Eye Irritation Test Research Articles

Rhamnolipid Nano-Micelles Inhibit SARS-CoV-2 Infection and Have No Dermal or Eye Toxic Effects in Rabbits.

Hand hygiene is considered to be the key factor in controlling and preventing infection, either in hospital care settings or in the community. Alcohol-based hand sanitizers are commonly used due to their rapid action and broad spectrum of microbicidal activity, offering protection against bacteria and viruses. However, their frequent administration during COVID-19 pandemic was associated with serious hazards, such as skin toxicity, including irritation, skin dermatitis, skin dryness or cracking, along with peeling redness or itching, with the higher possibility of getting infections. Thus, there is a need to find alternative and novel approaches for hand sanitation. In our previous publications, we reported that rhamnolipids nano-micelles had a comparable antibacterial activity to alcohol-based hand sanitizer and a lower cytotoxicity against human dermal fibroblast cells. In the current study, we investigated the antiviral activity of rhamnolipids nano-micelles against SARS-CoV-2. There was no cytotoxic effect on Vero cells noted at the tested concentrations of rhamnolipids nano-micelles. The rhamnolipids nano-micelles solution at 20, 78, and 312 µg/mL all demonstrated a significant (p < 0.05) decrease of virus infectivity compared to the virus only and the blank vehicle sample. In addition, an acute irritation test was performed on rabbits to further ascertain the biosafety of rhamnolipids nano-micelles. In the eye and skin irritation tests, no degree of irritation was recorded after topical application of rhamnolipids nano-micelles. In addition, histopathological, biomarker, and hematological analyses from animals treated with rhamnolipids nano-micelles were identical to those recorded for untreated animal. From the above, we can conclude that rhamnolipids nano-micelles are a good candidate to be used as a hand sanitizer instead of alcohol-based hand sanitizers. However, they must still be tested in the future among healthcare workers (HCW) in a health care setting to ascertain their antimicrobial efficacy and safety compared to alcohol-based hand sanitizers.

Evaluation of in vitro cornea models for quantifying destructive effects of chemicals

In vitro models are available as alternatives for the Draize eye irritation test. However, most of the alternative models are not quantitative nor designed to evaluate the effects of chemicals on the corneal barrier such as those encountered in pharmaceutical and cosmetic products. The objective of the present study was to investigate tissue electrical resistance to provide sensitive in vitro testing of tissue alteration caused by chemicals in pharmaceutical and cosmetic formulations as a potential eye irritation testing approach. The experimental protocols for effective tissue resistance measurements were examined using two in vitro eye models: porcine cornea and EpiCorneal. In these models, a test chemical was applied to the cornea or EpiCorneal tissue for 1 min, and tissue resistances/conductances were measured at 1–60 min after the application. The changes in conductance of the tissues after exposure to the chemicals were shown to provide quantitative evaluations to the influence of the chemicals. A correlation was found between the two in vitro models. The results suggest that these models can provide quantitative in vitro assessments of eye-irritating chemicals.

The user safety assessment of a selenized yeast feed additive

Purpose: Of the several selenized yeasts authorised for use as feed additives in the EU, Saccharomyces cerevisiae CNCM I-3060 inactivated’ (Sel-Plex®), was the first to be approved for use, in 2006. The additive has a concentration of selenium between 2000 and 2400 mg/kg and a selenomethionine content greater than 63%. Previous toxicological and safety studies have shown Sel-Plex® to be safe for use for target animal species, consumers, users and the environment. A new formulation of Sel-Plex® was recently developed however, with a minimum selenium content of 3000 mg/kg. The increase in selenium in this product, Sel-Plex® 3000, presented the need to assess the risk for workers and users and to establish if there would be any eye and/or skin irritancy and skin sensitisation effects associated with the product. The purpose of this paper is to present the methodology and results of the user safety skin and eye studies performed on Sel-Plex® 3000. Materials & Methods: In vitro skin and eye models were used to assess skin and eye irritancy, while skin sensitisation was examined using an in vivo method. The acute eye irritation was evaluated using a Reconstructed human Cornea-like Epithelium (RhCE) model, which followed the OECD guideline 492. The skin irritation was assessed based on its ability to induce cell death in a commercial reconstructed human epidermis (RhE) model (EPISKIN™) according to the OECD Guideline No. 439. The skin sensitising potential was evaluated in the Guinea pig in line with OECD Guideline 406, and measured the extent and degree of skin reaction to a challenge exposure following previous topical exposure of a substance on the skin. Results: The skin and eye irritation test results showed that Sel-Plex® 3000 was a non-irritant in both cases. The skin sensitisation study showed that the additive did not generate a sensitisation response in the guinea pig and should not be considered a skin sensitiser. Conclusion: These results indicate that Sel-Plex® 3000 is safe to use for workers in an industrial setting when handling the product and the studies may be further used to support regulatory compliance in respective markets.

