Bovine Cornea Research Articles

Cannabinoid CB2R receptors are upregulated with corneal injury and regulate the course of corneal wound healing

CB2R receptors have demonstrated beneficial effects in wound healing in several models. We therefore investigated a potential role of CB2R receptors in corneal wound healing. We examined the functional contribution of CB2R receptors to the course of wound closure in an in vivo murine model. We additionally examined corneal expression of CB2R receptors in mouse and the consequences of their activation on cellular signaling, migration and proliferation in cultured bovine corneal epithelial cells (CECs). Using a novel mouse model, we provide evidence that corneal injury increases CB2R receptor expression in cornea. The CB2R agonist JWH133 induces chemorepulsion in cultured bovine CECs but does not alter CEC proliferation. The signaling profile of CB2R activation is activating MAPK and increasing cAMP accumulation, the latter perhaps due to Gs-coupling. Lipidomic analysis in bovine cornea shows a rise in acylethanolamines including the endocannabinoid anandamide 1 h after injury. In vivo, CB2R deletion and pharmacological block result in a delayed course of wound closure. In summary, we find evidence that CB2R receptor promoter activity is increased by corneal injury and that these receptors are required for the normal course of wound closure, possibly via chemorepulsion.

Proton conductivity of glycosaminoglycans.

Proton conductivity is important in many natural phenomena including oxidative phosphorylation in mitochondria and archaea, uncoupling membrane potentials by the antibiotic Gramicidin, and proton actuated bioluminescence in dinoflagellate. In all of these phenomena, the conduction of protons occurs along chains of hydrogen bonds between water and hydrophilic residues. These chains of hydrogen bonds are also present in many hydrated biopolymers and macromolecule including collagen, keratin, chitosan, and various proteins such as reflectin. All of these materials are also proton conductors. Recently, our group has discovered that the jelly found in the Ampullae of Lorenzini- shark’s electro-sensing organs- is the highest naturally occurring proton conducting substance. The jelly has a complex composition, but we proposed that the conductivity is due to the glycosaminoglycan keratan sulfate (KS). Here we measure the proton conductivity of hydrated keratan sulfate purified from Bovine Cornea. PdHx contacts at 0.50 ± 0.11 mS cm -1, which is consistent to that of Ampullae of Lorenzini jelly at 2 ± 1 mS cm -1. Proton conductivity, albeit with lower values, is also shared by other glycosaminoglycans with similar chemical structures including dermatan sulfate, chondroitin sulfate A, heparan sulfate, and hyaluronic acid. This observation supports the relationship between proton conductivity and the chemical structure of biopolymers.

Formulation and Characterization of Glutathione-Loaded Bioadhesive Hydrogel for Ocular Delivery

Glutathione may prevent age-related, oxidative damage to ocular tissues but has poor corneal penetration. Hydrogel formulations were investigated to determine an optimized ocular delivery system. The rheology and texture profile of formulations were investigated at 25°C. The 1% w/v glutathione was incorporated into systems demonstrating desirable characteristics for ocular vehicles and physical characteristics re-determined. In vitro cumulative glutathione delivery across the bovine cornea was measured over 8 hours at 32°C in Franz diffusion cells. Carbopol-containing Poloxamer systems exhibited shear-thinning behavior desirable for ocular formulations whilst Polyvinyl alcohol (PVA) and Polyacrylic acid (PAA) systems exhibited Newtonian behavior. Of the glutathione-containing systems, 0.2% w/v Carbopol 1342 was the most viscous with a viscosity of 960 cP at a shear rate of 100 sec-1. All formulations significantly increased the amount of glutathione delivered across the cornea (relative to an aqueous solution of glutathione) with the exception of 0.1% w/v Carbopol 940 (p=0.12). Formulations containing 0.1% w/v Carbopol 934, 0.1% w/v Carbopol 1342 and 0.2% w/v Carbopol 940 improved penetration dramatically (ca. 30%); but were not significantly different from each other. Therefore, Carbopol and Poloxamer formulations demonstrated enhanced penetration of glutathione across the cornea. The 0.2% w/v C940-containing-Poloxamer formulation was determined to be the most promising drug delivery system for ocular delivery of glutathione.

