacterial viability and total biovolume were analyzed by confocal laser scanning microscopy (CLSM) using the Live/Dead technique. Nonparametric Kruskal-Wallis followed by Dunn tests were used to determine statistical differences (a = 5 %). The 17 % EDTA + 1 % BAK group showed significantly lower biovolume and bacterial viability values at the end of 1 hour (p < 0.05). After 7 days of contamination, the 17 % EDTA and 17 % EDTA + 1 % BAK groups showed similar results that differed statistically from those of the control group (p < 0.05). The saline solution group showed higher values. The use of BAK associated with EDTA on dentin blocks surfaces before exposure to contamination was able to interfere in the adhesion of E. faecalis to dentin. Also, dentin treatment by BAK associated with a chelating agent influences the secondary biofilm formation, which could have important effects on the long-term success of root canal treatment.

RESUMEN: El objetivo del estudio consistió en investigar in vitro, la influencia del ácido etilendiamino-tetraacético (EDTA) con cloruro de benzalconio (BAK) en la adhesión y formación de biopelículas de Enterococcus faecalis a la dentina. Discos de dentina bovina fueron tratadas con solución salina (control), 17 % de EDTA, 17% de EDTA asociado con 1 % de BAK durante 5 minutos y lavadas con solución salina. Las biopelículas de E. faecalis (ATCC 29212) se cultivaron sobre los discos de dentina durante intervalos de tiempo de 1 hora y 7 días (n = 20), lavados con solución salina tamponada con fosfato (PBS). La viabilidad bacteriana y el biovolumen total se analizaron mediante microscopía de barrido por láser (CLSM) utilizando la técnica Live / Dead. Se realizó prueba no paramétrica de Kruskal-Wallis, seguida por Dunn con una diferencia estadística (a = 5 %). El grupo de 17 % EDTA + 1 % BAK mostró valores significativamente menores de biovolumen y viabilidad bacteriana al final de 1 hora (p < 0,05). Después de 7 días de contaminación, los grupos de 17 % EDTA y 17 % EDTA + 1 % BAK mostraron resultados similares que diferían estadísticamente del grupo control (p < 0,05). La solución salina mostró valores más altos. La asociación de BAK con EDTA antes de la contaminación interfirió en la adhesión de E. faecalis. Además, el tratamiento de la dentina por BAK asociado con EDTA influye en la formación de biopelículas secundarias, lo que podría tener efectos importantes sobre el éxito a largo plazo del tratamiento del conducto radicular.

Studies on Antimicrobial Preservatives Screening and Antimicrobial Efficacy of Diclofenac Sodium Eye Drops / 中国药学杂志

OBJECTIVE: To evaluate the bacteriostatic efficacy of diclofenac sodium eye drops and to explore the reasonable dose of benzalkonium chloride, ethylparaben and thimerosal in diclofenac sodium eye drops. METHODS: According to the method of bacteriostatis effect test in 2015 edition of Chinese Pharmacopoeia (Ch.P),and Escherichia coli, Staphylococcus aureus, Pesudomonas aeruginosa, Candida albicans and Aspergillus niger as test strains,the bacteriostatis effect of diclofenac sodium eye drops from 10 batches of the samples was determined. Also, the concentration gradient of benzalkonium chloride, ethylparaben and thimerosal in diclofenac sodium eye drops was designed to investigate the optimum bacteriostatic concentration. RESULTS: The samples from 3 manufacturers could reach level B, no sample could reach level A,and those from 7 manufacturers did not comply with the specification. When the concentration of thimerosal was 0.01 mg•mL-1 and the concentration of ethylparaben was 0.3 mg•mL-1 in diclofenac sodium eye drops, the bacteriostatic efficacy could reach level B. When the concentration of benzalkonium chloride was 0.01 mg•mL-1, the bacteriostatic efficacy could reach level A. CONCLUSION: The bacteriostatis effect of diclofenac sodium eye drops from 10 batches of the samples is not good, It is recommended that these manufacturers should optimize the type and concentration of antimicrobial agents and optimize their formulation based on both biological tests and physical-chemical tests to ensure drug safety.