In-house performance assessment of 3D QobuR-Reconstructed Human Cornea-Like Epithelium (RhCE) for the evaluation of eye hazard

To replace the Draize eye irritation test (OECD Test Guideline 404), several test methods based on reconstructed cornea-like epithelium (RhCE) have been developed and adopted in the OECD TG 492. The objective of this study was to stablish the experimental procedures and evaluate the performance assessment of QobuR-RhCE, an in-house RhCE model to be used for the evaluation of eye hazard. We define the essential structural, functional and procedural elements of the test method components to help assuring that the proposed test method is based on the same concepts as the validated reference methods. Performance assessment was evaluated in accordance with the revised performance standards for the assessment of proposed similar or modified in vitro reconstructed human cornea-like epithelium and the minimum list of reference chemicals was evaluated. As result, the proposed method scored 93.3% sensibility, 60% specificity, 76.7% accuracy and 96.7% within-laboratory reproducibility (WLR), providing a similar performance in comparison to the validated reference methods. Additionally, we describe a secondary endpoint based on Transepithelial Electrical Resistance (TEER) that could be of use to better discriminate between irritants and non-irritants. Taken together the results indicate that the QobuR-RhCE test method is an accurate screening tool that can be used as a standalone alternative to evaluate ocular irritation.

Cytotoxicity assay to assess eye irritation – A comparison with other methods and possible strategies for use

Many in vitro methods can be used to classify eye irritation or damage caused by exposure to a substance. In this study, a recently described method called the Cytotoxicity Assay to Assess Eye Irritation (CEI) was compared with other selected in vitro methods adopted in the OECD guidelines. Furthermore, the influence of combining more than one in vitro method was investigated. Basic performance indices were considered and a risk assessment based on the number of correct and incorrect results (overestimated or underestimated) was performed. The CEI results were similar to those of other in vitro tests, however, the CEI can also directly classify substances irritating to the eyes. When the CEI preceded an eye irritation test based reconstructed human corneal-like epithelium, there were fewer underestimates compared with other method combinations. This combination can better protect human health and provides results comparable to those obtained in animal tests.

Ascorbic acid specifically reduces the misclassification of nonirritating reactive chemicals in the OptiSafe™ macromolecular eye irritation test

Recently, we showed that the addition of physiological concentrations of ascorbic acid, a tear antioxidant, to the OptiSafe™ macromolecular eye irritation test reduced the false-positive (FP) rate for chemicals that had reactive chemistries, leading to the formation of reactive oxygen species (ROS) and molecular crosslinking. The purpose of the current study was to 1) increase the number of chemicals tested to comprehensibly determine whether the antioxidant-associated reduction in OD is specific to FP chemicals associated with ROS chemistries and 2) determine whether the addition of antioxidants interferes with the detection of true positive (TP) and true negative (TN) ocular irritants. We report that when ascorbic acid is added to the test reagents, retesting of FP chemicals with reactive chemistries show significantly reduced OD values (P < 0.05). Importantly, ascorbic acid had no significant effect on the OD values of TP or TN chemicals regardless of chemical reactivity. These findings suggest that supplementation of ascorbic acid in alternative ocular irritation tests may help improve the detection of TN for those commonly misclassified reactive chemicals.

