Long-term effects of combined brilliant blue G and xenon light induced retinal toxicity following macular hole repair surgery.

BACKGROUND: The purpose of this study was to look at the long-term effects of retinal phototoxicity after macular hole repair surgery using xenon endolight illumination and Brilliant blue G (BBG) dye. CASE PRESENTATION: An elderly man in his late seventies underwent para plana vitrectomy with BBG dye to repair an idiopathic full-thickness macular hole (MH) in his right eye. Prior to macular hole surgery, his visual acuity in the right eye was 6/60, N24 at the time of presentation. The MH closed with type 1 closure immediately after surgery, but there was extensive damage to the outer retinal layers and retinal pigment epithelium (RPE) at the macula, resulting in a reduction in visual acuity to 2/60. We presumed that the combination of BBG and xenon light, is the probable reason of retinotoxicity in the current patient. There was a progressive increase in the area of retinal and RPE layer damage and choroidal thinning over a 4-year period. CONCLUSION: Due to combined BBG-induced dye and endoilluminator toxicity, a rare case of continuously progressing RPE layer damage with choroidal thinning over a long follow-up interval was described. Such long-term effects of BBG and endolight induced retinotoxicity have not been reported in the literature, to the best of our knowledge.

Oxidative Stress and Toxico-Pathic Branchial Lesions in Cyprinus carpio Exposed to Malachite Green.

Gill is the frontier tissue to come in direct contact with aquatic toxicants. Malachite green (MG) commercial textile dye was assessed for its impact on the gill cytoarchitecture. Cyprinus carpio were exposed to 0.087 and 0.146 mg/L of MG for 60 days. The tissue was processed, and HE stained slides revealed histo-pathic lesions such as lamellar curling, edema, necrosis, telangiectasia, aneurysm, and vacuolization. Scanning electron microscopy reported aberrations in lamellae and microridges of the epithelium. At the cellular level, transmission electron microscopy exhibited nuclear alterations in form of pyknosis and mitochondrial swelling followed by cristolysis. Pillar cells displayed cytoplasmic vacuolization and leukocyte infiltration, and goblet cell containing varied shaped and density mucous globules. The biochemical analysis supported the ultrastructural alterations and showed a negative impact of MG on the antioxidative enzymes (CAT, SOD, GSH), while levels of MDA were found to be significantly elevated. Thereby, concluding MG induced branchial toxicity in the fish.

Extracellular expression of mutant CotA-laccase SF in Escherichia coli and its degradation of malachite green.

In this study, mutant CotA-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase SF was 1257.22 U/L. Extracellularly expressed CotA-laccase SF exhibits enzymatic properties similar to intracellular CotA-laccase SF. CotA-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of CotA-laccase SF to MG were 39.6 mM and 18.36 s-1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotA-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, CotA-laccase SF had a good application prospect for degrading malachite green.

Microbial degradation, spectral analysis and toxicological assessment of malachite green by Streptomyces chrestomyceticus S20.

Malachite green (MG), a triphenylmethane dye is extensively used for coloring silk, aquaculture and textile industries, it has also has been reported toxic to life forms. This study aimed to investigate the biodegradation potential of MG by actinobacteria. The potent actinobacterial strain S20 used in this study was isolated from forest soil (Sabarimala, Kerala, India) and identified as Streptomyces chrestomyceticus based on phenotype and molecular features. Strain S20 degraded MG up to 59.65 ± 0.68% was studied in MSM medium and MG (300 mg l-1) and degradation was increased (90-99%) by additions of 1% glucose and yeast extract into the medium at pH 7. The treated metabolites from MG by S20 characterized by FT-IR and GC-MS. The results showed MG has been degraded into nontoxic compounds evaluated by (1) phytotoxic assay on Vigna radiata, (2) microbial toxicity on Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Streptococcus sp. and Escherichia coli, (3) cytotoxicity assay in a human cell line (MCF 7). The toxicity studies demonstrated that the byproducts from MG degradation by S. chrestomyceticus S20 were no toxic to plants and microbes and less toxic to human cells as compared to the parent MG. Perhaps this is the first work reported on biodegradation of MG by S. chrestomyceticus which could be a potential candidate for the removal of MG from various environments.

The gut microbiota: a new perspective on the toxicity of malachite green (MG).