Effects of benzalkonium chloride and cyclosporine applied topically to rabbit conjunctiva: a histomorphometric study / Efeitos do uso tópico do conservante cloreto de benzalcônio e ciclosporina na conjuntiva de coelhos: estudo histomorfométrico

ABSTRACT Purpose: Chronic instillation of benzalkonium chloride, a preservative, has inflammatory effects on the ocular surface. However, addition of the anti-inflammatory agent cyclosporine to a therapeutic protocol may mitigate these effects. This study compared the toxic effects of a 0.1% benzalkonium chloride solution and the possible protective effect of 0.05% cyclosporine when applied topically to the rabbit conjunctiva. Methods: Fifteen age- and weight-matched, female New Zealand white rabbits were categorized into three groups and treated for 30 consecutive days. Group 1, 2, and 3 - benzalkonium chloride received 0.1% every 24 h, 0.05% cyclosporine every 6 h, and both treatments, respectively. In each rabbit, the left eye was subjected to treatment and the right eye was a control. The rabbits were euthanized at after the experiment. Goblet cells and blood vessels were then enumerated in conjunctival tissues stained with periodic acid-Schiff and hematoxylin-eosin, respectively. Differences between treated and untreated eyes and between groups were compared using the t-test and analysis of variance. Results: Benzalkonium chloride treatment, with and without cyclosporine, significantly reduced (p≤0.05) in the number of goblet cells in treatment eyes compared with that in respective control eyes. Alternatively, adding cyclosporine to benzalkonium chloride did not prevent the loss of conjunctival goblet cells, and a significant reduction in the number of goblet cells was noted. Benzalkonium chloride-induced significant increase in the number of new blood vessels was mitigated significantly by the addition of cyclosporine. Conclusion: This study demonstrated the magnitude of conjunctival injury caused by chronic instillation of benzalkonium chloride. Although cyclosporine did not mitigate the effects on goblet cells, its addition minimized inflammatory angiogenesis induced by benzalkonium chloride.

RESUMO Objetivo: A instilação crônica de cloreto de benzal­cônio, um conservante, tem efeitos inflamatórios na superfície ocular. No entanto, a adição do agente anti-inflamatório ciclosporina a um protocolo terapêutico pode atenuar esses efeitos. Este estudo comparou os efeitos tóxicos de uma solução de cloreto de benzalcônio a 0,1% e o possível efeito protetor de ciclosporina a 0,05% quando aplicado topicamente à conjuntiva de coelho. Métodos: Quinze coelhos fêmeas brancos da raça Nova Zelândia, pareados por idade e peso, foram categorizados em três grupos e tratados por 30 dias consecutivos. Os grupos 1, 2 e 3 - receberam cloreto de benzalcônio 0,1% a cada 24h, ciclosporina a 0,005% a cada 6h e ambos os tratamentos, respectivamente. Em cada coelho, o olho esquerdo foi submetido a tratamento e o olho direito foi controle. Os coelhos foram submetidos à eutanásia após o experimento. Células caliciformes e vasos sanguíneos foram então enumerados em tecidos conjuntivais corados com ácido periódico-Schiff e hematoxilina-eosina, respectivamente. As diferenças entre os olhos tratados e não tratados e entre os grupos foram comparadas usando o teste t e análise de variância. Resultados: O tratamento com cloreto de benzalcônio, com e sem ciclosporina, reduziu significativamente (p£0,05) o número de células caliciformes nos olhos tratados em comparação com os olhos controle correspondentes. Alternativamente, a adição de ciclosporina ao cloreto de benzalcônio não impediu a perda de células caliciformes conjuntivais, e foi observada uma redução significativa no número de células caliciformes. O aumento significativo induzido pelo cloreto de benzalcônio no número de novos vasos sanguíneos foi significativamente mitigado pela adição da ciclosporina. Conclusão: Este estudo demonstrou a magnitude da lesão conjuntival resultante da instilação crônica de cloreto de benzalcônio. Embora a ciclosporina não tenha atenuado os efeitos nas células caliciformes, sua adição minimizou a angiogênese inflamatória induzida pelo cloreto de benzalcônio.