Acute toxicity and 28-day repeated dose studies of multi-walled carbon nanotubes

In the light of the increased production and use of multi-walled carbon nanotube (MWCNT) in consumer products, there is a need for screening the potential toxicity of this nanomaterial. In the present study, the researchers have investigated the acute dermal and acute eye irritation in rabbits and acute oral irritation and 28-day repeated administration of MWCNT in rats. A single dose of 5000 mg/kg was used to test Albino Rabbits for acute dermal and eye irritation test. Similar dose was given to Sprague Dawley Rats for the acute oral and 28-day repeated dose test. Clinical signs/manifestations were closely observed for the first four (4) hours post sample administration and was continued for 1,2,3,7 and 14-day observation period. In acute toxicity study, no treatment-related death or toxic signs were observed with MWCNT. In the 28-day repeated dose study, no significant differences in hematology and clinical biochemistry were detected between control and MWCNT. However, histopathology results revealed mild periarteriolar lymphoid cell depletion in the spleen and mild tubular cell degeneration on the cortex and medulla of both kidneys of the rats while no remarkable lesions were seen on the other organs of both female and male rats.

Safety assessment of a novel water-soluble extract of Boswellia serrata gum resin: acute toxicity, 90-day sub-chronic toxicity, Ames’ bacterial reverse mutation, and in vivo micronucleus assays

Boswellia serrata gum resin extracts have demonstrated potential benefits in alleviating joint pain and discomfort of osteoarthritis. The major objective of the present study was to assess the safety of a water-soluble B. serrata gum resin extract (LI51202F1) in diverse models of acute oral, acute dermal, primary dermal irritation, eye irritation, and 90-day sub-chronic repeated dose toxicity studies, as well as Ames’ bacterial reverse mutation assay and in vivo micronucleus assay. The acute oral and dermal toxicity studies in Sprague Dawley (SD) rats demonstrated that the median lethal dose (LD50) of LI51202F1 is >2000 mg/kg body weight (BW). The acute dermal and eye irritation tests in New Zealand white rabbits exhibited that LI51202F1 is non-irritating to the skin and mildly irritating to the eyes, respectively. The 90-days sub-chronic repeated oral dose study demonstrated that the LI51202F1-treated male and female SD rats did not show signs of toxicity on their BW, food intake, organ weights, thyroid hormones, and on the clinical pathology, gross pathology, and histopathological assessments. In male and female rats, the no-observed-adverse-effect level (NOAEL) of LI51202F1 was 500 mg/kg/day, the highest tested dose in the study. The results of the bacterial reverse mutation assay in Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2uvrA (pKM101) strains in the presence or absence of S9 metabolic activation system and a micro-nucleus assay in mouse bone marrow erythrocytes demonstrated that LI51202F1 is neither mutagenic nor clastogenic. In conclusion, under the conditions of these studies, LI51202F1 demonstrated broad-spectrum safety.

An overview of current alternative models in the context of ocular surface toxicity.

The 21st century has seen a steadily increasing social awareness of animal suffering, with increased attention to ethical considerations. Developing new integrated approaches to testing and assessment (IATA) strategies is an Organisation for Economic Co-operation and Development (OECD) goal to reduce animal testing. Currently, there is a lack of alternative models to test for ocular surface toxicity (aside from irritation) in lieu of the Draize eye irritation test (OECD guideline No. 405) performed in rabbits. Five alternative in vitro or ex vivo methods have been validated to replace this reference test, but only in combination. However, pathologies like Toxicity-Induced Dry Eye (TIDE), cataract, glaucoma, and neuropathic pain can occur after exposure to a pharmaceutical product or chemical and therefore need to be anticipated. To do so, new models of lacrimal glands, lens, and neurons innervating epithelia are required. These models must take into account real-life exposure (dose, time, and tear film clearance). The scientific community is working hard to develop new, robust, alternative, in silico, and in vitro models, while attempting to balance ethics and availability of biological materials. This review provides a broad overview of the validated methods for analyzing ocular irritation and those still used by some industries, as well as promising models that need to be optimized according to the OECD. Finally, we give an overview of recently developed innovative models, which could become new tools in the evaluation of ocular surface toxicity within the scope of IATAs.

