In vitro genotoxicity and cytotoxicity of benzalkonium chloride

Department of Otorhinolaryngology, University of Ulm, Medical School, Prittwitzstrasse 43, 89075 Ulm, Germany.
Toxicology in Vitro (Impact Factor: 2.9). 01/2007; 20(8):1472-7. DOI: 10.1016/j.tiv.2006.07.006
Source: PubMed


Benzalkonium chloride (BAC) acts as a preservative in numerous nasal preparations. Possible genotoxic and cytotoxic effects of BAC in human respiratory epithelial BEAS-2B cells should be investigated in vitro. Cell cultures were exposed for 2h to BAC in concentrations ranging from 0.002% to 0.05%. Methyl methanesulfonate served as positive control, PBS as negative control. The tail moment of single-cell gel-electrophoresis (SCGE) was used to assess BAC-induced DNA damage. Cell viability was measured by trypan blue dye exclusion staining. Additionally, the critical micellar concentration (CMC) of BAC in PBS was detected. The tail moment increased dose dependently with the maximum value at 0.02%, and declined for higher concentrations. Nearly all cells died at low BAC concentrations up to 0.01%. Above this concentration cell viability increased. The CMC of BAC in PBS was estimated to be 0.02%. BAC caused relevant DNA changes in respiratory epithelial cells in vitro at concentrations commonly employed in commercially available nasal preparations. Some of the exposed cells survived. In further studies it could be considered to look whether these cells would still be able to proliferate and possibly fix the damage that they have possibly accumulated into an actual mutation using for example the induction of micronuclei.