A new experimental model of intrinsic denervation in ileum from wistar rats through intramural microinjections of benzalkonium chloride / Um novo modelo experimental de desnervação intrínseca em íleo de ratos wistar através de micro injeções intramurais de cloreto de benzalcônio

Extensive literature is available about the intrinsic denervation of segments of the digestive tube through the application of CB in the serosa of the viscera. However, this technique has some disadvantages like causing peritonitis, flanges and high mortality, limiting its use in humans. The aim of the present study was to evaluate the feasibility of benzalkonium chloride (CB) to induce intrinsic chemical denervation, through applications of CB in the intramural ileum of wistar rats, as well as deepen the knowledge about the evolution of neuronal injury caused in the process. We used 40 rats, divided into two groups (control-GC and benzalkonium-GB) of 20 animals each, divided into four sub-groups according to the time of postoperative assessment of 24, 48 hours, 30 and 90 days. The animals were submitted to intramural microinjections of sterile saline solution 0.9% (GC) or benzalkonium chloride (GB) in ileal portion, and subsequent histopathological analysis and immunohistochemistry for evaluation of neuronal injury. A significant decrease (p<0.05) was found of the neuronal myenteric count over time in groups, GB3, GB4 and GB2. The specific positive immunolabeling for H2AX and Caspase-3 confirmed the results obtained in the histopathological evaluation, denoting the ignition of irreversible cell injury in 24 hours, evolving into neuronal apoptosis in 48 hours after application of the CB 0.3%. Under the conditions in which this work was conducted, it can be concluded that the application of CB 0.3% by means of microinjections intramural in the ileal wall is able to induce intrinsic chemical denervation of the diverticulum of wistar rats and that the main mechanism of neuronal death is induction of apoptosis.(AU)

Existe vasta literatura sobre a desnervação intrínseca de segmentos do tubo digestório através da aplicação de CB na serosa da víscera. Entretanto, essa técnica tem a desvantagem de causar peritonite, formação de bridas e alta mortalidade, não sendo factível para eventuais utilizações em humanos. O objetivo do presente estudo foi avaliar a viabilidade do Cloreto de benzalcônio (CB) induzir desnervação química intrínseca, por meio de aplicações intramurais em íleo de ratos wistar, além de aprofundar o conhecimento sobre a evolução da lesão neuronal causada neste processo. Foram utilizados 40 ratos, distribuídos em dois grupos (controle- GC e benzalcônio- GB) de 20 animais cada, subdivididos em quatro subgrupos de acordo com o tempo de avaliação pós-operatória de 24, 48 horas, 30 e 90 dias. Os animais foram submetidos à microinjeções intramurais de solução salina estéril 0,9% (GC) ou de cloreto de benzalcônio (GB) em porção ileal, e posterior análise histopatológica e imuno-histoquímica, para avaliação da lesão neuronal. Houve diminuição significativa (p<0,05) na contagem neuronal mientérica ao longo do tempo nos grupos GB2, GB3 e GB4. A imunomarcação específica positiva para H2AX e Caspase-3 confirmou os resultados obtidos na avaliação histopatológica, denotando início da lesão celular irreversível em 24 horas, evoluindo para apoptose neuronal em 48 horas após a aplicação do CB 0,3%. Nas condições em que este trabalho foi conduzido, é possível concluir que a aplicação de CB 0,3% por meio de microinjeções intramurais na parede ileal é capaz de induzir desnervação química intrínseca da porção ileal de ratos wistar e que o principal mecanismo de morte neuronal é a indução de apoptose.(AU)

Effect of Chronic Benzalkonium Chloride Exposure on Senescence in Trabecular Meshwork Cells

PURPOSE: To determine the possible effects of chronic exposure of low dose benzalkonium chloride (BAK) on trabecular meshwork cells, and to characterize the pathways involved in the effects. METHODS: Trabecular meshwork cells were treated with 0.0005%, 0.00075%, 0.001%, and 0.0025% BAK for 10 minutes; then, the cells were transferred to a new medium for 24 hours. This process was repeated three times. Cell survival was assessed using the MTT assay to determine the non-apoptotic BAK concentration. Senescence-associated (SA)-β-gal staining was performed to compare quantitatively the cellular senescence of BAK-treated cells with the control group. Cells treated with BAK were analyzed by western blot to determine whether the expressions of cell cycle regulators were affected. RESULTS: Two concentrations (0.0005% and 0.00075%) showed persistent cell viability and were chosen for further experiments. After SA-β-gal staining, cells treated with 0.0005% and 0.00075% BAK showed 28% (± 2.08), 37% (± 2.08) increases in cellular senescence expression, respectively, when compared with control cells (p < 0.05). To identify the molecular pathways involved in cell cycle arrest via BAK, western blot analysis was performed on trabecular meshwork cells, resulting in decreased expressions of cyclin E/CDK2, and increased expressions of the upper stream control molecules, p53 and p21. CONCLUSIONS: Chronic exposure to low dose BAK accelerated cell senescence through cell cycle arrest. Because senescent cells of the trabecular meshwork can inhibit its outflow pathway function and ultimately worsen the glaucomatous process, long-term usage of topical glaucoma medications containing BAK should be conducted with caution.