Performance Statistics of Defined Approaches for Eye Hazard Identification of Non-Surfactant Liquids to Distinguish Between the Three United Nations Globally Harmonized System Categories

Introduction: Cosmetics Europe develops two defined approaches (DAs) for eye hazard identification, that is, addressing serious eye damage (Cat. 1), eye irritation (Cat. 2), and the absence thereof (No Cat.), for nonsurfactant liquid test substances. DAL-1 combines four physicochemical properties, the reconstructed human cornea-like epithelium (EpiOcular™ eye irritation test [EIT]: VRM1 and SkinEthic™ human corneal epithelium EIT: VRM2) test method (OECD test guideline [TG] 492), and bovine corneal opacity and permeability (BCOP) test method (OECD TG 437). DAL-2 combines the short time exposure test method (OECD TG 491) and the BCOP test method (OECD TG 437). In both DAs, the BCOP with the laser light-based opacitometer is used, however, only opacity is used to identify liquids that cause serious eye damage. Materials and Methods: The performance of the DAs to distinguish between the three United Nations Globally Harmonized System of Classification (UN GHS) categories was compared against the minimum performance values proposed by Cosmetics Europe to the Organisation for Economic Co-operation and Development expert Group on eye/skin irritation/corrosion and phototoxicity and agreed upon by the experts. Furthermore, the class-specific performance metrics are also provided for each DA. Results: The balanced accuracy of DAL-1 with validated reference method—EpiOcular EIT (VRM1), DAL-1 with validated reference method—SkinEthic HCE EIT (VRM2), and DAL-2 was 69.2%, 75.2%, and 74.3%, respectively. DAL-1 with VRM1 identified 76.5% of Cat. 1 (N = 17), 59.1% of Cat. 2 (N = 22), and 72.1% of No Cat. (N = 55) correctly. DAL-1 with VRM2 identified 76.5% of Cat. 1 (N = 17), 68.7% of Cat. 2 (N = 23), and 80.4% of No Cat. (N = 46) correctly. DAL-2 identified 81.2% of Cat. 1 (N = 17), 56.3% of Cat. 2 (N = 24), and 85.3% of No Cat. (N = 123) correctly. Conclusion: The percentage of correct predictions was greater than the proposed minimum performance values of 75% for Cat. 1, 50% for Cat. 2, and 70% for No Cat. Therefore, DAL-1 and DAL-2 have shown to successfully distinguish between the three UN GHS categories for eye hazard identification.

In silico prediction of the full United Nations Globally Harmonized System eye irritation categories of liquid chemicals by IATA-like bottom-up approach of random forest method

ABSTRACT As an alternative to in vivo Draize rabbit eye irritation test, this study aimed to construct an in silico model to predict the complete United Nations (UN) Globally Harmonized System (GHS) for classification and labeling of chemicals for eye irritation category [eye damage (Category 1), irritating to eye (Category 2) and nonirritating (No category)] of liquid chemicals with Integrated approaches to testing and assessment (IATA)-like two-stage random forest approach. Liquid chemicals (n = 219) with 34 physicochemical descriptors and quality in vivo data were collected with no missing values. Seven machine learning algorithms (Naive Bayes, Logistic Regression, First Large Margin, Neural Net, Random Forest (RF), Gradient Boosted Tree, and Support Vector Machine) were examined for the ternary categorization of eye irritation potential at a single run through 10-fold cross-validation. RF, which performed best, was further improved by applying the ‘Bottom-up approach’ concept of IATA, namely, separating No category first, and discriminating Category 1 from 2, thereafter. The best performing training dataset achieved an overall accuracy of 73% and the correct prediction for Category 1, 2, and No category was 80%, 50%, and 77%, respectively for the test dataset. This prediction model was further validated with an external dataset of 28 chemicals, for which an overall accuracy of 71% was achieved.

