Ex Vivo Eye Irritation Test Research Articles

A Novel Ex Vivo Blinking Method for Comparing the Corneal Residence Time of New Shampoo Formulations.

In the cosmetics sector, many products such as shampoos have a probability of accidental ocular exposure during their routine use. One very specific safety parameter is the residence time of the substance on the corneal surface, as prolonged exposure may cause injury. In this study, we developed a system that simulates corneal exposure to blinking and tear flow, for comparing the corneal clearance times of viscous detergent formulations. The Ex Vivo Eye Irritation Test (EVEIT), which uses corneal explants from discarded rabbit eyes from an abattoir, was used as the basis for the new system. To simulate blinking, we developed a silicone wiping membrane to regularly move across the corneal surface, under conditions of constant addition and aspiration of fluid, to mimic tear flow. Six shampoo formulations were tested and were shown to differ widely in their corneal clearance time. Three groups could be identified according to the observed clearance times (fast, intermediate and slow); the reference shampoo had the shortest clearance time of all tested formulations. With this new system, it is now possible to investigate an important physicochemical parameter, i.e. corneal clearance time, for the consideration of ocular safety during the development of novel cosmetic formulations.

Novel Treatment Options for Corneal Edema

Introduction: The current treatment of corneal edema is limited to hyperosmotic saline mixtures. Usually the therapy consists of treatment with hypertonic 5% NaCl eye drops or 6% eye ointment. However, these only remain on the cornea for a short time because they are quickly removed by the tear film and blinking. This problem is partly addressed by adding hyaluronic acid to some pharmaceuticals. In this work, Sovermol as a carrier oil supplied with osmotically active substances, honey, icing sugar, Substance C188 and Prevor® substance X were investigated in an experimental setting as therapeutics for corneal edema. Material and Methods: In the Ex vivo Eye irritation Test (EVEIT), corneas can be examined under defined and stable biochemical conditions without blinking. Corneal edema can be simulated and examined using ultrasound pachymetry and Optical Coherence Tomography (OCT). Thus, we investigated the deswelling effect of the substances described above on 23 EVEIT corneas. Results: The mixtures, examined in this study, showed a significant reduction of corneal edema especially mixtures of Sovermol with Substance C188 were very promising as a new approach to treatment of corneal edema. As reference substance for corneal edema treatment ODM5® was used. Other mixtures, especially Honey and Prevor® substance X in DOL showed a sufficient reduction of corneal edema, while also damaging the corneal epithelium. While the mixture of DOL and powder sugar showed an increased corneal thickness and damaged Epithelium. Conclusion: The therapy of corneal edema with osmotically active substances as investigated here is a promising treatment for corneal edema. However, further pharmacological development is needed for application in patients.

Perspiratio Insensibilis of the Cornea

Introduction: The deswelling of the cornea via the natural pathways of perspiratio insensibilis and endothelial pump has been known for a long time. However, reliable data on the amount of perspiratio insensibilis, especially in edematous corneas, have been lacking until now. Material and Methods: In the Ex Vivo Eye Irritation Test (EVEIT), corneas can be observed purely physically under defined and stable biochemical conditions. The thickness and temperature of the corneas is measured by infrared thermography and Optical Coherence Tomography (OCT). Three corneas in stable MEM EVEIT culture and three corneas with induced and reversible edema in an EVEIT osmolar deficiency culture were placed in at 20°C and 60% humidity while tracking thickness and temperature for 30 minutes. Results: Over a period of 30 minutes, the temperature of both, healthy and edematous EVEIT corneas decreased from an initial 28°C to 21°C. The corneal thickness decreased by 51 ±12 μm in the healthy corneas and by 131 ±16 μm in the edematouscorneas. Conclusion: The considerable thickness differences between healthy and swollen corneas might give a physical and physiological explanation of some observable clinical effects in Fuchs’s endothelial dystrophy. The clinically observed effect of clearing of the cornea during the waking period. Furthermore, some phenomena of Dry Eye Syndrome (DES) can be explained by this difference of increased water release from the eye.