Antimicrobial effectiveness of domestic timolol maleate eye drops / 中国药学杂志

OBJECTIVE: To screen out suitable antibacterial agent and reasonable dosage for timolol maleate eye drops. METHODS: According to the method of bacteriostatic effect test in Chinese Pharmacopoeia (Ch.P) 2015, the antimicrobial effectiveness of antimicrobial preservatives at different concentrations was tested to screen out the optimal agent and dosage. RESULTS: The eye drops added with ethyl hydroxybenzoate met the B-level requirements of bacteriostatic efficacy, which was the same as the sample without antibacterial agent, so ethyl hydroxybenzoate was not suitable as the antimicrobial preservative. The growth of five kinds of test microorganisms was inhibited effectively by 0.03 mg·mL-1 benzalkonium bromide. The timolol maleate eye drops with 0.03 mg·mL-1 benzalkonium bromide met the requirement of the quality standard. CONCLUSION: Benzalkonium bromide can be used as the antimicrobial preservative for timolol maleate eye drops.

Benzalkonium chloride effectiveness as a disinfectant against hospital associated methicillin-resistant Staphylococcus aureus (HA-MRSA)

Aims@#Benzalkonium chloride is used to disinfect hospital instruments to prevent nosocomial infection caused by microorganisms, such as Methicillin Resistant Staphylococcus aureus (MRSA). There are strains of MRSA isolated from hospitals that were found to be resistant towards benzalkonium chloride. This research was aimed to compare the affectivity of different concentrations of benzalkonium chloride to inhibit the growth of Hospital-Associated MRSA (HA-MRSA) and determine the Minimum Inhibitory Concentration (MIC) of benzalkonium chloride against HA-MRSA. @*Methodology and results@#The samples were five HA-MRSA isolates obtained from Dr. Soetomo Hospital Surabaya. It was identified by amplification of SCCmec genes. The HA-MRSA with SCCmec type III was divided into six flasks based on the concentration of benzalkonium chloride in their inoculation media (0 μg/mL, 0.625 μg/mL, 1.25 μg/mL, 2.5 μg/mL, 5 μg/mL, and 10 μg/mL). The growth of HA-MRSA in media was determined by the number of colonies after treatment. The result showed that the MIC of benzalkonium chloride for HA-MRSA was 5 μg/mL, where no growth of bacterial was observed. There was significant difference in MRSA colony count between different groups of benzalkonium chloride concentrations (p = 0.001), and there was negative correlation between benzalkonium chloride concentration and HA-MRSA growth (p = 0.0001 and r = -0.880). @*Conclusion, significance and impact of study@#The concentration of benzalkonium chloride influences the growth of HA-MRSA. The higher the concentration, the fewer HA-MRSA growth. Application of benzalkonium chloride according to MIC will prevent HA-MRSA resistance towards benzalkonium chloride.

Effects of Preservative on the Meibomian Gland in Glaucoma Patients Treated with Prostaglandin Analogues / 전남의대학술지

This study compared the effect of preservative-containing (PC) and preservative-free (PF) prostaglandin analogue (PGA) formulations on the ocular surface, especially on the meibomian gland (MG) in patients with open-angle glaucoma (OAG). This is a retrospective study of treatment-naïve patients with OAG (n=80) and healthy controls (n=40). OAG patients were randomized into groups using either PC-PGA or PF-PGA for 12 months. All participants underwent ocular surface and MG examinations including their meibum score, meiboscore, and lid margin abnormality score (LAS). Eighty OAG patients were randomized into two groups (n=42 in PC, n=38 in PF). All PGA and control groups showed similar ocular surface and MG parameters at the baseline. Both PC- and PF-PGA groups showed increased meibum scores, meiboscores, and LASs at 12 months compared to the baseline (all p<0.05). At the 12-months visit, PC-PGA group showed severe OSDI, shorter TBUT, greater OSS, and worse MG parameters than those of the other two groups (all p<0.05). In addition, PF-PGA group showed worse meiboscores, meibum scores, and severe OSS scores than those of the control group (all p<0.05). Both PC and PF formulations can cause damage to the MG in patients using PGA. However, PC formulations induced more ocular discomfort, poorer ocular surface, and more severe MG loss compared to PF formulations. Therefore, it would be advisable to use PF formulations in patients with a preexisting or concomitant ocular surface disease or MGD.