Ocular microemulsion of brinzolamide: Formulation, physicochemical characterization, and in vitro irritation studies based on EpiOcular™ eye irritation assay

In recent years, the hydrophobic active substances have led researchers to develop new formulations to enhance bioavailability and dissolution rate; brinzolamide, a lipophilic drug belongs to carbonic anhydrase inhibitors, which cause reduction of intraocular pressure in patients suffering from glaucoma. Currently, the marketed product of brinzolamide is in the form of ocular drops; nonetheless, the conventional drops provide decreased therapeutic efficacy owing to their low bioavailability and pulsed drug release. Thus, the development of novel ocular formulations such as topical microemulsions is of high importance. In this work, the preparation of new microemulsions containing brinzolamide (0.2, 0.5 and 1% w/w) and comprised from isopropyl myristate, tween 80 and span 20 and Cremophor EL was performed. The obtained microemulsions were further characterized for their physicochemical properties. In addition, Fourier Transformed-Infrared spectroscopy was used touate the compatibility of active ingredients and components. In vitro release studies along with kinetic modeling were performed using the dialysis membrane method in simulated tear fluid. Bioadhesion studies were performed using Texture analysis. Finally, in vitro ocular irritation based on EpiOcular™ Eye Irritation Test and cytocompatibility studies was performed to examine any possible harm on ocular cells and predict in vivo safety profile.

Surfactant cocamide monoethanolamide causes eye irritation by activating nociceptor TRPV1 channels.

Cocamide monoethanolamide (CMEA) is commonly used as a surfactant-foam booster in cosmetic formulations. Upon contact with the eye or other sensitive skin areas, CMEA elicits stinging and lasting irritation. We hypothesized a specific molecular interaction with TRPV1 channels by which CMEA caused eye irritation. Eye irritancy was evaluated using eye-wiping tests in rabbits and mice. Intracellular Ca2+ concentrations and action potentials were measured using Ca2+ imaging and current clamp respectively. Voltage clamp, site-direct mutagenesis and molecular modelling were used to identify binding pockets for CMEA on TRPV1 channels. CMEA-induced eye irritation is ameliorated by selective ablation of TRPV1 channels.Rodents exhibit much stronger responses to CMEA than rabbits. In trigeminal ganglion neurons, CMEA induced Ca2+ influx and neuronal excitability, effects mitigated by a TRPV1 channel inhibition and absent in TRPV1 knockout neurons. In HEK-293 cells expressing TRPV1 channels, CMEA increased whole-cell currents by increasing channel open probability (EC50 = 10.2μM), without affecting TRPV2, TRPV3, TRPV4, and TRPA1 channel activities. Lauric acid monoethanolamide (LAMEA), the most abundant constituent of CMEA, was the most efficacious and potent TRPV1 channel activator, binding to the capsaicin-binding pocket of the channel. The T550I mutants of rabbit and human TRPV1 channels exhibit much lower sensitivity to LAMEA. CMEA directly activates TRPV1 channels to produce eye irritation. Rabbits, the standard animal used for eye irritancy tests are poor models for evaluating human eye irritants structurally related to CMEA. Our study identifies potential alternatives to CMEA as non-irritating surfactants.

Topical Delivery of Levocarnitine to the Cornea and Anterior Eye by Thermosensitive in-situ Gel for Dry Eye Disease.