Defining corneal chemical burns: A novel exact and adjustable ocular model.

Live-animal-free ocular toxicity models and tests are a necessity in multiple branches of medicine, industry and science. Corneal models with adjustable ranges of injury severities do not exist. In this work, a novel and precise and dose - response method to induce and observe ex vivo corneal chemical burns has been established. The EVEIT (Ex Vivo Eye Irritation Test) is based on an ex vivo corneal organ model for rabbit corneas from food industry. Further, a highly precise three - axis workstation has been employed to apply liquid corrosive, sodium hydroxide (NaOH), droplets in a nanolitre (nL) range onto the corneal surface. Optical Coherence Tomography (OCT) has been used to observe and quantify the elicited changes in the corneal layers. The speed and intervals of single nanodroplet application played a crucial role in the extent of the corneal changes. Similar total volumes applied at low frequencies elicited deep and extensive changes in the corneal layers whereas high application frequencies elicited comparatively superficial changes. Increasing NaOH concentrations effected measurably increasing corneal changes. Increasing the volume of applied NaOH also showed an increase in corneal changes. OCT imaging proved to be effective in observing, documenting and quantifying the changes in the corneal layers. The ex vivo model, in conjunction with the novel application method was able to induce and display distinctive and consistent correlations between NaOH volume, concentration and elicited corneal changes. This ex vivo ocular chemical burn model provides a consistent in vitro basis for pharmaceutical and toxicological experiments and investigations into corneal chemical burn mechanisms and treatment.

Irrigation with phosphate-buffered saline causes corneal calcification during treatment of ocular burns

Corneal calcification is a vision-threatening manifestation of calcium containing agents in ocular burn. As we previously reported, our interest was sparked by a particular discrepancy of a case: A patient treated for a non-calcium containing agent in eye burn from exposure to an alkaline mixture of NaOH and KOH, who unexpectedly developed corneal calcification. This current study aims to elucidate whether the 2min lasting irrigation with a phosphate-buffered saline itself, regardless of rinsing regimen, triggers corneal calcification. The Ex Vivo Eye Irritation Test (EVEIT) system was used on rabbit corneas to replicate the very same phosphate-buffered saline solution the patient was treated with. The rabbit corneas were first burned with 1 M NaOH, rinsed with 4.9% phosphate-buffered saline for 2 min, and were then moisturized with an artificial tear solution for 48 h. All corneas were fluorescein-stained for photo documentation, snap-frozen, lyophilizated, and the electrolyte content was analyzed by Energy-Dispersive X-ray spectroscopy (EDX). The EDX analysis revealed pathological phosphorous in corneal stroma after a single rinsing with phosphate-buffered saline. Ongoing application of artificial tears containing physiological 14.581 mmol Ca2+ /l led to macroscopically visible calcification, but only in areas of induced corneal erosion. Regardless of the rinsing protocol neither 2 or 15 min of eye rinsing with phosphate containing rinsing solutions, we have given proof that corneal calcification is a foreseeable effect of the phosphate-buffered saline rinsing of mechanically epithelial damaged and chemically burnt eyes. Thus, it is crucial to legally restrict the formulations of phosphate-buffered salines in the medical treatment of eye burns, corneal erosions or chemical splashes of the eye.

Safety and Tolerability of Overdosed Artificial Tears by Abraded Rabbit Corneas.