Dermatite de contato por cloreto de benzalcônio / Contact dermatitis from benzalkonium chloride

O cloreto de benzalcônio é um conservante encontrado em produtos de higiene pessoal e preparações farmacêuticas há mais de seis décadas. Antes tido como irritante; porém, evidências crescentes apontam que ele possa induzir alergia de contato em uma taxa mais elevada do que o previsto. Os autores apresentam um caso de um adulto de 71 anos, com reação alérgica ao cloreto de benzalcônio contido em solução tópica oftalmológica. Pretende-se, com este caso, alertar sobre as possíveis hipersensibilidades aos conservantes de medicamentos e cosméticos.

Benzalkonium chloride is a preservative found in personal care products and pharmaceutical preparations for over six decades. It was previously thought to be an irritant, but a growing body of evidence suggests that it may induce contact allergy at a higher rate than anticipated. The authors describe the case of a 71-yearold male patient with allergic reaction to benzalkonium chloride present in a topical ophthalmic solution. With this case report we intend to warn about possible hypersensitivity reactions to preservatives contained in medicines and cosmetics.

Preventive effect of Angelica gigas Nakai extract oral administration on dry eye syndrome

Objective:To identify the preventive effect of Angelica gigas Nakai (A. gigas Nakai) extract in a benzalkonium chloride-induced dry eye model.Methods:A total of 28 mice were divided into 4 groups: 1) Normal group: mice received only saline; 2) positive control group: mice received an oral solution without A. gigas Nakai extract at 10:00 a.m. and 0.2% benzalkonium chloride eye drops at 2:00 p.m.; 3) A. gigas Nakai extract (5 mg); 4) A. gigas Nakai extract (10 mg). Both group 3) and group 4) received an oral solution with A. gigas Nakai extract (either 5 mg/kg or 10 mg/kg) at 10:00 a.m. and 0.2% benzalkonium chloride eye drops at 2:00 p.m. After 14 d of follow-up, tear volume measurement and fluorescein staining were evaluated for the recovery effects on ocular surface. Histologic analysis was conducted by hematoxylin and eosin staining. Apoptosis on ocular epithelium layer was examined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Expression of TNF- α was also measured using western blot analysis.Results:An increase in both the tear volume and the sustained fluorescein staining scores was observed, demonstrating the preventive effects of A. gigas Nakai extract. Structure changes such as irregularity of the epithelial layer and corneal epithelial cell death were inhibited in the A. gigas Nakai extract groups. Expression of TNF- α moderately declined; however, its expression level was still higher, compared to the normal group.Conclusions:Results from the current study show the significant preventive effect of A. gigas Nakai extract in a mouse model of benzalkonium chloride-induced dry eye syndrome. Thus, A. gigas Nakai extract could be considered as an oral preventive agent for dry eye syndrome in the future.

Effects of benzalkonium bromide and citalopram on the thickness of corneal epithelium and full-thickness of the cornea in mice / 眼科新进展