Development and investigation of timolol maleate niosomal formulations for the treatment of glaucoma

The objective of the present study was to develop niosomal gels loaded with Timolol Maleate (TM), Non-selective beta-adrenergic receptor antagonist, for prolonged duration and improved bioavailability for glaucoma treatment. TM niosomes were prepared by film hydration method with various mixtures of different non-ionic surfactants including Span 20, 40, 60 and Tween 20, 40, along with cholesterol. The prepared vesicles were evaluated for entrapment efficiency, in vitro drug release, particle size, zeta potential and morphology by optical and transmission electron microscopy (TEM). The selected formulations were incorporated into Sodium carboxymethyl cellulose (CMC Na) and Carbopol 934 gels. Gel formulations were characterized for in vitro drug permeation and ex vivo drug permeation through bovine cornea. In addition, stability study, isotonicity test and in-vivo evaluation of the selected formulations were done. The results showed that, the niosomes formed were white and spherical in shape with uniform particle size. Formulations containing span 60 and that containing mixture of span 60 and tween 40 gave the highest entrapment efficiency (94.6% and 98.8%, respectively) and a sustained release of timolol maleate from the niosomes within 24 h (96% and 97.10%, respectively). The in vitro and ex vivo drug release studies showed that there was a slow and prolonged release of drug from niosomal gel formulations. Considering the in-vitro release, niosomal gel formulae GN5 and GN6 (containing CMC Na 3% w/w) were the best among the studied formulations. Draize test was carried out and the intra-ocular pressure lowering activity of prepared formulations were detected and compared with marketed Timogel. Formula containing 3% CMC Na showed relative bioavailability 1.6 times more than bioavailability of marketed Timogel.

Brinzolamide–loaded nanoemulsions: ex vivo transcorneal permeation, cell viability and ocular irritation tests

The aim of this study was to investigate the corneal penetration of brinzolamide (BZ) nanoemulsions (NEs) and evaluate their in vitro and ex vivo irritancy potential. Twelve BZ NEs were prepared by the spontaneous emulsification method and ex vivo permeability studies were conducted using excised bovine corneas fixed onto Franz diffusion cells. To confirm the safety of the formulations for ophthalmic use, preparations were examined for potential ocular irritancy using a cell viability assay on retinal cells, the Hen's Egg Test-Chorio-Allantoic Membrane (HET-CAM) and the bovine corneal opacity-permeability (BCOP) test. Seven BZ NEs exhibited superior penetration across isolated bovine cornea compared to the marketed BZ suspension. The half maximal inhibitory concentration (IC50) values of various surfactants and oils determined using the sulforhodamine B cell viability assay on retinal cells showed that Transcutol P, Cremophor RH40 and Triacetin were the least toxic excipients and may be safely used in the eye at various concentrations. HET-CAM and BCOP tests revealed that NE6B and NE4C did not result in any irritation and were thus considered safe for ocular use. Our finding suggests that optimized NEs can be a safe and effective vehicle for ocular delivery of BZ.

In Vitro Models of Eye Infection with Neisseria gonorrhoeae.

Prophylaxis with silver nitrate and later antibiotics has significantly reduced the cases of infant blindness from gonococcal infection at birth to the point where it has all but been forgotten in the developed world as the devastating disease that it was in the pre-antibiotic era. As a result, while it is known that the bacteria are transmitted to the eyes during passage through the infected birth canal, little is known about Neisseria gonorrhoeae colonization of the eye and the establishment and progression of keratitis. Treatment failures due to rising antimicrobial resistance necessitate investigations into all aspects of gonococcal disease, including eye infections, so that new treatment strategies can be developed. Here we present models for N. gonorrhoeae eye infection using excised bovine corneas and coculture of gonococci with primary human corneal epithelial cells. These models can be used to explore the interactions of the bacteria with corneal tissues and cells and to investigate novel therapeutics against infection.

Evaluation of 2 ex vivo Bovine Cornea Storage Protocols for Drug Delivery Applications

The use of corneal tissue for ex vivo therapeutic evaluations is limited due to its rapid loss of viability after excision. Optimization of storage conditions may allow prolonged retention of physical tissue properties. In this study, we evaluated how storage in optimized organ culture (OC) medium at 37°C or phosphate-buffered saline (PBS) at 2–8°C impacted physical properties of bovine corneas. Tissue hydration, permeability and histology were monitored at baseline and following 1, 4 and 7 days of storage. Corneas stored in OC demonstrated significantly higher hydration and permeability when compared to those stored in PBS. Histology revealed that storage in OC consistently caused detachment of the epithelial layer by day 4 of storage, whereas both storage conditions caused a significant increase in stromal thickness and tissue vacuolation. This study highlights the limitations of currently available corneal tissue storage approaches for ex vivo drug permeation studies.