Purpose: Preservative-free cationic emulsion-based artificial tear (AT) is an innovative eye drop based on the Novasorb® technology with cetalkonium chloride (CKC) as the cationic agent. The cationic emulsion Cationorm is designed for the management of mild-to-moderate dry eye disease (DED) patients that present cornea epithelium alterations. The aim of the present study was to evaluate the safety and tolerability of overdosed ATs by altered corneal epithelium in vivo and assess the usefulness of the ex vivo eye irritation test (EVEIT) as a predictive alternate toxicity test method.Methods: The experimental procedure, treatment duration, and instillation frequency closely mimic in vivo the ex vivo protocol described by Pinheiro et al. and discussed in the Discussion and Conclusion section of this article. Two to 3-month-old female New Zealand white rabbits, n = 6 per group, were treated with ATs (21 instillations/day over 3 days) following corneal abrasion. Corneal fluorescein staining, in vivo confocal microscopy (IVCM), and slit lamp examinations were performed to assess corneal epithelium recovery and the ocular tolerability of the overdosed ATs.Results: All abraded eyes experienced almost complete epithelium recovery within 3 days following treatments with Cationorm, Optive, Vismed, and Saline. Benzalkonium chloride (BAK, 0.02%) treatment resulted in 82.4% reepithelialization. IVCM data illustrated corneal epithelium normal recovery. Acute local tolerability of the overdosed ATs was confirmed using Draize and McDonald–Shadduck's test scales.Conclusions: The different ATs were demonstrated to be well tolerated by abraded corneas in vivo, and the extreme overdosing regimen did not hamper the wound healing process of the rabbit eye in comparison to saline. These data did not confirm the ones obtained with the nonvalidated ex vivo eye irritation test.

The Ex vivo Eye Irritation Test (EVEIT) model as a mean of improving venom ophthalmia understanding

Snakes belonging to the genus Naja (Elapid family), also known as “spitting cobras”, can spit venom towards the eyes of the predator as a defensive strategy, causing painful and potentially blinding ocular envenoming. Venom ophthalmia is characterized by pain, hyperemia, blepharitis, blepharospasm and corneal erosions. Elapid venom ophthalmia is not well documented and no specific treatment exists. Furthermore, accidental ejection of venom by non-spitting vipers, as Bothrops, also occurs. The Ex vivo Eye Irritation Test model (EVEIT) has enabled important progress in the knowledge of chemical ocular burns. Considering the lack of experimental animal model, we adapted the EVEIT to study venom ophthalmia mechanisms. Ex vivo rabbit corneas were exposed to venoms from spitting (Naja mossambica, Naja nigricollis) and non-spitting (Naja naja, Bothrops jararaca and Bothrops lanceolatus) snakes, and rinsed or not with water. The corneal thickness and the depth of damage were assessed using high-resolution optical coherence tomography (HR-OCT) imaging and histological analysis. All Naja venoms induced significant corneal edema, collagen structure disorganization and epithelial necrosis. Corneas envenomed by African N. mossambica and N. nigricollis venoms were completely opaque. Opacification was not observed in corneas treated with venoms from non-spitting snakes, such as the Asian cobra, N. naja, and the vipers, B. jararaca and B. lanceolatus. Moreover, Bothrops venoms were able to damage the epithelium and cause collagen structure disorganization, but not edema. Immediate water rinsing improved corneal status, though damage and edema could still be observed. In conclusion, the present study shows that the EVEIT model was successfully adapted to set a new experimental ex vivo animal model of ophthalmia, caused by snake venoms, which will enable to explore new therapies for venom ophthalmia.

The Ex Vivo Eye Irritation Test as an alternative test method for serious eye damage/eye irritation.

Ocular irritation testing is a common requirement for the classification, labelling and packaging of chemicals (substances and mixtures). The in vivo Draize rabbit eye test (OECD Test Guideline 405) is considered to be the regulatory reference method for the classification of chemicals according to their potential to induce eye injury. In the Draize test, chemicals are applied to rabbit eyes in vivo, and changes are monitored over time. If no damage is observed, the chemical is not categorised. Otherwise, the classification depends on the severity and reversibility of the damage. Alternative test methods have to be designed to match the classifications from the in vivo reference method. However, observation of damage reversibility is usually not possible in vitro. Within the present study, a new organotypic method based on rabbit corneas obtained from food production is demonstrated to close this gap. The Ex Vivo Eye Irritation Test (EVEIT) retains the full biochemical activity of the corneal epithelium, epithelial stem cells and endothelium. This permits the in-depth analysis of ocular chemical trauma beyond that achievable by using established in vitro methods. In particular, the EVEIT is the first test to permit the direct monitoring of recovery of all corneal layers after damage. To develop a prediction model for the EVEIT that is comparable to the GHS system, 37 reference chemicals were analysed. The experimental data were used to derive a three-level potency ranking of eye irritation and corrosion that best fits the GHS categorisation. In vivo data available in the literature were used for comparison. When compared with GHS classification predictions, the overall accuracy of the three-level potency ranking was 78%. The classification of chemicals as irritating versus non-irritating resulted in 96% sensitivity, 91% specificity and 95% accuracy.