Objective To explore the effects of benzalkonium bromide and citalopram on the corneal epithelium and corneal thickness of mice using optical coherence tomography angiography (OCTA).Methods Together 60 mice were randomly divided into 5 groups (group A,B,C,D and E;n =12),with group A left untreated,group B receiving PBS eye drops,group C given benzalkonium bromide eye drops,group D undergoing intraperitoneal administration of citalopram suspension,and group E treated with combination of benzalkonium bromide eye drops and citalopram suspension.After 2 weeks,OCTA was applied for corneal subarea,followed by measurement of the thickness of corneal epithelium and full-thickness of the cornea of all mice,and then the mean values were calculated.Results The thickness of corneal epithelium and fullthickness of the cornea was (66 ±7) μm and (141 ± 11) μm in the group A,(66 ± 8) μm and (140 ± 12) μm in the group B and D,(73 ± 10) μm and (141 ± 14) μm in the group C,(76 ± 12) μm and (141 ± 15) μm in the E group,respectively.And there was no significant difference in the thickness of corneal epithelium and full-thickness of the cornea before treatment and 2 weeks after treatment in the group A,B and D (all P ＞ 0.05),but both variables were markedly thickened in group C and E 2 weeks after treatment,and the difference was statistically significant (all P ＜0.05).Moreover,the increased levels on the both variables in the group E was higher than those in the group C 2 weeks after treatment,and the difference was statistically significant (both P ＜ 0.05).The average thickness of corneal epithelium and full-thickness of the cornea in the group C and E were significantly thickened after treatment,and the difference was statistically significant (all P ＜ 0.05).The average values of both variables in the group C and E were obviously larger than those in the group A,and the difference was statistically significant (all P ＜ 0.05).Conclusion Citalopram alone has no significant effects on the corneal thickness by OCTA,whereas both the thickness of corneal epithelium and fullthickness of the cornea tend to thicken by benzalkonium bromide treatment,which has a synergistic effect on corneal thickening with citalopram.

Effects of Several Cosmetic Preservatives on ROS-Dependent Apoptosis of Rat Neural Progenitor Cells

Benzalkonium chloride, diazolidinyl urea, and imidazolidinyl urea are commonly used preservatives in cosmetics. Recent reports suggested that these compounds may have cellular and systemic toxicity in high concentration. In addition, diazolidinyl urea and imidazolidinyl urea are known formaldehyde (FA) releasers, raising concerns for these cosmetic preservatives. In this study, we investigated the effects of benzalkonium chloride, diazolidinyl urea, and imidazolidinyl urea on ROS-dependent apoptosis of rat neural progenitor cells (NPCs) in vitro. Cells were isolated and cultured from embryonic day 14 rat cortices. Cultured cells were treated with 1–1,000 nM benzalkonium chloride, and 1–50 μM diazolidinyl urea or imidazolidinyl urea at various time points to measure the reactive oxygen species (ROS). PI staining, MTT assay, and live-cell imaging were used for cell viability measurements. Western blot was carried out for cleaved caspase-3 and cleaved caspase-8 as apoptotic protein markers. In rat NPCs, ROS production and cleaved caspase-8 expression were increased while the cell viability was decreased in high concentrations of these substances. These results suggest that several cosmetic preservatives at high concentrations can induce neural toxicity in rat brains through ROS induction and apoptosis.

Preventive effect of Angelica gigas Nakai extract oral administration on dry eye syndrome

Objective: To identify the preventive effect of Angelica gigas Nakai (A. gigas Nakai) extract in a benzalkonium chloride-induced dry eye model. Methods: A total of 28 mice were divided into 4 groups: 1) Normal group: mice received only saline; 2) positive control group: mice received an oral solution without A. gigas Nakai extract at 10:00 a.m. and 0.2% benzalkonium chloride eye drops at 2:00 p.m.; 3) A. gigas Nakai extract (5 mg); 4) A. gigas Nakai extract (10 mg). Both group 3) and group 4) received an oral solution with A. gigas Nakai extract (either 5 mg/kg or 10 mg/kg) at 10:00 a.m. and 0.2% benzalkonium chloride eye drops at 2:00 p.m. After 14 d of follow-up, tear volume measurement and fluorescein staining were evaluated for the recovery effects on ocular surface. Histologic analysis was conducted by hematoxylin and eosin staining. Apoptosis on ocular epithelium layer was examined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Expression of TNF- α was also measured using western blot analysis. Results: An increase in both the tear volume and the sustained fluorescein staining scores was observed, demonstrating the preventive effects of A. gigas Nakai extract. Structure changes such as irregularity of the epithelial layer and corneal epithelial cell death were inhibited in the A. gigas Nakai extract groups. Expression of TNF- α moderately declined; however, its expression level was still higher, compared to the normal group. Conclusions: Results from the current study show the significant preventive effect of A. gigas Nakai extract in a mouse model of benzalkonium chloride-induced dry eye syndrome. Thus, A. gigas Nakai extract could be considered as an oral preventive agent for dry eye syndrome in the future. http://www.apjtm.org/article.asp?issn=1995-7645;year=2018;volume=11;issue=6;spage=369;epage=375;aulast=Lee;type=2.