Mucoadhesive PEGylated inulin-based self-assembling nanoparticles: In vitro and ex vivo transcorneal permeation enhancement of corticosteroids

As transcorneal drug delivery is still a challenge, the scope of the present study was to prepare useful nanosystems able to enhance transcorneal permeation/penetration of drugs. Moreover, this work aims to evaluate the effectiveness of inulin-based nanosystems in the specific field of ocular drug delivery and the effect of PEG chains to promote mucoadhesion, stability and transcorneal penetration/permeation enhancer effect of self-assembling nanoparticles in vitro (transwell systems and HCE) and ex vivo (Franz cells and bovine cornea). In particular, inulin was chosen as the starting natural polysaccharide polymer to design a novel amphiphilic derivative named INU-EDA-RA-PEG capable of self-assembling to form self-assembling nanoparticles and corticosteroids-loaded self-assembling nanoparticles. As observed, self-assembling nanoparticles show appropriate particle size values, mucoadhesivity and cytocompatibility. Moreover, self-assembling nanoparticles are able to act efficiently as permeation/penetration enhancer. Additionally, the presence of PEG has positive influence. Thus, the developed inulin-based nanosystems represent a promising tool to improve transcorneal delivery of corticosteroids.

Soluplus micelles for acyclovir ocular delivery: Formulation and cornea and sclera permeability

Nearly 20% of people affected by the herpes simplex virus (HSV) suffer from vision problems. The virus can infect all layers of the cornea or cause inflammatory diseases of the sclera. The aim of this work was to test whether encapsulation of acyclovir in Soluplus or Solutol polymeric micelles increases its solubility, corneal permeability and sclera penetration. The aqueous solubility of acyclovir is known to be low, and therefore approaches that increase both its solubility and ability to penetrate through the eye may favor the efficacy of the treatments. Copolymer dispersions (covering wide range of concentrations) were prepared in water and PBS 7.4 and characterized regarding size and Z-potential (close to zero). Solutol micelles increased their size when the drug was incorporated (135 vs. 19 nm), while Soluplus micelles showed little difference (137 nm). Only Soluplus micelles significantly enhanced acyclovir solubility and withstood dilution stability tests. Soluplus (12–20%) formulations showed a progressive increase in viscoelasticity as temperature rose, which may allow for easy dropping onto the eye and subsequent retention in the gel form. Drug permeability through bovine cornea and sclera was investigated in detail. Although similar permeability coefficients were recorded for the drug when applied as the free drug in solution or formulated in Soluplus micelles, the micelle formulation significantly shortened the permeation lag time through the cornea. Moreover, Soluplus micelles were advantageous compared to the drug solution in terms of greater amount of acyclovir accumulated in both cornea and sclera, and higher steady state flux. If compared with cornea, the amounts of drug permeated through the sclera were approx. 10 times greater, which opens the possibility of drug delivery to the posterior eye segment.

Fundamental pharmacological expressions on ocular exposure to capsaicin, the principal constituent in pepper sprays

Eye irritation assessment is compulsory to anticipate health risks in military personnel exposed to riot control agents such as capsaicin, the principal constituent of oleoresin capsicum, or pepper sprays. The present work investigates certain fundamental yet unaddressed pharmacological manifestations on ocular exposure to capsaicin. Ocular pharmacology of capsaicin was studied using acute eye irritation (AEI), bovine corneal opacity and permeability (BCOP) assay, corneal fluorescein staining and indirect ophthalmoscopy studies, transcorneal permeation, Schirmer tear secretion test, nerve conduction velocity study and enzyme-linked immunosorbent assay (ELISA). Additionally, histopathology and scanning electron microscopy (SEM) of bovine corneas and rat optic nerves were done to further estimate capsaicin induced morphological variations. Our findings demonstrated that AEI, BCOP, corneal fluorescein staining and indirect ophthalmoscopy were useful in assessing capsaicin induced ocular irritation; AEI and BCOP also contributed towards indicating the eye irritation potential of capsaicin as per the United Nations Globally Harmonized System of Classification and Labelling of Chemicals categorization. Additional experimental observations include considerable transcorneal permeation of capsaicin, capsaicin induced reduction in tear secretions and nerve conduction velocity and increased expression of proinflammatory cytokines by ELISA. Histopathology and SEM were favourable techniques for the detection of capsaicin induced ocular physiological modifications.

