Draize Eye Test Research Articles

Development of a defined approach for eye hazard identification of solid chemicals according to the three UN GHS categories.

Currently there are two OECD-adopted defined approaches (DA) for eye hazard identification of non-surfactant liquids (OECD TG 467). The current study aimed to develop a DA for eye hazard identification of solid chemicals according to the three UN GHS categories (Cat.1, Cat. 2, No Cat.): the DAS. The DAS combines two test methods described in OECD TG 437 and TG 492. The DAS was developed based on in-depth statistical analysis of a database on solids containing in vitro and historically curated in vivo Draize eye test data. The performance of the DAS was assessed by comparing the predictions with the classification based on in vivo Draize eye test data, on the one hand, and with the performance criteria established by the OECD expert group, on the other hand. In a first tier of the DAS, the SkinEthic™ HCE EIT method (TG 492) is used to distinguish No Cat. from classified substances. For classified substances, the BCOP LLBO method (TG 437) is used to identify Cat. 1, and the remaining solids are predicted Cat. 2. In summary, 77.4% Cat. 1 (N=31), 52.3% Cat. 2 (N=18), and 70.0% of No Cat. (N=60) solids were correctly identified compared to the classification based on the Draize eye test. The percentage of correct predictions met the minimum OECD performance values of 75% Cat. 1, 50% Cat. 2, and 70% No Cat., and the percentage of mispredictions was below the established maximum values. Therefore, inclusion of the DAS in OECD TG 467 has been achieved.

Ethosomal Nanoformulations for Combinational Photothermal Therapy of Fungal Keratitis

AbstractFungal keratitis/mycotic keratitis or keratomycosis, is characterized by the inflammation of underlying stroma of corneal epithelium due to fungal infection. Multi‐drug resistance and subsequent recurrence of infection limits the clinical outcomes of anti‐fungal agents such as Amphotericin B and Natamycin; and therefore, demands research on newer anti‐microbial strategies. In this study, the authors have synthesized and characterized ethosomes (IAEs) encapsulating a NIR dye, Indocyanine green (ICG), and an anti‐fungal drug, Amphotericin B, for a combinational photothermal therapy of fungal keratitis. The in vitro anti‐fungal efficacy of the IAEs is validated against Candida albicans by colony formation assay, calcofluor staining, and SEM analysis. The results indicate that the IAEs, upon NIR irradiation, displays a synergistic anti‐fungal efficacy. The IAEs are highly biocompatible when tested using a human corneal epithelial (HCE) cell line model (in vitro) and a Draize eye test model in rabbits (in vivo). Further, it is observed that the IAEs,upon NIR laser exposure significantly eliminate the fungal infection in rat model of occular fungal keratitis. The research indicates that the IAEs nano‐formulation exhibits a synergistic and sustained anti‐fungal effect paving its way for further translation from bench to bedside for the efficient management of fungal keratitis.

Gallic Acid Ameliorates Aspergillus Fumigatus Keratitis Through Reducing Fungal Load and Suppressing the Inflammatory Response.

The purpose of this study was to explore the antifungal and anti-inflammatory effects of gallic acid (GA) on Aspergillus fumigatus (A. fumigatus) keratitis. CCK-8 assay and Draize eye test were used to determine the non-cytotoxic concentration of GA in RAW264.7 cells and an A. fumigatus keratitis mouse model. The antifungal effects of GA were analyzed using minimal inhibitory concentration (MIC), biofilm formation test, fungal adherence assay, calcofluor white staining, and propidium iodide staining. The therapeutic effects of GA were estimated by slit lamp photographs, clinical score, hematoxylin and eosin (H&E) staining, and Periodic acid-Schiff staining in vivo. Immunofluorescence staining and myeloperoxidase assay were conducted to identify neutrophil infiltration and activity. RT-PCR, ELISA, and Western blot were performed to detect the expression of pro-inflammatory cytokines and Nrf2/HO-1. In HCECs and A. fumigatus keratitis mouse model, GA at 100 µg/mL did not affect cell viability, thus this concentration was applied to subsequent experiments. In vitro, GA significantly inhibited A. fumigatus growth, biofilm formation, and adhesion. In vivo, 100 µg/mL GA alleviated the severity of fungal keratitis (FK) by repressing fungal load, reducing neutrophil infiltration, and lowering MPO activity. Besides, the expression of IL-1β, TNF-α, LOX-1, and COX-2 was inhibited, whereas Nrf2 and HO-1 expression was enhanced at both mRNA and protein levels in the 100 µg/mL GA treated group in comparison to PBS control. GA ameliorates FK severity through inhibiting A. fumigatus load, reducing neutrophils infiltration, downregulating the expression of pro-inflammatory cytokines, and enhancing the Nrf2/HO-1 pathway, which provides new insight into A. fumigatus keratitis treatment.