PurposeTo prepare the levocarnitine thermosensitive in situ gel (LCTG) and evaluate its effect on dry eye disease (DED).MethodsDraize eye irritation test and other examinations were used to evaluate the eye irritation after multiple administration of LCTG. The Schirmer test, fluorescein sodium staining, HE staining and TUNEL staining were used to detect the tear secretion, corneal injury, histopathological changes of the cornea and lacrimal gland, and the apoptosis rate of cornea epithelial cells after 3 days of the administration. The conjunctival goblet cell density was detected by PAS staining, and the expression levels of matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-9 (MMP-9) of corneal epithelial cells were detected by immunofluorescence staining after 7 days of the administration.ResultsLCTG is non-irritating to rabbit eyes and has good biocompatibility. LCTG administration for 3 days can significantly increase the amount of tear secretion in mice with DED, promote corneal epithelial integrity and central corneal epithelium thickness recovery, and improve the pathological morphology and structure of corneal and lacrimal gland tissues, and reduce the apoptosis rate of the corneal epithelial cells. After 7 days of the administration, the preparation can promote the proliferation of conjunctival goblet cells and down-regulate the cornea expression levels of MMP-3 and MMP-9 in epithelial cells.ConclusionThe LCTG has a good curative effect on mice with DED, and the overall curative effect is better than that of levocarnitine solution.

Effect of Poly(L-lysine) and Heparin Coatings on the Surface of Polyester-Based Particles on Prednisolone Release and Biocompatibility.

A plethora of micro- and nanoparticle types are currently investigated for advanced ocular treatment due to improved drug retention times, higher bioavailability and better biocompatibility. Yet, comparative studies of both physicochemical and toxicological performance of these novel drug delivery systems are still rare. Herein, poly(L-lactic acid)- and poly(ε-caprolactone)-based micro- and nanoparticles were loaded with prednisolone as a model drug. The physicochemical properties of the particles were varied with respect to their hydrophilicity and size as well as their charge and the effect on prednisolone release was evaluated. The particle biocompatibility was assessed by a two-tier testing strategy, combining the EpiOcularTM eye irritation test and bovine corneal opacity and permeability assay. The biodegradable polyelectrolyte corona on the particles’ surface determined the surface charge and the release rate, enabling prednisolone release for at least 30 days. Thereby, the prednisolone release process was mainly governed by molecular diffusion. Finally, the developed particle formulations were found to be nontoxic in the tested range of concentrations.

Application of in vitro methods to evaluate the safety of baby care products

Draize rabbit eye irritation and skin irritation tests are widely used in the chemical industry as traditional methods to evaluate the safety of cosmetics. However, great differences among laboratories have caused great doubt in the industry. In addition, with vigorous development of the global animal protection movement, developed countries have launched the “3R” campaign, and various kinds of in vitro alternative methods have emerged. Hen's egg test on the chorioallantoic membrane (HET-CAM), which is similar to the structure of the human cornea and has a clear and complete vascular system, is based on the characteristics of the chorioallantoic membrane (CAM) in mid-term SPF egg embryos. The reconstructed human epidermis (EpiSkin®) is composed of normal human keratinocytes that are histologically similar to human epidermises seen in vivo, and it is cultured on a collagen matrix. Similar to EpiSkin®, Human Corneal Epithelium (SkinEthic™) is another reconstructed 3D human-corneal structure that is an alternative to the traditional eye irritation test. Three in vitro methods were conducted to evaluate the safety of 12 baby care products, which included the most common types. In addition, a consumer research study was also carried out for two weeks to evaluate the safety. The results of the reconstructed human epidermis model, human corneal epithelium model and consumer research showed that no irritation was found in any test products; however, HET-CAM tests showed positive results.

Formulation and evaluation of thermosensitive flurbiprofen in situ nano gel for the ocular delivery