Anti‐glaucoma drugs and their additives, testing the influence of excipients on the corneal healing process in the EX‐Vivo Eye Irritation Test (EVEIT) system

AbstractPurpose This study examined the impact of anti‐glaucoma medications and their additives on the corneal healing process and corneal toxicity.Methods BAK‐free PG analogues (Lumigan UD, Monoprost, Taflotan Sine), soft preserved Travatan BAK free, as well as polyquaternium‐1 (0.001%) and Macrogolglycerolhydroxystearate 40 (5%) were tested regarding corneal irritability and effect on corneal healing against HYLO COMOD (1 mg/mL sodium hyaluronate) as positive and 0.02% benzalkonium chloride (BAK) as negative control. Formulas were applied over three days, six times daily on rabbit corneas cultured on an artificial anterior chamber the Ex Vivo Eye Irritation Test (EVEIT) system. Initially, four corneal abrasions (2.5–4.5 mm2) were induced. All defects were monitored by fluorescein stains and photographs.Results For BAK, the corneal erosion size increased from 14.16 mm² to 88.89 mm². A delay of corneal healing is evident for Monoprost (14.84 mm² to 9.45 mm²) and Macrogolglycerolhydroxystearate 40 (11.58 mm² to 1.56 mm²) in comparison to a complete healing for HYLO COMOD, Lumigan, Taflotan Sine, Travatan and polyquaternium‐1®. For both Monoprost and macrogolglycerolhydroxystearate 40, a bright opaque halo around the remaining erosion area could be noticed. Histology revealed severe alterations of the superficia stroma. Cornea tested without erosions show no corneal toxicity of macrogol 4000.Conclusion Macrogolglycerolhydroxystearate 40, an additive found in Monoprost® shows corneal toxicity solely in case of corneal erosions. This excipient is a known skin irritant. Therefore, the use of Monoprost® in patients with epithelial defects should be questioned. Commercial interest

Comparison of the lubricant eye drops Optive®, Vismed® and Cationorm® on the corneal healing process in an ex‐vivo model of the anterior ocular segment, the Ex Vivo Eye Irritation Test (EVEIT) System.

AbstractPurpose Evaluation of the impact of lubricant eye drops on the corneal healing process and corneal toxicity.Methods Optive and Cationorm were tested regarding corneal irritability and effect on corneal healing against Vismed and 0.01% benzalkonium chloride (BAK) as negative and positive control. Formulas were applied hourly over 3 days on rabbit corneas cultured on artificial anterior chambers (Ex‐Vivo Eye Irritation Test (EVEIT) system). Five corneae were used for each substance except for BAK (n=1). Initially, four corneal abrasions (2 ‐ 5.4 mm²) were induced. All defects were monitored by fluorescein stains and photographes.Results Optive and Vismed showed a complete corneal healing on day 2. In one cornea (Optive) erosion reoccurred on day 3. Erosion sizes of Cationorm treated corneae increased significantly from 12.20 mm² to a subtotal erosion of 51.89 mm² on day 3. Histology revealed epithelial loss and severe alterations of the superficial stroma for Cationorm. Glucose and lactate concentrations were about unchanged after application of Optive and Vismed. In contrast, Cationorm and BAK treated cornea showed a significant increase in lactate concentrations.Conclusion Vismed application led to a rapid corneal healing. Whether the toxicity seen for Optive in one cornea is a valid result should be examined in a long term application study. Cationorm shows considerable corneal toxicity that could be caused by its additive, cetalkonium chloride. Otherwise, Cationorms electrostatic properties lead to a drug film on the area of epithelial loss that could hinders epithelial cell migration and adhesion to heal the lesion. Commercial interest