Laccase-mediated functionalization of natamycin by gallic acids for the therapeutic effect on Aspergillus fumigatus keratitis

To improve the therapeutic effect of natamycin on fungal keratitis (FK), the grafted derivatives of natamycin and gallic acid were obtained, and the effects of the grafted derivatives on Aspergillus fumigatus (A. fumigatus) keratitis were investigated. The structure of natamycin grafted with gallic acid was identified by FT-IR and UV–Vis, and the successful synthesis of Gallic-Natamycin (GA-NAT) was proved. CCK-8 and the Draize eye test showed that GA-NAT had less cytotoxicity. Then, through in vitro antibacterial experiments such as minimum inhibitory concentration (MIC), adhesion, biofilm formation, and calcium fluorescence staining and in vivo experiments such as clinical score and plate counting, the results showed that GA-NAT had similar antifungal activity to natamycin, but had a better therapeutic effect than natamycin. Myeloperoxidase assay and immunofluorescence staining also showed that GA-NAT significantly inhibited neutrophil recruitment and activity. Moreover, It was further found that GA-NAT could inhibit the mRNA and protein expressions of LOX-1, TNF-α, and IL-1β. These results indicated that GA-NAT inhibited the fungal growth, reduced the neutrophil infiltration into cornea, and down-regulated the expression of inflammatory factors in lesions, which provides a new choice for FK treatment.

The therapeutic potential of chondroitin sulfate in Aspergillus fumigatus keratitis

PurposeTo explore the therapeutic effect of chondroitin sulfate (CS) on Aspergillus fumigatus (A. fumigatus) keratitis. MethodsThe nontoxic concentration of CS was determined using the Draize eye test and cell counting kit-8. Cell scratch test and cell proliferation test were evaluated the impact of CS on the proliferation and migration of human corneal epithelial cells (HCECs). Adherence assay and plate counting were used to detect fungal load in vivo and in vitro, respectively. Clinical score and hematoxylin-eosin (HE) staining were applied to assess the therapeutic effects of CS in an A. fumigatus keratitis mice model. The neutrophil infiltration and activity were detected by flow cytometry (FCM), immunofluorescence staining, and myeloperoxidase (MPO) assay. Toll-like receptor 4 (TLR-4) expression in RAW 264.7 cells and mouse cornea was detected by immunofluorescence staining. Real-time PCR (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to examine the expression of inflammatory mediators. ResultsCS at 400 μg/mL (non-cytotoxic) significantly promoted proliferation and migration of HCECs. In an A. fumigatus keratitis mice model, CS treatment alleviated fungal keratitis (FK) severity by decreasing corneal fungal load and inhibiting neutrophil infiltration. In RAW 264.7 cells, the mRNA and protein levels of TLR-4, phosphorylated nuclear factor (NF)-κB (p-NF-κB), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), cyclooxygenase 2 (COX-2), and macrophage inflammatory protein-2 (MIP-2) were remarkably lower in the siTLR-4 treated group, while higher in rTLR-4 treated group than in the corresponding control group. CS treatment suppressed rTLR-4 induced the mRNA and protein expression of TLR-4, p-NF-κB, IL-1β, IL-6, COX-2, TNF-α, and MIP-2 in RAW cells. ConclusionCS may ameliorate the prognosis of A. fumigatus keratitis by promoting corneal epithelial proliferation, inhibiting the recruitment and activity of neutrophils, and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling.

Development and Evaluation of Herbal Hair Serum: A traditional way to Improve Hair Quality

