Comparative toxicity of preservatives on immortalized corneal and conjunctival epithelial cells.

PURPOSE: Nearly all eye drops contain preservatives to decrease contamination. Nonpreservatives such as disodium-ethylene diamine tetra-acetate (EDTA) and phosphate-buffered saline are also regularly added as buffering agents. These components can add to the toxicity of eye drops and cause ocular surface disease. To evaluate the potential toxicity of these common components and their comparative effects on the ocular surface, a tissue culture model utilizing immortalized corneal and conjunctival epithelial cells was utilized. METHODS: Immortalized human conjunctival and corneal epithelial cells were grown. At confluency, medium was replaced with 100 microL of varying concentrations of preservatives: benzalkonium chloride (BAK), methyl paraben (MP), sodium perborate (SP), chlorobutanol (Cbl), and stabilized thimerosal (Thi); varying concentrations of buffer: EDTA; media (viable control); and formalin (dead control). After 1 h, solutions were replaced with 150 microL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazonium bromide). After 4 h, solutions decanted, 100 microL of acid isopropanol added, and the optical density determined at 572 nm to evaluate cell viability. RESULTS: Conjunctival and corneal cell toxicity was seen with all preservatives. Depending upon concentration, BAK exhibited from 56% to 89% toxicity. In comparison, Cbl exhibited from 50% to 86%, MP from 30% to 76%, SP from 23% to 59%, and Thi from 70% to 95%. EDTA with minimal toxicity (from 6% to 59%) was indistinguishable from SP. CONCLUSIONS: Generally, the order of decreasing toxicity at the most commonly used concentrations: Thi (0.0025%) > BAK (0.025%) > Cbl (0.25%) > MP (0.01%) > SP (0.0025%) approximately EDTA (0.01%). Even at low concentration, these agents will cause some degree of ocular tissue damage.

Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells.

PURPOSE: The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. METHODS: Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. RESULTS: HCEC survival was 20-30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. CONCLUSIONS: The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative.

Epithelial activity of hexokinase and glucose-6-phosphate dehydrogenase in cultured bovine lenses recovering from pharmaceutical-induced optical damage.

PURPOSE: In a previous toxicological study, cultured bovine lenses exposed to three topical anesthetics displayed distinct patterns of optical damage and recovery. This work investigated the epithelial activity of the metabolic enzymes hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PD) in lenses recovering from anesthetic-induced damage. METHODS: Cultured bovine lenses were exposed to the anesthetics Alcaine, Fluress and Fluoracaine for 2 h. An automated laser scanner was used to determine the focal length variability (FLV) of the lenses at time-points up to 24 h following their return to fresh culture medium. The epithelial enzyme activities for HK and G6PD were then assayed at the 24 h time-point. RESULTS: Lenses exposed to Alcaine displayed an abrupt increase in FLV, while Fluoracaine treated lenses exhibited optical damage at a slower rate. The FLV in these two groups recovered to near-control levels after 24 h. Fluress treated lenses did not differ in FLV from controls at any time. The activities of both HK and G6PD were significantly reduced in epithelial samples from each of the three anesthetic treatment groups, relative to controls. CONCLUSIONS: These results show that lens optical quality can recover despite a severe reduction in epithelial HK and G6PD activity, indicating that the optical function of the lens may not be directly related to epithelial metabolic activity. The ScanTox In Vitro Assay System provides an objective measure of lens optical quality, enabling a direct comparison of optical damage and recovery to lens biochemical changes.

Chlorobutanol: maternal serum levels and placental transfer in the mouse.

Chlorobutanol (CB) is a pharmaceutical preservative and the active ingredient in certain sedatives and anesthetics and produces adverse effects in adult tissues and mouse embryos in vitro. Chlorobutanol is slowly eliminated in humans, but little is known about its serum levels in other species or placental transfer in any species. Pregnant mice gavaged with 80 mg CB/kg on gestational day (gd) 9.5 provided serum at 10 min to 36 h post-dosing for a time course study. Additional mice gavaged with 0, 8, 40 or 80 mg CB/kg on gd 9.5 provided maternal serum and embryos 2 h post-dosing for a placental transfer study, and CB levels were measured using capillary gas chromatography with electron capture detection. Dosing with 80 mg CB/kg produced maternal serum levels between 30.8 micrograms/mL (10 min) and < 1 microgram/mL (36 h), with a half-life of 7.45 h. Embryonic CB levels increased with maternal dose and were correlated with maternal serum levels. Serum levels of CB in the mouse appeared to follow a time course similar to humans, with rapid absorption and slow elimination. Placental transfer, as demonstrated here in the mouse, may allow embryonic accumulation of CB to potentially toxic levels.

Chlorobutanol toxicity.

Although chlorobutanol is available in a large number of preparations, the incidence of toxicity appears to be low. However, with continued exposure to high-dose chlorobutanol, significant toxicity may occur. It seems prudent to use caution in the continued administration of products containing chlorobutanol. The lack of pharmacokinetic data warrant that extra care be used when administering chlorobutanol-containing products to patients with hepatic or renal dysfunction. Patients should be monitored for possible increased somnolence, alterations in speech pattern, dysarthria, and hemodynamic changes. Chlorobutanol toxicity should be ruled out by serum analysis if the patient exhibits any of the above reactions.