An Experimental Study of Femto-Laser in Assisting Xenograft Acellular Cornea Matrix Lens Transplantation

BackgroundThe aim of this study was to evaluate the feasibility of using a femto-laser in assisting xenograft cornea matrix lens transplantation in correcting ametropia, along with evaluating the effectiveness and predictability of this procedure.Material/MethodsA corneal matrix pouch was prepared on the right eyes on 8 healthy New Zealand rabbits by a femto-laser that was also employed to perform small incision lenticule extraction (SMILE) on 8 bovine cornea matrix lenses (+6D). A lens was treated acellular and implanted into a right rabbit cornea matrix pouch. Surface inflammation was observed at 1, 2, 4, 8, 12, and 24 weeks after surgery. Anterior ocular segment optical coherence tomography (OCT), corneal topography, retinoscopy, and cornea endothelial cell enumeration were performed.ResultsAll the surgeries were successfully performed without any complications. The hyperopia condition of the rabbit eyes transformed into myopia status at an early stage and gradually developed hyperopia. Diopter at 24 weeks after surgery was 1/3 of that before surgery. Central corneal thickness stabilized at 4 weeks after surgery. Anterior segment OCT showed a clear lens edge at early post-operative stage, and blurred edge at 24 weeks later, indicating gradual fusion with the rabbit corneal matrix.ConclusionsFemto-laser assisted xenograft corneal matrix lens transplantation is safe and effective in correcting ametropia, with satisfactory predictability, thus providing novel choice for correcting ametropia.

Average refractive index of tendon as a function of water content

Experimental data on the dependence of the average refractive index of rat tail tendon (RTT) on water content are reported. Using optical coherence tomography, the average group refractive index (at a wavelength of 930 nm) and cross-section area of rat tail tendon fascicle specimens during their air-drying and rehydration were monitored. The dependence of the average group refractive index of RTT ( n g ) on the volume fraction of water ( C w ) has been found to be nonlinear and to be well approximated by the quadratic polynomial n g = 1.5713 – 0.1969 C w – 0.0328( C w ) 2 . The reported data are shown to be in good agreement with previously published data for bovine cornea.

Development and Evaluation of a Novel Microemulsion of Dexamethasone and Tobramycin for Topical Ocular Administration.

The purpose of this study was to develop and evaluate a novel dexamethasone- and tobramycin-loaded microemulsion for its potential for treating anterior segment eye infections. The microemulsion was evaluated for pH, particle size, zeta potential, light transmittance, morphology, and in vitro drug release. Sterility of the microemulsion was evaluated by direct as well as plate inoculation methods. Anti-inflammatory activity of dexamethasone, bactericidal activity of tobramycin, and cytotoxicity of the microemulsion were assessed and compared to that of the marketed eye drop suspension (Tobradex®). Histological evaluation was performed in bovine corneas to assess the safety of microemulsion in comparison to Tobradex suspension. In addition, the stability of the microemulsion was studied at 4°C, 25°C, and 40°C. The pH of the microemulsion was close to the pH of tear fluid. The microemulsion displayed an average globule size under 20 nm, with light transmittance around 95%-100%. The aseptically prepared microemulsion remained sterile for up to 14 days. The cytotoxicity of the microemulsion in bovine corneal endothelial cells was comparable to that of the Tobradex suspension. The anti-inflammatory activity of dexamethasone and the antibacterial activity of tobramycin from the microemulsion were significantly higher than those of the Tobradex suspension (P < 0.05). Histological evaluation showed an intact corneal epithelium without any signs of toxicity, and the developed microemulsion was found to be stable at 4°C and 25°C for 3 months. In conclusion, the developed microemulsion could be explored as a suitable alternative to the marketed suspension for treating anterior segment eye infections.