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    • "Montmorillonite is the main constituent of Bentonite, the weathering product of volcanic ash. A limitation to the use of clays is the incompatibility between the hydrophilic clay and hydrophobic polymers that could cause agglomeration of clay-mineral polymer matrices [6] [8]. Therefore, to enhance the intercalation/exfoliation process in a polymer matrix, clay surfaces are chemically modified by treatment with organic compounds, so that the clays become hydrophobic and thereby more compatible with polymers [9]. "
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    ABSTRACT: Because of the increasing use of clays and organoclays in industrial applications it is of importance to consider the toxicity of these materials. Recently it was reported that the commercially available Montmorillonite clay, Cloisite® 30B, which is surface-modified by organic quaternary ammonium compounds, was genotoxic in vitro. In the present study the in-vivo genotoxic and inflammatory potential of Cloisite® 30B was investigated as a follow-up of the in-vitro studies. Wistar rats were exposed to Cloisite® 30B twice 24 h apart by oral gavage, at doses ranging from 250 to 1000 mg/kg body weight [indicate duration of treatment; Ed.]. There was no induction of DNA strand-breaks in colon, liver and kidney cells and there was no increase in inflammatory cytokine markers in blood-plasma samples. In order to verify the possible absorption of Cloisite®30B from the gastrointestinal tract, inductively coupled plasma mass-spectrometry (ICP-MS) analysis was performed on samples of liver, kidney and faeces, with aluminium as a tracer element characteristic to clay. The results showed that aluminium could be detected in faeces, but not in the liver or kidneys. This indicated that there was no systemic exposure to clay particles from Cloisite®30B. Detection and identification of free quaternary ammonium modifier in the highest dose of Cloisite® 30B was carried out by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). This analysis revealed a mixture of three quaternary ammonium analogues. The detected concentration of the organomodifier corresponded to an exposure of rats to about 5 mg quaternary ammonium analogues/kg body weight.
    Mutation Research/Genetic Toxicology and Environmental Mutagenesis 08/2014; 770. DOI:10.1016/j.mrgentox.2014.04.023 · 2.42 Impact Factor
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    • "␮g/ml, which is comparable with the genotoxic concentration range in the Ferk et al. study [24]. Deutschle et al. [26] investigated genotoxic effects of BAC in human respiratory epithelial BEAS-2B cells after 2 h of exposure. This study showed a clear concentration-related increase in DNA strand-breaks in the comet assay at concentrations up to 0.02%. "
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    ABSTRACT: The natural clay mineral montmorillonite (Cloisite) Na+) and an organo-modified montmorillonite (Cloisite 30B) were investigated for genotoxic potential as crude suspensions and as suspensions filtrated through a 0.2-microm pore-size filter to remove particles above the nanometre range. Filtered and unfiltered water suspensions of both clays did not induce mutations in the Salmonella/microsome assay at concentrations up to 141microg/ml of the crude clay, using the tester strains TA98 and TA100. Filtered and unfiltered Cloisite) Na+ suspensions in culture medium did not induce DNA strand-breaks in Caco-2 cells after 24h of exposure, as tested in the alkaline comet assay. However, both the filtered and the unfiltered samples of Cloisite 30B induced DNA strand-breaks in a concentration-dependent manner and the two highest test concentrations produced statistically significantly different results from those seen with control samples (p<0.01 and p<0.001) and (p<0.05 and p<0.01), respectively. The unfiltered samples were tested up to concentrations of 170microg/ml and the filtered samples up to 216microg/ml before filtration. When tested in the same concentration range as used in the comet assay, none of the clays produced ROS in a cell-free test system (the DCFH-DA assay). Inductively coupled plasma mass-spectrometry (ICP-MS) was used to detect clay particles in the filtered samples using aluminium as a tracer element characteristic to clay. The results indicated that clay particles were absent in the filtered samples, which was independently confirmed by dynamic light-scattering measurements. Detection and identification of free quaternary ammonium modifier in the filtered sample was carried out by HPLC-Q-TOF/MS and revealed a total concentration of a mixture of quaternary ammonium analogues of 1.57microg/ml. These findings suggest that the genotoxicity of organo-modified montmorillonite was caused by the organo-modifier. The detected organo-modifier mixture was synthesized and comet-assay results showed that the genotoxic potency of this synthesized organo-modifier was in the same order of magnitude at equimolar concentrations of organo-modifier in filtrated Cloisite) 30B suspensions, and could therefore at least partly explain the genotoxic effect of Cloisite) 30B.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 04/2010; 700(1-2):18-25. DOI:10.1016/j.mrgentox.2010.04.021 · 3.68 Impact Factor
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    • "Firstly, BKC is a mixture of alkylbenzyldimethylammonium chloride with the formula [C 6 H 5 CH 2 N(CH 3 ) 2 R]Cl, where R is an alkyl group varying from C 8 H 17 to C 18 H 37 (Rojsitthisak et al., 2005) (Fig.1). It was reported that homologs of C 12 and C 14 were the most common components suitable for ophthalmic formulations (Deutschle et al., 2006). A complete method should allow for the homologs of BKC to be quantified. "
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    ABSTRACT: A high-performance liquid chromatography (HPLC) system was used in the reversed phase mode for the determination of benzalkonium chloride (BKC) in azithromycin viscous ophthalmic drops. A Venusil-XBP(L)-C(18) (150 mmx4.6 mm, 5 microm) column was used at 50 degrees C. The mobile phase consisted of a mixture of methanol-potassium phosphate (16:5, v/v). Two sample preparation methods were compared. The results suggested that, compared with an extraction procedure, a deproteinization procedure was much quicker and more convenient. Using the deproteinization procedure for sample preparation, calibration curves were linear in the range 5.0 to approximately 50 microg/ml. The within-day and inter-day coefficients of variation were less than 10%. The average recoveries were determined as 96.70%, 98.52%, and 97.96% at concentrations of 10.0, 30.0, and 50.0 microg/ml, respectively. Variability in precision did not exceed 5%. In conclusion, this HPLC method using a simple sample treatment procedure appears suitable for monitoring BKC content in azithromycin viscous ophthalmic drops.
    Journal of Zhejiang University SCIENCE B 12/2009; 10(12):877-82. DOI:10.1631/jzus.B0920229 · 1.28 Impact Factor
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