The contemporary research implicates the formulation and evaluation of a thermosensitive in situ nano gelling method to improve solubility and ocular residence time of flurbiprofen. This study was carried out in two phases. In the first phase, an insolubility drug has been formulated in the form of a nanoparticulate system and evaluated for its characteristics. The nanoparticles obtained demonstrated an average size array of 150 to 250 nm in diameter, up to 79.35% encapsulation efficiency, and up to 93.42% drug release throughout 2 h. In the second phase, nanoparticulate systems were dispersed in aqueous solutions of Pluronic F 127 (14%) and various concentrations of Carbopol 934 in combination to form an in situ nano gel. The prepared in situ gel was investigated for its physicochemical properties like pH, flowability, sol-gel transition temperature, gelling capacity, and rheological properties. Carbopol 934 did not significantly affect sol-gel transition temperature in optimized concentration (<0.3%) but altered gelling capacity, pH, and transparency of the formulations. In vivo resident time and eye irritation test was evaluated in the rabbit eye. In optimized in situ gelling formulation (NIGF3), approximately 95% of in vitro drug release was observed after 6 h. NIGF3 increased precorneal residence time and high concentration in aqueous humor when paralleled to flurbiprofen eye drops. Greater concentration of drug in aqueous humor was due to its improved saturation solubility of the drug, and amplified residence time was attributed to the formation of gel matrix-embedded nanoparticles. This demonstrated that in situ nano gels (NIGF3) comprehending aqueous solutions of 0.3% w/v concentrations of Carbopol 934 with Pluronic F 127 may ominously persist the residence time and mend bioavailability of a water-insoluble drug.

In vitro reconstructed 3D corneal tissue models for ocular toxicology and ophthalmic drug development.

Testing of all manufactured products and their ingredients for eye irritation is a regulatory requirement. In the last two decades, the development of alternatives to the in vivo Draize eye irritation test method has substantially advanced due to the improvements in primary cell isolation, cell culture techniques, and media, which have led to improved in vitro corneal tissue models and test methods. Most in vitro models for ocular toxicology attempt to reproduce the corneal epithelial tissue which consists of 4-5 layers of non-keratinized corneal epithelial cells that form tight junctions, thereby limiting the penetration of chemicals, xenobiotics, and pharmaceuticals. Also, significant efforts have been directed toward the development of more complex three-dimensional (3D) equivalents to study wound healing, drug permeation, and bioavailability. This review focuses on in vitro reconstructed 3D corneal tissue models and their utilization in ocular toxicology as well as their application to pharmacology and ophthalmic research. Current human 3D corneal epithelial cell culture models have replaced in vivo animal eye irritation tests for many applications, and substantial validation efforts are in progress to verify and approve alternative eye irritation tests for widespread use. The validation of drug absorption models and further development of models and test methods for many ophthalmic and ocular disease applications is required.

Strategy Combining Nonanimal Methods for Ocular Toxicity Evaluation.

Historically, the ocular toxicity of manufactured consumer materials has been evaluated using the rabbit in vivo Draize rabbit eye test. The animal data obtained were used by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) to define the classification and labelling (C&L) for eye damage/irritation endpoint. However, the Draize test, a method which was never formally validated, has been widely criticized because of its technical limitations. In addition, ethical and economic issues and advances in scientific knowledge, and political and public pressures have made animal experimentation unsustainable. This scenario has consequently led to the development of nonanimal testing and protocols/approaches with considerable predictive value and relevance for humans. It is widely accepted that one single nonanimal method cannot cover all the criteria of damage/inflammation assessed by regulatory adopted in vivo animal testing. Thus, integrated testing strategies (ITS) have been proposed, including a tiered testing approach combining different nonanimal testing with different endpoints, which have been used for regulatory purposes, on a case-by-case basis and within integrated approaches to testing and assessment (IATA), to identify materials according to their ability to trigger eye damage. In particular, the top-down and bottom-up approaches have been recommended for the C&L of materials, which cause serious eye damage or eye irritation, respectively. This chapter describes detailed protocols for eye irritation testing based on cells (Short Time Exposure-STE, OECD No. 491/2017), a vascularized membrane (the Hen's Egg Test-Chorioallantoic Membrane-HET-CAM) and corneal tissue (Bovine Corneal Opacity and Permeability-BCOP, OECD No. 437/2017), which can be applied using top-down or bottom-up approaches. In addition, it suggests making a corneal histomorphometric evaluation as an additional parameter in the BCOP method to differentiate materials that cause serious eye tissue damage (UN GHS Cat. 1) from materials that have reversible eye irritation effects (UN GHS Cat. 2).

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.