Background: In the mammalian system, the hair follicle is known to be the most significant organ that determines appearance, gender distinction, provides intense temperature protection, and plays a role in self-defense. The younger generations have begun to suffer from extreme hair loss problems due to many lifestyle-related changes such as fatigue, anxiety, intake of junk foods, use of different hairstyling/coloring methods, etc. The loss of hair is not temporary in most cases, but it results in alopecia. Many people suffering from hair loss are in search of multiple treatments due to extreme anxiety and tension, from mythology to traditional and therapeutic healing to the use of minoxidil and finasteride. To improve hair growth and to prevent hair loss, hair root activation is required. Objective: The present study was intended to use different herbs to formulate herbal hair serum for general purposes (hair application). Methods: Crude herbs are obtained from nearby regions. In order to obtain the extract, the required part of herbs, such as Citrus sinensis peel, Zingiber officinale roots, and Linum usitatissimum seeds, Nigella sativa, and Trigonella foenum-graecum were specifically weighed and dispensed in water, boiled, allowed to cool, and then filtered. To the filtrate, castor oil and vitamin E were added. The formulated herbal hair serum was tested, and different criteria were determined and recorded in this text, such as physical appearance, viscosity, pH, homogeneity, eye sensitivity (Draize eye test), hair growth activity, hair weight, stability test, etc. Results: Prepared herbal hair serum was found to be pale brown in color with pseudoplastic behavior. The texture of the formulation was smooth and lubricative, and the pH was within the acceptable limit. Herbal hair serum revealed good hair growth as well as hair weight after application with minor sensitivity for the first time. The formulation was found to be stable for seven days. Conclusion: Herbal cosmetics are still commonly used by average citizens because of fewer side effects and greater protection and safety profile. Current research has revealed that herbal formulations are effective in enhancing hair consistency.

Cellular viability and death biomarkers enables the evaluation of ocular irritation using the bovine corneal opacity and permeability assay

The BCOP assay is used in the identification of chemicals that cause no ocular irritation or serious damage. However, this method has not been found to adequately discriminate between mild from moderate ocular irritation (category 2A/2B), based upon the animal data. In this study, we aimed to establish methods for discerning ocular irritation by chemicals. We used the BCOP assay and the fluorescence staining methods based on biomarkers for cellular viability and death. The potential for ocular irritation by 12 chemicals from different UN GHS categories was assessed by the BCOP assay. Cryosections of bovine corneas were obtained. The necrotic nucleus was TUNEL labeled, cytoplasmic f-actin was stained by phalloidin while the nucleus was stained by DAPI. The depth of injury (DOI) was then measured. According to BCOP assay, in vivo data of Draize eye test and DOI, the results showed that category NC irritants caused ≤ 10 % epithelial DOI, irritants of category 2B caused >10 % epithelial DOI and showed no stromal damage, while category 2A showed damage to the stroma. Based on these results, the GHS prediction model could distinguish between GHS 2A and 2B. Authenticating the viability of BCOP by DOI measurements can provide a more reliable basis for classifying ocular irritants.

Positively charged polymeric nanoparticles improve ocular penetration of tacrolimus after topical administration

Tacrolimus (TAC) is an immunosuppressant with potential use in ophthalmology, especially as a steroid sparing agent. The efficacy of TAC has been investigated in the treatment of different anterior segment inflammatory disorders. However, its use is limited by its poor corneal penetration, which has led to the development of different nanocarriers. In order to overcome this limitation, we prepared positively charged polymeric nanoparticles loaded with tacrolimus (TAC-NPs) for topical ocular application. TAC-NPs were prepared by interfacial polymer deposition following solvent displacement method, using Eudragit® RL100, a cationic polymer. The formulation was characterized and the ocular safety was assessed in vivo by Draize eye test and histopathological analysis. TAC was quantified in aqueous humor to determine the in vivo release of the nanoparticles. TAC-NPs presented suitable physical-chemical characteristics for ocular application, including monomodal size distribution with a mean diameter of 104 ± 1 nm, positive zeta potential and mucoadhesive properties. The monitoring of the physical-chemical characteristics and the multiple light scattering analysis of TAC-NPs suggested their long-term stability. The in vivo safety investigation of TAC-NPs and blank-NPs showed no signs of ocular irritation on the ophthalmological examination and histopathological study, indicating the biocompatibility of this carrier. The nanoparticles were able to deliver TAC for over 36 h with a better performance than the drug suspension. The in vivo release profile of TAC-NPs indicated slower elimination with 1.7-fold higher maximum concentration of TAC and 5.3-fold higher AUC, suggesting increased ocular penetration and bioavailability in addition to extended delivery. TAC-NPs improved the ocular delivery of tacrolimus after topical administration, without causing any ocular irritation. It shows the potential of this carrier as an alternative for ocular delivery of tacrolimus in the treatment of anterior segment inflammatory conditions.