Semifluorinated alkanes as a liquid drug carrier system for topical ocular drug delivery

Semifluorinated alkanes (SFA, e.g. perfluorobutylpentane F4H5, perfluorohexyloctane F6H8) are inert, non-toxic fluids capable of dissolving lipophilic drugs. The aim of this study to assess the bioavailability and safety of SFAs as drug solvents for the topical ocular application of Cyclosporin A (CsA). A commercially available CsA formulation (Restasis®, 0.05% CsA in castor oil) was tested against two novel formulations of 0.05% CSA in (a) F4H5 containing Ethanol (0.5w/w%) and (b) F6H8 containing Ethanol (0.5w/w%) with 0.05% CsA. Formulations were tested on rabbit corneas cultured on an artificial anterior chamber with a constant flow of an aqueous humour supplement (Ex Vivo Eye Irritation Test (EVEIT) system). Anterior chamber fluids were sampled at multiple time points to analyse the CsA concentration following single and repeated application regimes by HPLC. Photographs of fluorescein sodium-stained corneas were recorded for corneal toxicity evaluation. The impact of the formulations on the integrity of the corneal barrier function was tested after drug application by fluorescein sodium corneal diffusion experiments. The influence on the corneal metabolism was evaluated by analysis of the metabolic markers glucose and lactate.Restasis® did not pass the corneal barrier after short term application, CsA in ethanolic F4H6 reached a maximum of 152.95ng/ml in anterior chamber fluid samples whilst CsA in ethanolic F6H8 reached a maximum of 15.12ng/ml. After repeated applications for 8h, Restasis® reached 21.07ng/ml compared to 247.62ng/ml and 174.5ng/ml for F4H5 and F6H8, respectively. No corneal toxicity was observed in following application of any of the formulations.In contrast to the commercially available castor oil-based formulation, CsA dissolved in SFAs reached therapeutic inner ocular concentrations after topical administration, possibly leading to the replacement of systemic applications of CsA for inflammatory ocular disease.

The Ex Vivo Eye Irritation Test (EVEIT) in evaluation of artificial tears: Purite®-preserved versus unpreserved eye drops

Preservatives in artificial tears cause controversy. New developments such as the Purite system have been introduced into the market, with the promise of little damage to the corneal surface. We wanted to give insight into the differences in the effect of preserved and unpreserved artifical tears on rabbit corneas cultured with the Ex Vivo Eye Irritation Test (EVEIT) system. We compared the two artifical tears products Hylo Comod and Optive being dropped for 72 hours each hour one drop onto the corneal surface. Each cornea was mechanically wounded with four epithelial defects on each cornea with a size of 3 to 4.5 mm(2). With n = 4 corneas in the Hylo-Comod and n = 4 corneas in the Optive group, we exposed the corneal surfaces to repeated doses of these artificial tears for 3 days. We observed healing of corneal erosions and surface epithelial integrity with sodium-fluoresceine staining under cobalt blue light illumination. We found nearly complete healing of epithelial defects with both artificial tears. The Hylo-Comod group healed significantly faster. After 72 hours, the vast majority of epithelial defects were closed. All corneas exposed to Purite showed superficial stippling, whereas the HyloComod group did not show any stippling of the cornea; this difference was significant. Epithelial healing and recovery in the EVEIT system is observed in both groups, confirming the concept of artificial tears as a supporting factor of corneal health and healing. The superficial stippling of the corneal epithelium was observed only in the Optive group. This effect is considered as a marker of dry eye syndrome, and should be prevented by the application of artificial tears. Preservative-free eye drops such as HyloComod improve healing, and prevent symptoms of dry eye syndrome in the EVEITsystem. Compared to EVEIT results of former experiments with benzalconium chloride-preserved eye drops, Optive promoted healing of corneal erosions.

Changing the composition of buffered eye-drops prevents undesired side effects

PurposeThe Ex Vivo Eye Irritation Test (EVEIT) is used to analyse the clinical observations of corneal calcification attributed to the presence of phosphate within applied eye-drops used in treating glaucoma,...