Acute effects of chlorobutanol- or benzalkonium chloride-containing artificial tears on the surface features of rabbit corneal epithelial cells.

I examined the epithelial surface, by high resolution scanning electron microscopy, after its recovery from the instillation of artificial tears containing chlorobutanol or benzalkonium chloride. Is the mild epithelial surface compromise observed after occasional use of a chlorobutanol-containing artificial tear more substantial after use of a benzalkonium-containing artificial tear? Two drops of a chlorobutanol- or benzalkonium chloride-containing artificial tear were instilled into the right eye of 6 female gray rabbits (2 kg) at 9:00 p.m. and 9:00 a.m. At the same time six control animals received no eyedrops. All animals were euthanized at 3:00 p.m. and the central region of the corneal epithelium quantitatively assessed using a digitizer pad/computer system. There were up to 5% exfoliating cells evident at the ocular surface in treated rabbits but with no difference between the two products. Controls had no cell exfoliation (< 0.5%). The distribution of surface areas of the squamous cells in the treated eyes was shifted to slightly larger values than in the controls after use of the chlorobutanol-containing product but the number of epithelial cell craters/cell was unchanged from that of the controls. Cell surface areas were shifted to significantly smaller values than controls after use of the benzalkonium chloride-containing product and there were much fewer epithelial cell craters/cell. The results reveal differences in the effects of preservative-containing artificial tears on the squamous cells of the corneal epithelium in a clinically relevant situation.

Embryotoxic effects of chlorobutanol in cultured mouse embryos.

Chlorobutanol (CB) is a commonly used preservative which is added to numerous pharmaceutical preparations, and it is the active ingredient in certain oral sedatives and topical anesthetics. Chlorobutanol has demonstrated adverse effects in adult tissues, but CB has not been previously investigated for its effect on the developing whole embryo. The method of whole-embryo culture was used in this study to expose mouse embryos during two stages of organogenesis to CB at final concentrations of 0 (control), 10, 25, 50, 100, and 200 micrograms/ml. Embryos were evaluated for heart rate (HR), malformations, and somite number, and embryos and visceral yolk sacs (VYSs) were assayed for total protein content as a measure of overall growth. Neurulating (3-6 somite) embryos were malformed and growth retarded by exposure to CB concentrations > or = 25 micrograms/ml, with decreased VYS growth at > or = 50 micrograms/ml and decreased HR at > or = 100 micrograms/ml CB. Early limb-bud stage (20-25 somite) embryos were malformed at CB concentrations > or = 50 micrograms/ml and growth retarded at > or = 100 micrograms/ml, with decreased VYS growth at 200 micrograms/ml and decreased HR at > or = 100 micrograms/ml CB. Thus, CB produces dysmorphogenesis in mouse embryos in vitro, and neurulating embryos are somewhat less sensitive than early limb-bud stage embryos. The concentrations of CB that interfere with normal embryonic development are within the range of human blood levels measured following multiple doses of CB. Preparations containing CB should be used with caution during pregnancy, particularly when repeated dosing may allow accumulation of CB to potentially embryotoxic levels.

Is ketamine or its preservative responsible for neurotoxicity in the rabbit?

BACKGROUND: Although ketamine has been administered spinally in humans, previous neurotoxicity studies have shown that it can induce spinal cord lesions in various animal models. The aim of this work was to evaluate by histologic and blood-brain barrier studies whether different components of the commercial ketamine solution might be responsible for the microscopic lesions observed. METHODS: Forty white New Zealand rabbits were randomly assigned to four groups of 10. One-percent preservative-free ketamine (0.3 ml), 1% d-ketamine, 0.05% chlorobutanol, and 1% lidocaine were intrathecally injected through the atlantooccipital membrane. Laminectomy was performed on day 8, and the dura was preserved using paraformaldehyde-glutaraldehyde fixative. Light and fluorescence microscopy were performed on transverse spinal cord sections by a neuropathologist unaware of injected agents used. Specimens were then graded as normal or abnormal as compared with a control group receiving lidocaine. RESULTS: Isomers of ketamine did not induce spinal cord lesions in either study, but chlorobutanol (the preservative used in the ketamine solution) induced significant severe spinal cord lesions in both studies. CONCLUSIONS: The appearance of spinal cord lesions after intrathecal chlorobutanol strongly suggests that this preservative is responsible for apparent toxicity of ketamine and therefore should not be used in any solution intrathecally injected into humans.

Twice-daily use of a chlorobutanol-preserved artificial tear on rabbit corneal epithelium assessed by scanning electron microscopy.

Female pigmented rabbits received two drops of a chlorobutanol 0.5%-preserved, polyvinyl alcohol-based artificial tear at 21.00 h and 09.00 h for 1, 2, 3, 6 or 12 consecutive days. The animals were killed at 15.00, 6 h after the last treatment. Scanning electron microscopy of the corneal epithelial surface at x 200 magnification revealed only occasional cell exfoliation averaging < 8% of the surface analysed at central, mid-peripheral and peripheral sites, showing minimal cytotoxic effects. The level of exfoliation was maximal after 2 to 3 days treatment and then returned to close to zero, so the eye adapts to repeated use of the artificial tears. Evaluation of the surface microplicae of the cells at x 15,000 indicated subtle, but distinct, changes in what might be the mucus material associated with the cell surface, especially after 2 and 3 days treatment.