Replacing the Draize eye test: Impedance spectroscopy as a 3R method to discriminate between all GHS categories for eye irritation

Highly invasive animal based test procedures for risk assessment such as the Draize eye test are under increasing criticism due to poor transferability for the human organism and animal-welfare concerns. However, besides all efforts, the Draize eye test is still not completely replaced by alternative animal-free methods. To develop an in vitro test to identify all categories of eye irritation, we combined organotypic cornea models based on primary human cells with an electrical readout system that measures the impedance of the test models. First, we showed that employing a primary human cornea epithelial cell based model is advantageous in native marker expression to the primary human epidermal keratinocytes derived models. Secondly, by employing a non-destructive measuring system based on impedance spectroscopy, we could increase the sensitivity of the test system. Thereby, all globally harmonized systems categories of eye irritation could be identified by repeated measurements over a period of 7 days. Based on a novel prediction model we achieved an accuracy of 78% with a reproducibility of 88.9% to determine all three categories of eye irritation in one single test. This could pave the way according to the 3R principle to replace the Draize eye test.

A simplified numerical scoring system to quantify skin and eye irritation/corrosion

In 1944, Draize et al., published a paper entitled “Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes”. The Organization for Economic Co-operation and Development published their first guideline on eye irritation in 1981, using rabbits. In the early eighties the development of alternative non-animal tests to replace the Draize eye test started. The first attempts to validate alternative tests for eye irritation were considered to be relatively simple by comparing in vitro and in vivo irritation index scores. In the early nineteen-eighties, we introduced the use of isolated eyes as an alternative test for the Draize eye irritation test. What was expected to be a process of several years, however, turned out to be a decades spanning process still not fully completed. For a large part, this can be attributed to the nature of the in vivo test in rabbits, which is more complicated and compromised than originally believed. This paper describes, most chronologically, the development, performance, validation and application of the Isolated Eye Test and, in broader perspective, the international validation and acceptance of this alternative test by regulatory authorities and agencies.

Ocular irritation and cyclosporine A distribution in the eye tissues after administration of Solid Lipid Microparticles in the rabbit model

The aim of this study was to investigate the in vivo effect of Solid Lipid Microparticles (SLM), proposed for topical ocular administration of cyclosporine, on the rabbit eye.SLM carrier is an aqueous dispersion of lipid microparticles (20% w/w) with a size up to 15 μm. Cyclosporine was dissolved in the formulation in the concentration of 0.5 or 2.0% (w/w). Ocular tolerance of microsphere dispersion was assessed in rabbit model by the Draize eye test (SLM was compared with emulsion and oily solution), and cyclosporine distribution in ocular tissues was evaluated after multiple application of tested formulations (SLM dispersions, emulsions and oily solution) for 7 days.Good tolerance of cyclosporine-SLM formulation was demonstrated in the rabbit model. Concentration of cyclosporine in the precorneal tissues, such as cornea and conjunctiva, was much higher than the therapeutic value (8.4 ng/mg and 3.2 ng/mg, respectively). After SLM administration, the cyclosporine concentrations determined in the anterior ocular tissues, were also significantly higher compared to those obtained after the application of other tested carriers (emulsions and oily solution).The obtained results prove that the recognized SLM dispersions are safe formulations for ophthalmic use. It can be concluded that lipid microparticles are highly promising for an efficient ophthalmic drug delivery, when compared to other conventional dosage forms.

Methods for Testing Ocular Toxicity: Current Status

The developed ophthalmic formulations should be tested for ocular toxicity testing like draize eye test. Rapid growth in the field of ocular toxicity testing replaces the usage of animals and to adopt the modified protocols. The researchers have to develop properly validated and regulatory approved options to animal testing. Alternative ocular toxicity testing is based on corneal structure and mechanism through which the specific ocular formulation causes irritation to the eyes. Several alternative assay methods have been introduced in the field of ocular toxicity testing like in vitro organotypic model for testing ocular toxicity and several other methods like Bovine Corneal Opacity and Permeability Assay (BCOP Assay), isolated chicken eye test, isolated rabbit eye test and Hen's egg-test on the chorioallantoic membrane. The ultimate aim of alternate methods of ocular toxicity testing is to develop properly validated and regulatory approved best practices. Hence this review highlights the development in the field of alternative ocular toxicity testing methods.

Expansion of the applicability domain for highly volatile substances on the Short Time Exposure test method and the predictive performance in assessing eye irritation potential.