The antibacterial activity and release of quaternary ammonium compounds in an orthodontic primer

The aim of this study was to evaluate the impact of 10 wt% benzalkonium chloride (TBBAC) or 10 wt% cetylpyridinium chloride (TBCPC) on the antimicrobial properties of the orthodontic adhesive primer, Transbond XT™ (TB). Antimicrobial activity was assessed using a zone of inhibition diffusion test and the release of the antimicrobial compounds was monitored by high performance liquid chromatography (HPLC). Shear bond strength (SBS) was tested using bovine enamel. Control, TB, specimens failed to demonstrate intrinsic antibacterial activity at 1, 7 and 14 days; whereas, TBBAC and TBCPC showed antibacterial effects at all times. HPLC analysis indicated no significant differences in the release behaviour of TBBAC and TBCPC (ttest, p > 0.05), except for the 7day release which was higher for TBBAC (p < 0.05). By 14 days the extents of release were 27 ± 2% and 25 ± 5% of the total initial loading for TBBAC and TBCPC, respectively. The incorporation of 10 wt% BAC or CPC in Transbond XT™ adhesive primer also resulted in superior shear bond strength at 7 and 14 days (Fisher's LSD, p < 0.05) with no significant change in the mode of bracket failure under shear stress (Pearson's chisquared, p > 0.05) (AU)

El objetivo de este estudio fue evaluar el impacto del cloruro de benzalconio al 10% en peso del peso (TBBAC) o de cloruro de cetilpiridinio al 10% del peso (TBCPC) con propiedades antimicrobianas presentes en el adhesivo acondicionador ortodóncico, Transbond XT ™ (TB). La actividad antimi crobiana se evaluó usando una zona de prueba de difusión de inhibición y la liberación de los compuestos antimicrobianos se controló mediante cromatografía líquida de alta resolución (HPLC). La resistencia de adhesión al corte (SBS) se probó usando esmalte bovino. Las muestras control, TB no lograron demostrar actividad antibacteriana intrínseca a 1, 7 y 14 días; mientras que TBBAC y TBCPC mostraron efectos antibac terianos en todo momento. El análisis por HPLC no indicó diferencias significativas en el comportamiento de liberación de TBBAC y TBCPC (prueba t, p> 0,05), excepto en la liberación a los 7 días que fue más alta para TBBAC (p <0,05). A los 14 días, los grados de liberación fueron de 27 ± 2% y de 25 ± 5% de la carga inicial total para TBBAC y TBCPC, respectivamente. La incorpora ción de 10% en peso de BAC o CPC en el imprimador adhesivo Transbond XT ™ también dio como resultado una resistencia superior corte a los 7 y 14 días (Fisher's LSD, p <0.05) sin cambios significativos en el modo de falla del bracket bajo tensión de corte (Pearson's chicuadrado, p> 0.05) (AU)

Simultaneous Determination of 4 Preservatives in Marketed Eye Drops by HPLC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of residual methylparaben,ethylparaben,nipa-sol and benzalkonium chloride in marketed eye drops. METHODS:HPLC method was adopted. The determination was performed on Hypersil GOLD C18 column with mobile phase consisted of 0.005 mol/L ammonium acetate(10 mL triehtylamine in 1 L solu-tion,pH adjusted to 5.0±0.5 with glacial acetic acid)-acetonitrile(45:55,V/V)at the flow rate of 1.0 mL/min. The detection wave-length was 262 nm(methylparaben,ethylparaben,nipasol)and 214 nm(benzalkonium chloride),respectively. The column tem-perature was 30 ℃ and sample size was 20 μL. RESULTS:The linear range were 1.2350-15.4380 μg/mL for methylparaben(r=0.9999),1.3170-16.3836 μ g/mL for ethylparaben (r=0.9997),1.2072-15.0894 μ g/mL for nipasol (r=0.9996) and 17.776-222.0 μg/mL for benzalkonium chloride(r=0.9999),respectively. Limits of quantitation were 2.0,2.0,2.0,1.11 μg,re-spectively;limits of determination were 0.375,0.375,0.375,0.333 μg,respectively. RSDs of precision,stability and reproducibili-ty tests were all lower than 2.0%. The average recoveries were 98.14%-102.48%(RSD=1.6%,n=9),98.79%-102.42%(RSD=1.3%,n=9),98.19%-102.49%(RSD=1.5%,n=9)and 98.76%-100.53%(RSD=0.6%,n=9),respectively. CONCLUSIONS:The method is accurate,reproducible,simple and suitable for the determination of residual methylparaben,ethylparaben,nipasol and benzalkonium chloride in marketed eye drops.