Gut microbiome critically contributes to host health status. Thus, investigating the relationship between the gut microbiome and toxic chemicals is a hot topic in toxicology research. Exposure to malachite green (MG) has been linked to various health disorders. Thus, exploring the gut microbiota changes in response to MG would provide a new perspective on the toxicity effects of this chemical substance. MG exposure resulted in the significantly lower alpha diversity (Mann-Whitney U test, z = - 6.83, p = 0.00) but higher beta diversity (Mann-Whitney U test, z = - 1.98, p = 0.04) of gut microbiota, and significantly decreased ecosystem stability (alpha and beta variability; Mann-Whitney U test, all p < 0.05) of gut microbial communities. Gut bacterial networks showed that the interactions became more complex and stronger after MG exposure, which could decrease the stability of the network. Changes in gut microbiota composition were mainly reflected in the enrichment of opportunistic bacteria (i.e., Aeromonas and Vibrio) and the depression of fermentative bacteria (i.e., Bacteroides and Paludibacter). MG exposure leads to a significantly increased gut permeability (lipopolysaccharide-binding protein; Mann-Whitney U test, z = - 6.92, p = 0.00), which could reduce the host selective pressures on particular bacterial species (such as members in Aeromonas and Vibrio). This result was further supported by the weakened importance of a deterministic microbial assembly after MG exposure. All these findings indicated that MG exposed fishes might have more possibilities to be infected, as demonstrated by the enrichment of opportunistic pathogenic bacteria, high-level immune responses, and increased gut permeability. These findings greatly improve our understanding of the toxicity effects of MG.

Malachite Green Induced Ultrastructural Corneal Lesions in Cyprinus carpio and Its Amelioration Using Emblica officinalis.

Malachite green, a multi-purpose dye induces cyto-toxicity upon its entry and bioaccumulation in tissues. A semi-static chronic (60 days) bioassay was conducted by exposing Cyprinus carpio to sublethal concentration of the dye and Emblica officinalis in four experimental groups viz control, malachite green, E. officinalis, and malachite green + E. officinalis. Effect of dye on the cornea was investigated considering ultra-structural alterations owing to its direct contact to the pollutant in the aquatic medium. SEM studies on corneal epithelium revealed broken continuity of pavement cells, shrunk microplicae, increased intra-microplicae distance, globularization and epithelial uplifting, thereby affecting the integrity of corneal surface and tear film adherence. Whereas dietary supplementation with the plant extract served to restore cytoarchitecture with appearance of large number of regenerating cells. Both lesions and restoration were found to be duration dependent. Thus, E. officinalis can be considered as an effective ameliorant against malachite green induced toxicity.

Foetal bovine serum can reduce toxicity of indocyanine green, brilliant blue G and trypan blue in ARPE-19 cellular model that suggests new surgical staining protocols for internal limiting membrane peeling procedure.

BACKGROUND: To investigate and compare the cytotoxicity of indocyanine green (ICG), brilliant blue G (BBG) and trypan blue (TB) using ARPE-19 cells that have been pre-treated/post-treated with balanced salt solution (BSS) or foetal bovine serum (FBS). METHODS: The cultured human retina pigment epithelium ARPE-19 cells were pre-treated/post-treated with BSS or FBS (represent the autologous serum in clinic) in parallel with cells being soaked with various concentrations of ICG, BBG and TB. The cells were then assessed for viability, growth rate, reactive oxygen species (ROS) level, mitochondrial membrane potential (Δψ) and mitochondrial mass as cytotoxic indices. For the FBS pre-treated cells, only ROS was examined. RESULTS: Using the MTT assay, cytotoxicity seemed to appear when the dye concentration was above 2.5 mg/mL for ICG but no cytotoxicity for BBG and TB at the concentrations used. Cell growth was arrested at a concentration 1 mg/mL when ICG or BBG were present but no arrest at any of the tested concentrations was found for TB with the cell-growth curve was slowest for ICG. Cellular ROS levels increased at all concentrations of all dyes, but the increasing slopes were decreased after FBS post-treatment washout. CONCLUSIONS: As a rinse buffer FBS performs much better than BSS in terms of cell rescue, which agrees with a clinical report when autologous whole blood was applied to macular hole surgery. However, FBS pre-treatment seems to be much better than FBS use as washout buffer in post-treatment.