Ophthalmic Econazole Hydrogels for the Treatment of Fungal Keratitis

Econazole is a feasible alternative treatment in the management of fungal keratitis. Nevertheless, its low water solubility is considered the main limitation to the incorporation into ophthalmic formulations. In this work, econazole nitrate is solubilized by using cyclodextrins to achieve an optimum therapeutic concentration. Phase solubility diagrams suggest α-cyclodextrin as the most effective cyclodextrin and later the inclusion complex formed with this one was characterized in solution by 1D, 2D-NMR, and molecular modeling. Econazole-α-cyclodextrin inclusion complex was included in 2 types of ocular hydrogels: a natural polysaccharides ion-sensitive hydrogel and a hyaluronic acid hydrogel. Both of them show no ocular irritation in the hen's egg test on chorioallantoic membrane assay and a controlled econazole release over time. Permeability studies suggest that hydrogels do not modify the econazole nitrate permeability through bovine cornea in comparison with an econazole-α-cyclodextrin inclusion complex solution. Finally, ocular biopermanence studies performed using positron emission tomography show these hydrogels present a high retention time on the eye. Results suggest the developed formulations have a high potential as vehicles for the econazole topical ocular administration as fungal keratitis treatment.

Optimizing Genipin Concentration for Corneal Collagen Cross-Linking: An ex vivo Study

Purpose: Studying genipin variable concentrations, treatment durations, and delivery methods as a substance to increase corneal stiffness by inducing corneal collagen cross-linking (CXL). Materials and Methods: 100 bovine corneas treated with different genipin concentrations (0.1, 0.5, and 1%) and treatment durations (15 min, 40 min, 2 h, and 3 days) through different delivery methods compared to 10 controls treated with riboflavin/UV. Histology examination, enzymatic digestion with collagenase and thermal differential scanning calorimetry were performed on the different samples. Results: Bovine corneas soaked in 0.5% genipin morphologically showed 4.7% CXL in comparison to 5.6% in controls (p < 0.05). Corneas treated with topical 0.5% genipin, by a 140-µL drop applied hourly for 2 h, showed 7% corneal CXL. Corneas treated with topical genipin 0.5% for 30 min, 1 and 2 h showed 54 ± 6, 40 ± 7, and 39 ± 9% enzymatic degradation, respectively, in comparison to controls (74%). Corneas treated with 0.5% genipin for 1, 2, and 8 h showed higher thermal denaturation resistance (Td values of 64.9 ± 0.3, 64.7 ± 0.0 and 67.3 ± 0.9), respectively, in comparison to the control group (64.6 ± 0.5) (p < 0.05). Conclusions: Genipin 0.5%, in a 140-µL drop applied hourly for 2 h, showed better potential to enhance corneal stiffness and stability through inducing CXL.

Enhancement and inhibition effects on the corneal permeability of timolol maleate: Polymers, cyclodextrins and chelating agents

This study investigates how both bioadhesive polymers (chitosan, hyaluronic acid and alginate) and permeability enhancers (ethylene glycol- bis(2-aminoethylether)- N, N, N', N'- tetraacetic acid (EGTA) and hydroxypropyl-ß-cyclodextrin) influence the permeability of the anti-glaucoma drug timolol maleate through ex vivo bovine corneas. Our results showed that only the permeability enhancers alone were able to increase drug permeability, whereas the polymers significantly reduced drug permeation, and however, they increased the pre-corneal residence of timolol. Ternary systems (polymer-enhancer-drug) showed a reduced drug permeability compared to the polymers alone. Fluorescence microscopy analysis of the epithelium surface confirmed there was no evidence of epithelial disruption caused by these formulations, suggesting that polymer-enhancer interactions reduce drug solubilization and counteract the disruptive effect of the permeability enhancers on the surface of the cornea. Further mucoadhesive tests, revealed a stable interaction of chitosan and hyaluronic acid with the epithelium, while alginate showed poor mucoadhesive properties. The differences in mucoadhesion correlated with the permeability of timolol maleate observed, i.e. formulations containing mucoadhesive polymers showed lower drug permeabilities.The results of the present study indicate polymers acting as an additional barrier towards drug permeability which is even more evident in the presence of permeability enhancers like EGTA and hydroxypropyl-ß-cyclodextrin. Then, this study highlights the need to adequately select additives intended for ocular applications since interactions between them can have opposite results to what expected in terms of drug permeability.