Detection Method for Three Ultraviolet Absorbers in Cosmetics / 中国药师

Objective: To develop an HPLC method for the simultaneous determination of 3 ultraviolet absorbers (benzylidene camphor sulfonic acid, camphor benzalkonium methosulfate, 4-methylbenzylidene camphor) in cosmetics.Methods: After extracted by acetonitrile-methanol-ammonium acetate aqueous solution-tetrahydrofur(30∶35∶30∶5, v/v)(for lotion and milk) or acetonitrile-methanol-ammonium acetate aqueous solution(30∶20∶50, v/v)(for cream and powder), the UV absorbers were separated on a Thermo scientific C18 column (250 mm×4.6 mm,5 μm) at 35 ℃ with methanol, acetonitrile and 0.02 mol·L-1ammonium acetate aqueous solution (pH 5.15 adjusted by glacial acetic acid) as the mobile phase with gradient elution at the flow rate of 1.0 ml·min-1, and then analyzed by a DAD detector at the wavelength of 288 nm and 300 nm.Results: Benzylidene camphor sulfonic acid, camphor benzalkonium methosulfate and 4-methylbenzylidene camphor showed good linearity within the range of 12.26-98.06 μg·ml-1(r=0.999 9), 9.31-74.48 μg·ml-1(r=0.999 9) and 6.86-54.88 μg·ml-1(r=0.999 9), respectively.The limit of detection (LOD) and the limit of quantitation (LOQ) were 4.6, 6.4 and 1.6 ng and 17.4, 15.9 and 5.5 ng, respectively.The average recovery was 99.2%(RSD=1.58%), 99.8%(RSD=2.38%) and 99.2%(RSD=2.03%)(n=36), respectively.Conclusion: The method is simple, rapid, reproducible, accurate and reliable, and can be applied in the determination of ultraviolet absorbers in cosmetics.

High-throughput determination of preservatives in eye drops and antimicrobial effectiveness assessment / 中国药学杂志

OBJECTIVE: To establish an HPLC method for rapid screening and simultaneous determination of 10 preservatives in eye drops and assess their antimicrobial effectiveness. METHODS: A Waters XBridge C18 column (4.6 mm × 150 mm, 5 μm) was used, and the mobile phase was 0.1% phosphoric acid-methanol-THF eluted in gradient mode at a flow rate of 1.0 mL·min-1. The detection wavelength was set at 214 nm. The antimicrobial effectiveness of the preservatives was assessed according to Ch. P 2015. RESULTS: The calibration curves were linear in the range of the corresponding test concentrations (r=0.999 3 -1.000 0). The recoveries were between 96.1%-101.8%. One of the eye drops products did not meet the requirement of Ch. P 2015. CONCLUSION: The established method is rapid and inexpensive. And it ensures excellent simplicity, sensitivity, specificity, and reproducibility and can be used for rapid screening and determination of the contents of preservatives in eye drops. The amount of preservatives should be established during the R&D period or determined according to the results of antimicrobial effectiveness test rather than using empirical values.

Comparison of Standards and Related Methods of the Quality Control of Benzalkonium Chloride in Phar-macopoeias of China and Foreign / 中国药房

OBJECTIVE:To provide ideas for revise and improve the standard and related method of the quality control of ben-zalkonium chloride in Chinese Pharmacopoeia (2015 edition,Ⅱ). METHODS:The standards and related methods of the quality control of benzalkonium chloride in Chinese Pharmacopoeia(2015 edition,Ⅱ),British Pharmacopoeia(2013 edition),European Pharmacopoeia (7.0 edition) and United States Pharmacopoeia (36 edition) were comprehensively compared. RESULTS:Com-pared with Chinese Pharmacopoeia(2015 edition,Ⅱ),the standards abroad provided the component and the ratio of the benzalko-nium chloride substituted homolog,the method for ammonia compound test had higher sensibility,it also added the test for benzyl alcohol,benzaldehyde and benzyl chloride impurity,as well as the component ratio test and average relative molecular mass calcu-lation. CONCLUSIONS:The standard and related method of the quality control of benzalkonium chloride in Chinese Pharmacopoe-ia(2015 edition,Ⅱ)still need to be further improved.