Chemical Exacerbation of Light-induced Retinal Degeneration in F344/N Rats in National Toxicology Program Rodent Bioassays.

Retinal degeneration due to chronic ambient light exposure is a common spontaneous age-related finding in albino rats, but it can also be related to exposures associated with environmental chemicals and drugs. Typically, light-induced retinal degeneration has a central/hemispherical localization whereas chemical-induced retinal degeneration has a diffuse localization. This study was conducted to identify and characterize treatment-related retinal degeneration in National Toxicology Program rodent bioassays. A total of 3 chronic bioassays in F344/N rats (but not in B6C3F1/N mice) were identified that had treatment-related increases in retinal degeneration (kava kava extract, acrylamide, and leucomalachite green). A retrospective light microscopic evaluation of the retinas from rats in these 3 studies showed a dose-related increase in the frequencies of retinal degeneration, beginning with the loss of photoreceptor cells, followed by the inner nuclear layer cells. These dose-related increased frequencies of degenerative retinal lesions localized within the central/hemispherical region are suggestive of exacerbation of light-induced retinal degeneration.

Electrochemical degradation of malachite green: Multivariate optimization, pathway identification and toxicity analysis.

Application of a newly developed electrode material, PbO2 coated on mild steel plate (MS-PbO2), for the degradation of malachite green (MG) by photocatalytic oxidation (PCO), electrochemical oxidation (ECO) and photoelectrochemical oxidation (PEC) was explored. PEC performed marginally better at lower current density. However, the performances of PEC and ECO were equally good at higher current densities. One variable at a time optimization was carried out to identify the major parameters influencing ECO. Multivariate optimization was carried out with NaCl concentration, current density and pH as the variables and chemical oxygen demand (COD) removal efficiency and current efficiency (CE) as the responses. Increasing the current density aided the COD removal efficiency, but decreased the CE. Low NaCl concentration and acidic pH were beneficial for both. The optimum condition for maximizing the COD removal efficiency and CE of MG (50 mg L(-1)) was obtained as NaCl concentration of 1.56 g L(-1), a current density of 1.91 mA cm(-2) and pH 5. The maximum predicted and experimental COD removal efficiencies were 89.41% and 90.8%, and CEs were 21.52% and 21.1%, respectively. Degradation intermediates were identified and a possible pathway of degradation was proposed. Disc inhibition study showed that the degraded samples are non-toxic. The efficacy of the method was tested for treating wastewater collected from dyebath having a COD of about 2000 mg L(-1). COD removal efficiency of greater than 90% was achieved within 12 h at a current density of 7.2 mA cm(-2).

Toxicity induced by Basic Violet 14, Direct Red 28 and Acid Red 26 in zebrafish larvae.

Basic Violet 14, Direct Red 28 and Acid Red 26 are classified as carcinogenic dyes in the European textile ecology standard, despite insufficient toxicity data. In this study, the toxicity of these dyes was assessed in a zebrafish model, and the underlying toxic mechanisms were investigated. Basic Violet 14 and Direct Red 28 showed acute toxicity with a LC50 value at 60.63 and 476.84 µg ml(-1) , respectively, whereas the LC50 of Acid Red 26 was between 2500 and 2800 µg ml(-1) . Treatment with Basic Violet 14, Direct Red 28 and Acid Red 26 resulted in common developmental abnormalities including delayed yolk sac absorption and swimming bladder deflation. Hepatotoxicity was observed in zebrafish treated with Basic Violet 14, and cardiovascular toxicity was found in zebrafish treated with Acid Red 26 at concentrations higher than 2500 µg ml(-1) . Basic Violet 14 also caused significant up-regulation of GCLC gene expression in a dose-dependent manner whereas Acid Red 26 induced significant up-regulation of NKX2.5 and down-regulation of GATA4 at a high concentration in a dose-dependent manner. These results suggest that Basic Violet 14, Direct Red 28 and Acid Red 26 induce developmental and organ-specific toxicity, and oxidative stress may play a role in the hepatotoxicity of Basic Violet 14, the suppressed GATA4 expression may have a relation to the cardiovascular toxicity of Acid Red 26.

Fish erythrocytes as biomarkers for the toxicity of sublethal doses of an azo dye, Basic Violet-1 (CI: 42535).