Cysteamine polysaccharide hydrogels: Study of extended ocular delivery and biopermanence time by PET imaging

Cystinosis is a rare autosomal recessive disorder in which cystine crystals accumulate within the lysosomes of various organs, including the cornea. Ocular treatment is based on the administration of cysteamine eye drops, requiring its instillation several times per day. We have introduced the cysteamine in two types of previously developed ocular hydrogels (ion sensitive hydrogel with the polymers gellan gum and kappa-carrageenan and another one composed of hyaluronic acid), aiming at increasing the ocular retention in order to extend the dosing interval. The biopermanence studies (direct measurements and PET/CT) show that these formulations present a high retention time on the ocular surface of rats. From the in vitro release study we determined that both hydrogels can control the release of cysteamine over time, showing a zero order kinetics during four hours. At the same time, these hydrogels could act as corneal absorption promoters, as they allow a higher permeation of cysteamine through bovine cornea compared to a solution. HET-CAM test and cytotoxicity assays show no irritation on the ocular surface. These results demonstrate that the developed formulations present a high potential as vehicles for the topical ocular administration of cysteamine.

Delivery of Riboflavin-5′-Monophosphate Into the Cornea: Can Liposomes Provide Any Enhancement Effects?

Keratoconus is a progressive condition caused by the thinning of the cornea, which eventually deforms the front surface of the eye into a cone shape leading to ghosting, multiple images, glare, and several other vision problems. Currently, keratoconus is treated with UV-induced riboflavin (Rb)-mediated collagen cross-linking, which requires a physical removal of the corneal epithelium under topical anesthesia. This study reports the penetration of Rb and its more water-soluble form, riboflavin-5′-monophosphate (RbP), into the bovine cornea ex vivo. Using ex vivo bovine corneal tissues and 0.8 mg/mL drug solutions in phosphate buffer, it was established that RbP penetration into the cornea within 3 h of diffusion experiment was greater (17.3 ± 0.8 μg) compared with Rb (10.4 ± 4.2 μg). In the cornea, RbP was found to convert to Rb, which is mediated with enzymes present in this tissue. Several formulations including the conventional and propylene glycol–containing liposomes with encapsulated RbP have been developed, and their effect on the drug penetration into the bovine cornea was evaluated. Encapsulation of RbP into the liposomes did not provide any statistically significant improvement in the penetration of RbP into the cornea.

Nanodispersion-loaded mucoadhesive polymeric inserts for prolonged treatment of post-operative ocular inflammation

Mucoadhesive polymeric films incorporated with ketorolac tromethamine-loaded nanodispersion aiming the sustained delivery of the drug to the cornea have been developed and characterised for the treatment of post-operative ocular inflammation. Nanodispersions were prepared by ionic gelation method with various concentrations of chitosan and sodium tripolyphosphate. The developed nanodispersions were analysed for morphology, particle size, dispersion homogeneity, zeta potential, entrapment efficiency and drug release. The nanodispersion that showed the smallest particle size and the highest entrapment efficiency was incorporated in optimised HPMC E15 and Eudragit RL100/HPMC K4m films. The formulation with optimum physicomechanical properties was selected to study its ex vivo transcorneal permeation through freshly excised bovine cornea in comparison with the nanodispersion and the marketed eye drops (Acular®). The polymeric ocular film showed greater permeation than aqueous eye drops. Moreover, the ocular film revealed a prolonged anti-inflammatory effect compared to eye drops when applied to inflamed rabbit’s eyes.

Lacking applicability of in vitro eye irritation methods to identify seriously eye irritating agrochemical formulations: Results of bovine cornea opacity and permeability assay, isolated chicken eye test and the EpiOcular™ ET-50 method to classify according to UN GHS

In vitro methods have gained regulatory acceptance for the prediction of serious eye damage (UN GHS Cat 1). However, the majority of in vitro methods do not state whether they are applicable to agrochemical formulations. This manuscript presents a study of up to 27 agrochemical formulations tested in three in vitro assays (three versions of the bovine corneal opacity and permeability test (BCOP, OECD TG 437) assay, the isolated chicken eye test (ICE, OECD TG 438) and the EpiOcular™ ET-50 assay). The results were compared with already-available in vivo data. In the BCOP only one of the four, one of five in the ICE and six of eleven tested formulations in the EpiOcular™ ET-50 Neat Protocol resulted in the correct UN GHS Cat 1 prediction. Overpredictions occurred in all assays. These data indicate a lack of applicability of the three in vitro methods to reliably predict UN GHS Cat 1 of agrochemical formulations. In order to ensure animal-free identification of seriously eye damaging agrochemical formulations testing protocols and/or prediction models need to be modified or classification rules should be tailored to in vitro testing rather than using in vivo Draize data as a standard.