Effect of the use of artificial tears on the size of squamous cells of the rabbit corneal epithelium evaluated by scanning electron microscopy.

Female pigmented rabbits (2 to 2.3 kg) received 2 drops of a chlorobutanol 0.5%-preserved, polyvinyl alcohol-based artificial tear at 21.00 h and 09.00 h for 1, 2, 3, 6, or 12 days. The animals were euthanized at 15.00 h and the corneas fixed with 2.0% glutaraldehyde in 80 mM cacodylate buffer. Scanning electron micrographs were taken close to the corneal center at 500 x magnification and normal to the epithelial surface. The surface areas of the squamous cells were measured with a digitizer pad. As a result of the treatment, up to 9% of the cells were observed to be in the process of exfoliation with the effect being greatest after 3 days. Control (nontreated) corneas showed only 0.44% exfoliating cells. Over this time period there was a progressive shift of cell sizes to larger values followed by recovery back to control values by 12 days. The results confirm the lack of gross cytotoxic effect of chlorobutanol-preserved artificial tears and indicate that the corneal epithelium can adapt to the use of a polymer-containing artificial tear.

The airways effects of inhaled chlorbutol in asthmatic subjects.

Chlorbutol is an antibacterial and antifungal agent incorporated in terbutaline (Bricanyl) nebulizer solution. Ten stable atopic asthmatic subjects undertook bronchial challenge testing, according to a double-blind protocol. Patients inhaled doubling concentrations of either methacholine (0.13-4.0 mg.ml-1) or chlorbutol (0.16-5.0 mg.ml-1) for 2 min until the forced expiratory volume in one second (FEV1) had fallen by 20% from baseline. If this had not occurred following the administration of the final concentration, then this highest concentration was repeated for 4 min. The nine subjects completing the study had a geometric mean provocation concentration producing a 20% fall from baseline FEV1 (PC20) methacholine of 0.16 mg.ml-1 (range less than 0.125-0.475 mg.ml-1). After inhalation of 2.5 mg.ml-1 chlorbutol one subject experienced a fall in FEV1 greater than 20%. In the remaining eight subjects, inhalation of chlorbutol did not affect airway calibre. We conclude that chlorbutol, in the concentration present in Bricanyl nebulizer solution, has no clinically significant effect on airway calibre.

Cytotoxic effects of benzalkonium chloride and chlorobutanol on human corneal epithelial cells in vitro.

By exposing primary cultures of human corneal epithelial cells to a single dose of either benzalkonium chloride or chlorobutanol, evaluated the cytotoxicity of these two preservatives which are commonly used in artificial tear solutions and other topical ophthalmic medications. Control and experimental cultures were analyzed by continuous time-lapse videomicrographic recordings as well as by sequential phase-contrast microscopy. Both benzalkonium chloride at a concentration of 0.01% and chlorobutanol at 0.5% caused immediate cessation of normal cytokinesis and mitotic activity, and epithelial cells degenerated within two hours and eight hours, respectively.

Allergic and toxic reactions of soft contact lens wearers.

The allergic and toxic effects of preservatives found in soft contact lens solutions are reviewed. Thimerosal, a preservative commonly found in soft contact lens solutions, may cause ocular delayed hypersensitivity. Patients with delayed hypersensitivity to thimerosal may develop conjunctival hyperemia, corneal infiltrates and intolerance to lens wear with the use of soft contact lens solutions containing thimerosal. Delayed hypersensitivity to thimerosal can be demonstrated by an occlusive patch test or intradermal injection. Discontinuation of chemical disinfection with substitution of thermal disinfection using unit-dose, non-preserved saline causes resolution of signs and symptoms. Another cause of lens wear intolerance is contact lens-associated giant papillary conjunctivitis. Protein deposits on the lens may act as antigens and initiate an allergic condition which disappears when the lens is discontinued. Alternatively, the giant papillae may be the result of mechanical irritation induced by the contact lenses. The chemical preservatives that are found in soft contact lens solutions cause epithelial toxicity when applied to isolated rabbit corneas. However, corneal toxicity toxicity in a clinical setting has not been demonstrated.

Commonly used intraocular medications and the corneal endothelium.

Forty-one cats had balanced salt solution, chymotrypsin, epinephrine hydrochloride with preservatives in varying concentrations, acetylcholine chloride, and carbachol injected intracamerally to determine the toxic effects on the endothelium. Specular microscopy was performed both before and after injection, and for each of the medications, the cell densities were compared with the results obtained with the balanced salt solution. The mean postinjection density as compared with the preinjection density for the medications varied between 98% and 108%, and there were no statistically significant differences between each medication group and the balanced salt solution results. From the results of this study, it is believed that when indicated, these medications probably do not produce substantial toxic effects on the corneal endothelium.