The aim of the present study was to investigate poikilocytosis in Labeo rohita (an important food fish) as an early indicator of stress due to an azo dye, Basic Violet-1 (CI: 42535). This dye was observed to be very toxic to test fish (96 h LC50 as0.45 mg/L dye). Fish were given short-term (96 h) and subchronic (150 days) exposures to the dye, and poikilocytosis was recorded under light and scanning electron microscopy (SEM). Light microscopy helped in identification of micronuclei along with irregularities, notches, blebs, lobes, crenation, clumps, chains, spherocytes, vacuolation, and necrosis in erythrocytes. However, SEM indicated shrinkage, oozing of cytoplasm, and several new abnormal shapes including marginal foldings, discocytes, keratocytes, dacrocytes, degmacytes, acanthocytes, echinocytes, protuberances, stomatocytes, drepanocytes, holes in the membrane, stippling/spicules, crescent-shaped cells, triangular cells, and pentagonal cells. Earlier studies speculated changes in the membrane to be responsible for clumping and chaining of erythrocytes, whereas the present SEM study clearly indicates that oozing out of cytoplasm is also responsible for the formation of chains and clumps. This study also shows that erythrocytes exhibit pathological symptoms before the appearance of other external symptoms such as abnormal behavior or mortality of fish. There was a dose- and duration-dependent increase; therefore, poikilocytosis, especially echinocytes, spherocytes, and clumps, can act as a biomarker for the stress caused by azo dyes.

Malachite green bioremoval by a newly isolated strain Citrobacter sedlakii RI11; enhancement of the treatment by biosurfactant addition.

Citrobacter sedlackii RI11, isolated from acclimated textile effluent after selective enrichment on synthetic dyes, was assessed for malachite green (MG) biotreatment potency. Results indicate that this bacterium has potential for use in effective treatment of MG contaminated wastewaters under shaking conditions at neutral and alkaline pH value, characteristic of typical textile effluents. Also, the newly isolated strain can tolerate higher doses of dye and decolorize up to 1,000 mg/l of dye. When used as microbial surfactant to enhance MG biodecolorization, Bacillus subtilis SPB1-derived lipopeptide accelerated the decolorization rate and maximized the decolorization efficiency at an optimal concentration of biosurfactant of about 0.075%. Studies ensured that MG removal by this strain could be due to biodegradation and/or adsorption. Results on germination potencies of different seeds using the treated dyes under different conditions favor the use of SPB1 biosurfactant for the treatment of MG.

Genetic characterization of plasmid-associated triphenylmethane reductase in Listeria monocytogenes.

The enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into colorless, nontoxic derivatives, and TMR-producing microorganisms have been proposed as bioremediation tools. Analysis of the genome of Listeria monocytogenes H7858 (1998-1999 hot dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette (bcrABC) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium disinfectants also harbored a gene (tmr) highly homologous to TMR-encoding genes from diverse Gram-negative bacteria. The pLM80-associated tmr was located two genes downstream of bcrABC as part of a putative IS1216 composite transposon. To confirm the role of tmr in triphenylmethane dye detoxification, we introduced various tmr-harboring fragments of pLM80 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed the resistance of the constructs to the triphenylmethane dyes crystal violet (CV) and malachite green. Transcriptional and subcloning data suggest that the regulation of TMR is complex. Constructs harboring fragments spanning bcrABC and tmr were CV resistant, and in such constructs tmr transcription was induced by sublethal levels of either BC or CV. However, constructs harboring only tmr and its upstream intergenic region could also confer resistance to CV, albeit at lower levels. Screening a panel of BC-resistant L. monocytogenes strains revealed that all those harboring bcrABC and adjacent pLM80 sequences, including tmr, were resistant to CV and decolorized this dye. The findings suggest a potential role of TMR as a previously unknown adaptive attribute for environmental persistence of L. monocytogenes.

Testing the effects of the dye acid violet-17 on retinal function for an intraocular application in vitreo-retinal surgery.