The Short Time Exposure (STE) test method is an in vitro method for assessing the eye irritation potential of chemicals and is used to classify the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Category 1 and No Category (NC). The method has been adopted by the Organisation for Economic Co-operation and Development (OECD) as test guideline (TG) 491 since 2015. While this method can be used to classify GHS NC, it is not suitable for testing highly volatile substances and solids other than surfactants. Here we evaluated highly volatile substances to expand the applicability domain. According to TG 491, acetone, ethanol, iso-propanol, and methyl acetate as highly volatile substances resulted in false negatives. Saline was selected as a solvent of these false negatives. In this study, mineral oil was used as the solvent, because these false negatives were amphiphilic. Based on this change, four highly volatile substances were correctly evaluated. The predictive performance for classifying GHS NC was then verified using a substance dataset constructed in reference to the Draize eye test Reference Database and STE Summary Review Document. The accuracy and false-negative rate were 86.6% (194/224) and 3.8% (3/80), respectively. Collectively, the applicability domain was expanded by changing the solvent to mineral oil for highly volatile substances, and the predictive performance for the new applicability domain including highly volatile substances was excellent. The STE test method is suitable to classify GHS NC, indicating its applicability as a test method in a bottom-up approach.

The Isolated Chicken Eye test to replace the Draize test in rabbits

In 1944, Draize et al., published a paper entitled “Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes”. The Organization for Economic Co-operation and Development published their first guideline on eye irritation in 1981, using rabbits. In the early eighties the development of alternative non-animal tests to replace the Draize eye test started. The first attempts to validate alternative tests for eye irritation were considered to be relatively simple by comparing in vitro and in vivo irritation index scores. In the early nineteen-eighties, we introduced the use of isolated eyes as an alternative test for the Draize eye irritation test. What was expected to be a process of several years, however, turned out to be a decades spanning process still not fully completed. For a large part, this can be attributed to the nature of the in vivo test in rabbits, which is more complicated and compromised than originally believed. This paper describes, most chronologically, the development, performance, validation and application of the Isolated Eye Test and, in broader perspective, the international validation and acceptance of this alternative test by regulatory authorities and agencies.

Influence of freeze-drying and γ-irradiation in preclinical studies of flurbiprofen polymeric nanoparticles for ocular delivery using d-(+)-trehalose and polyethylene glycol.

This study investigated the suspension of poly(ε-caprolactone) nanoparticles as an ocular delivery system for flurbiprofen (FB-PεCL-NPs) in order to overcome the associated problems, such as stability, sterility, tolerance, and efficacy, with two different FB-PεCL-NP formulations. The formulations were stabilized with poloxamer 188 (1.66% and 3.5%) and submitted individually for freeze-drying and γ-irradiation with polyethylene glycol 3350 (PEG3350) and d-(+)-trehalose (TRE). Both formulations satisfied criteria according to all physicochemical parameters required for ocular pharmaceuticals. The FB-PεCL-NP formulations showed non-Newtonian behavior and sustained drug release. Ex vivo permeation analysis using isolated ocular pig tissues suggested that the presence of PEG3350 results in a reduction of FB transcorneal permeation. Moreover, TRE improved the penetration of FB across the cornea, especially after γ-irradiation. In addition, both formulations did not show a significant affinity in increasing FB transscleral permeation. Both formulations were classified as nonirritating, safe products for ophthalmic administration according to hen’s egg test-chorioallantoic membrane and Draize eye test. Furthermore, an in vivo anti-inflammatory efficacy test showed that irradiated FB-PεCL-NPs prepared with PEG3350 (IR-NPsPEG) have longer anti-inflammatory effects than those presented with irradiated FB-PεCL-NPs prepared with TRE (IR-NPsTRE). IR-NPsPEG showed a suitable physical stability after an aqueous reconstitution over >30 days. This study concludes that both formulations meet the Goldman’s criteria and demonstrate how irradiated nanoparticles, with innovative permeation characteristics, could be used as a feasible alternative to a flurbiprofen solution for ocular application in clinical trials.

Analysis of Draize eye irritation testing and its prediction by mining publicly available 2008-2014 REACH data.