PURPOSE: To facilitate epiretinal or inner limiting membrane peeling, dyes like Indocyanine Green (ICG) as well as Trypan Blue (TB) were used so far. However, toxic effects on the retina were described for both dyes. The aim of our study was to investigate the effects of a novel vital dye Acid violet-17 (AV-17) on retinal histology and function to assess a possible application in vitreo-retinal surgery. METHODS: AV-17 was dissolved in a solvent with heavy water. An electroretinogram was recorded on perfused bovine retina. After reaching stable b-wave amplitudes, AV-17 (0.125-0.5 mg/ml) or the solvent was applied epiretinally for 30-300 seconds. The b-wave amplitudes were recorded before, during, and after treatment. Cultures of bovine retina were incubated for 30 or 300 seconds with the dye or solvent and processed for live/dead staining, immunohistochemistry, and immunoblotting. RESULTS: Reductions of the b-wave amplitudes were observed directly after the exposure to AV-17, which were rapidly and completely reversible within the recovery period for all exposure times at the concentrations of 0.125 and 0.25 mg/ml as opposed to the partial recovery after exposure to 0.5 mg/ml. A high degree of damage in the ganglion cell layer (GCL) and glial reactivity were detected at the concentrations of 0.25 and 0.5 mg/ml but not after exposure to lower concentrations or the solvent. CONCLUSION: Application of AV-17 at a concentration of up to 0.125 mg/ml was well tolerated in terms of retinal function, survival in the GCL, and glial reactivity whereas higher concentrations are not recommended.

Toxicity of imine-iminium dyes and pigments: electron transfer, radicals, oxidative stress and other physiological effects.

Although conjugation is well known as an important contributor to color, there is scant recognition concerning involvement of imine and iminium functions in the physiological effects of this class of dyes and pigments. The group includes the dyes methylene blue, rhodamine, malachite green, fuchsin, crystal violet, auramine and cyanins, in addition to the pigments consisting of pyocyanine, phthalocyanine and pheophytin. The physiological effects consist of both toxicity and beneficial aspects. The unifying theme of electron transfer-reactive oxygen species-oxidative stress is used as the rationale in both cases. Toxicity is frequently prevented or alleviated by antioxidants. The apparent dichotomy of methylene blue action as both oxidant and antioxidant is rationalized based on similar previous cases. This mechanistic approach may have practical benefit. This review is important in conveying, for the first time, a unifying mechanism for toxicity based on electron transfer-reactive oxygen species-oxidative stress arising from imine-iminium.

The effect of toxic malachite green on the bacterial community in Antarctic soil and the physiology of malachite green-degrading Pseudomonas sp. MGO.

The effects of malachite green (MG) on the bacterial community in Antarctic soil were assessed. Culture-independent community analysis using 16S rRNA gene pyrosequencing showed that, in the presence of MG, the relative abundance of Pseudomonas dramatically increased from 2.2 % to 36.6 % (16.6-fold), and Pseudomonas became the predominant genus. The reduction in bacterial biodiversity was demonstrated by diversity indices and rarefaction curves. MG-degrading Pseudomonas sp. MGO was isolated from Antarctic soil. MG tolerance and decolorization activity were confirmed by growth, spectrophotometric, high-performance liquid chromatography, and thin-layer chromatography analyses in high MG concentrations. Our data showed that the decolorization process occurred via biodegradation, while biosorption also occurred after some time during the fed-batch decolorization process. Significant inductions in laccase, nicotinamide adenine dinucleotide-2,6 dichlorophenol indophenol reductase, and MG reductase activities suggested their involvement in the decolorization process. We also showed that the high tolerance of strain MGO to toxic MG might be mediated by upregulation of oxidative stress defense systems such as superoxide dismutase and protease. Collectively, these results demonstrated the response of the Antarctic soil bacterial community to MG and provided insight into the molecular mechanism of MG-tolerant Pseudomonas strains isolated from Antarctic soil.

Investigation of the retinal biocompatibility of acid violet for chromovitrectomy.