SummaryPublic data from ECHA online dossiers on 9,801 substances encompassing 326,749 experimental key studies and additional information on classification and labeling were made computable. Eye irritation hazard, for which the rabbit Draize eye test still represents the reference method, was analyzed. Dossiers contained 9,782 Draize eye studies on 3,420 unique substances, indicating frequent retesting of substances. This allowed assessment of the test’s reproducibility based on all substances tested more than once. There was a 10% chance of a non-irritant evaluation after a prior severe-irritant result according to UN GHS classification criteria. The most reproducible outcomes were the results negative (94% reproducible) and severe eye irritant (73% reproducible).To evaluate whether other GHS categorizations predict eye irritation, we built a dataset of 5,629 substances (1,931 “irritant” and 3,698 “non-irritant”). The two best decision trees with up to three other GHS classifications resulted in balanced accuracies of 68% and 73%, i.e., in the rank order of the Draize rabbit eye test itself, but both use inhalation toxicity data (“May cause respiratory irritation”), which is not typically available.Next, a dataset of 929 substances with at least one Draize study was mapped to PubChem to compute chemical similarity using 2D conformational fingerprints and Tanimoto similarity. Using a minimum similarity of 0.7 and simple classification by the closest chemical neighbor resulted in balanced accuracy from 73% over 737 substances to 100% at a threshold of 0.975 over 41 substances. This represents a strong support of read-across and (Q)SAR approaches in this area.

Morphological Characterization of a Reconstructed Human Corneal Epithelial Model (LabCyte CORNEA-MODEL) as an Alternative to the Draize Eye Test for the Assessment of Eye Irritation

Morphological Characterization of a Reconstructed Human Corneal Epithelial Model (LabCyte CORNEA-MODEL) as an Alternative to the Draize Eye Test for the Assessment of Eye Irritation

Pharmacokinetics and local safety profile of propranolol eye drops in rabbits.

Oral propranolol, a nonselective β-blocker, is able to reduce the progression of retinopathy of prematurity in newborns, but it appeared unsafe. This study aimed to find, in rabbits, a propranolol eye drop concentration able to induce lower plasma but higher retinal concentrations than those obtained after oral administration. Male New Zealand white rabbits were treated with oral propranolol (0.25 mg/kg/6 h) for 5 d, and propranolol concentrations were measured after 1, 2, 3, and 6 h in plasma, aqueous humor, vitreous humor, and retina. These concentrations were compared with those obtained after the administration of one drop of 25 μl of propranolol 0.1% prepared in saline, applied every 6 h to both eyes for 5 d. A Draize eye test and histological analyses were performed to assess eye drop tolerability. The administration of eye drops produced retinal concentrations similar to, but plasma concentrations significantly lower than, those measured after oral administration. The local tolerability profile was excellent. Propranolol eye drops are able to ensure high retinal and low plasma concentrations of propranolol, and this finding opens the perspective of possible topical treatment with propranolol in newborns with retinopathy of prematurity.

Retrospective analysis of the Draize test for serious eye damage/eye irritation: importance of understanding the in vivo endpoints under UN GHS/EU CLP for the development and evaluation of in vitro test methods

For more than two decades, scientists have been trying to replace the regulatory in vivo Draize eye test by in vitro methods, but so far only partial replacement has been achieved. In order to better understand the reasons for this, historical in vivo rabbit data were analysed in detail and resampled with the purpose of (1) revealing which of the in vivo endpoints are most important in driving United Nations Globally Harmonized System/European Union Regulation on Classification, Labelling and Packaging (UN GHS/EU CLP) classification for serious eye damage/eye irritation and (2) evaluating the method’s within-test variability for proposing acceptable and justifiable target values of sensitivity and specificity for alternative methods and their combinations in testing strategies. Among the Cat 1 chemicals evaluated, 36–65 % (depending on the database) were classified based only on persistence of effects, with the remaining being classified mostly based on severe corneal effects. Iritis was found to rarely drive the classification (<4 % of both Cat 1 and Cat 2 chemicals). The two most important endpoints driving Cat 2 classification are conjunctiva redness (75–81 %) and corneal opacity (54–75 %). The resampling analyses demonstrated an overall probability of at least 11 % that chemicals classified as Cat 1 by the Draize eye test could be equally identified as Cat 2 and of about 12 % for Cat 2 chemicals to be equally identified as No Cat. On the other hand, the over-classification error for No Cat and Cat 2 was negligible (<1 %), which strongly suggests a high over-predictive power of the Draize eye test. Moreover, our analyses of the classification drivers suggest a critical revision of the UN GHS/EU CLP decision criteria for the classification of chemicals based on Draize eye test data, in particular Cat 1 based only on persistence of conjunctiva effects or corneal opacity scores of 4. In order to successfully replace the regulatory in vivo Draize eye test, it will be important to recognise these uncertainties and to have in vitro tools to address the most important in vivo endpoints identified in this paper.

Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model, LabCyte CORNEA-MODEL

Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1min for liquid chemicals or 24h for solid chemicals, and the post-exposure incubation periods were set at 24h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test.