PURPOSE: The primary objective was to investigate the retinal biocompatibility of acid violet (AV) as a vital dye for chromovitrectomy. The secondary objective was to evaluate the capacity of AV to stain the anterior capsule of the lens. METHODS: An amount of 0.05 ml of 0.25 g/l and 0.5 g/l AV was injected intravitreally in the OD, while balanced salt solution (BSS) was applied in the OS for control. Clinical examination and histology with light microscopy (LM) were performed after 7 days. Retinal cell layers were evaluated for morphologic alterations and number of cells. The electroretinographic (ERG) changes were assessed at baseline and 7 days. In another part of the study, 0.1 ml of 0.25 g/l AV was injected into the anterior chamber of ex-vivo porcine eyes, and its capacity to stain the anterior capsule was determined. Cadaveric eyes were used to test the capacity of AV to stain the internal limitant membrane (ILM) during vitrectomy. RESULTS: The gross histopathologic appearance of the retina, choroids, sclera, and optic nerve was within normal limits, without any signs of severe retinal necrosis or cystic degeneration. AV caused no substantial retinal alterations in retinal layers by LM at either the lower or higher dose when compared with the control eye. The injection of AV did not induce considerable ERG alterations. The violet dye stained the anterior capsule after anterior chamber injection and the ILM, allowing a safer capsulorrhexis and vitrectomy. CONCLUSION: Acid violet may be safe for the retina at concentrations of 0.25 and 0.50 g/l after intravitreous injection, and may be used as a vital dye for staining the anterior capsule and the ILM.

The retinal biocompatibility of dyes in the ex vivo model of the isolated superfused vertebrate retina.

BACKGROUND: Peeling of the internal limiting membrane or epiretinal membranes is a successful principle in macular surgery to achieve a functional benefit. Different dyes are used to facilitate the identification of intraocular tissues. The aim of our work was to investigate the retinal tolerance to the different dyes and their solvent carriers to provide valuable data for surgeons in handling for an optimal intraoperative use. METHODS: Using the ex vivo model of the isolated superfused vertebrate retina technique, the effects of the dyes were tested on human and bovine retinal function. The retinas were perfused with an oxygen preequilibrated standard solution. The electroretinogram (ERG) was recorded using Ag/AgCl electrodes. After recording stable ERG amplitudes, the dyes brilliant blue G, indocyanine green, trypan blue, patent blue, triamcinolone and their solvent carriers were investigated. RESULTS: Reductions of the ERG amplitudes were found for each tested dye. The effects after application of the dyes were dependent on time and concentration of the applied dyes, which were different for each dye. CONCLUSION: In part, the ERG has shown strong effects already after a short period of dye application. Surgeons who rely on the intraocular use of the dyes should keep in mind our findings, and the use of some dyes should be limited to selected cases. The well-considered use of the dyes by the surgeons could lead to a better functional outcome and avoid a possible harmful outcome of the surgery after mishandling.

Effect of novel vital dyes on outer blood-retina barrier function in cultured human retinal pigment epithelium.

PURPOSE: To assess tight junction (TJ) integrity in cultured human fetal retinal pigment epithelium (HFRPE) after exposure to clinically relevant novel vital dyes. METHODS: HFRPE floater cells were harvested from RPE primary cultures of 4 donor eyes and seeded on polyester Transwell® for 4-6 weeks. The apical compartments of well-differentiated cultures were exposed to 0.005 mg/ml Coomassie violet R200 (CVR), methyl 2B (M2B) or Orange II. Periods of 30-300 s were chosen to mimic surgical exposure times, while 3 h was used for toxicity assays, with subsequent washout. Cell-cell junctions were studied by immunofluorescence (zonula occludens-1, ZO-1). Transepithelial electrical resistance (TER) was measured regarding blood-retina barrier (BRB) function. RESULTS: At 4-6 weeks after confluence, HFRPE had grown into pigmented hexagonal monolayers with stable TER values (451-1,520 Ω·cm(2)). After 300-second dye treatments, a continuous ZO-1 signal was detected in all vital dye-treated groups 1.5 h after exposure, whereas trypsin controls showed patchy loss of the TJ stain. TER of CVR-, M2B- and Orange-II-treated groups had dropped 1.5 h after exposure to 148 ± 58.4, 162 ± 23.7 and 164 ± 18.5 Ω·cm(2), respectively, compared to 73 ± 44.9 Ω·cm(2) in positive controls. After 3 h of exposure to 0.005 mg/ml vital dyes in thick drops, TER maintained similar levels to those prior to exposure (90.8 ± 4.7% of the original values, 93.8 ± 6.5 and 91.9 ± 3.6%, respectively), together with no difference from the vehicle controls (94.8 ± 6.6%). TER values recovered in all groups to prior levels within 3 days. CONCLUSION: Novel vital dyes (CVR, M2B and Orange II) caused no outer BRB function alteration.