Draize Rabbit Eye Research Articles

Ocular irritation reversibility assessment of a laundry detergent using the Porcine Corneal Opacity Reversibility Assay (PorCORA): a focused study

Consumer product manufacturers utilise a spectrum of alternative ocular irritation assays, as these tests do not require the use of live animals. Despite their usefulness, no regulatory-accepted assay assesses the reversibility of ocular damage, a key criterion of GHS ocular classification, like the rabbit eye test (i.e., Draize Rabbit Eye Test [DRET]) . The Porcine Corneal Opacity Reversibility Assay (PorCORA), an ex vivo intact corneal tissue culture model, predicts the reversibility of damage by ocular irritants. Inclusion of the damage reversibility endpoint in the PorCORA supplements other alternative test methods for ocular irritation, by assessing induced eye damage and the ability of this damage to reverse (heal) without the use of live animals to distinguish between Globally Harmonised System of Classification and Labelling of Chemicals (GHS) ocular classifications. In this focused study, results of a Bovine Corneal Opacity and Permeability (BCOP) test of a laundry detergent, neat and 10% dilution, (product mixture from S.C. Johnson & Son, Inc. [SCJ]) classified the product into GHS Category 1; however, the BCOP test cannot assess the reversibility of ocular damage. The laundry detergent was evaluated using the PorCORA, where ocular damage induced by the detergent was fully reversed within seven days. Evaluation of the reversibility of ocular damage using the PorCORA in this focused study can add strength to the weight-of-evidence (WoE) analysis approach in ocular hazard assessment. This WoE approach strengthens the argument that the PorCORA can be used to supplement BCOP data, and that this laundry detergent is not an irreversible eye irritant.

Irritant toxicity and lectin content of different processed products of Pinelliae Rhizoma

The present study aims to investigate the correlation between irritant toxicity variation and lectin content variation during the processing of Pinelliae Rhizoma products and to explore the feasibility of Western blot as a method for the detection of lectin. We processed Pinelliae Rhizoma Praeparatum Cum Alumine, Pinelliae Rhizoma Praeparatum, and Pinelliae Rhizoma Praeparatumcum Zingibere et Alumine to different degrees and then analyzed their irritant toxicity via Draize rabbit eye test. Western blot was employed to determine the lectin content in Pinelliae Rhizoma products processed with different methods. The correlation between toxicity variation and lectin content variation was then analyzed. Different decoction pieces of Pinelliae Rhizoma were collected for the determination of lectin content. The three processed products of Pinelliae Rhizoma showed gradually decreased toxicity and lectin content as the processing continued. The decreasing trend of lectin content was consistent with that of irritant toxicity during processing, which indicated that the change in lectin content could reflect the trend of irritant toxicity. No band of lectin appeared in the Western blot of processed products of Pinelliae Rhizoma, which suggested that western blotting can be used for the detection of toxic lectin in the processed products of Pinelliae Rhizoma. Lectin should not be detected in the Pinelliae Rhizoma products processed according to the methods in the Chinese Pharmacopoeia.

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.

Mechanistic-based non-animal assessment of eye toxicity: Inflammatory profile of human keratinocytes cells after exposure to eye damage/irritant agents

Eye toxicity is a mandatory parameter in human risk and safety evaluation for products including chemicals, pesticides, medicines and cosmetics. Historically, this endpoint has been evaluated using the Draize rabbit eye test, an in vivo model that was never formally validated. Due to advances in scientific knowledge, economic and ethical issues, non-animal methods based on mechanisms of toxicity are being developed and validated for increasing the capability of these models to predict eye toxicity. In this study, the Cytometric Bead Array (CBA) and ELISA assays were used to evaluate the inflammatory cytokine profile produced by HaCaT human keratinocytes after exposure to chemicals with different UN GHS eye toxicity classifications, aiming to stablish a correlation between inflammatory endpoints and eye toxicity (damage/irritation) potential. As a first step, cytotoxic profile of the chemicals, including 3 non-irritants and 10 eye toxicants (GHS Category 1, 2A and 2B), was evaluated after 24 h exposure using MTT assay and Inhibitory Concentration of 20% of cell viability (IC20) was calculated for each chemical. Then, the cells were exposed to these chemicals at IC20 for 24 h and supernatants and cell lysates were analyzed by CBA assay for quantification of the following cytokines: IL-6, IL-8, IL-10, IL-1β, TNF and IL-12p70. Regarding cytotoxicity evaluation, chemicals showed different cytotoxicity profiles and data demonstrated no correlation with their UN GHS classification. Among the cytokines evaluated, IL-1β production has changed after exposure and such alterations were confirmed by quantification employing ELISA method. The higher intracellular levels of IL-1β were found in GHS Category 1 chemicals, followed by Category 2A and 2B, while non irritants did not induce such increase. Thus, these findings show that IL-1β measurement, using HaCaT model, can be a considerable biomarker to identify chemicals according to their potential in promote eye toxicity, differentiating damage from irritation potential.

Controlled drug delivery for glaucoma therapy using montmorillonite/Eudragit microspheres as an ion-exchange carrier.

BackgroundGlaucoma is a serious eye disease that can lead to loss of vision. Unfortunately, effective treatments are limited by poor bioavailability of antiglaucoma medicine due to short residence time on the preocular surface.Materials and methodsTo solve this, we successfully prepared novel controlled-release ion-exchange microparticles to deliver betaxolol hydrochloride (BH). Montmorillonite/BH complex (Mt-BH) was prepared by acidification-intercalation, and this complex was encapsulated in microspheres (Mt-BH encapsulated microspheres [BMEMs]) by oil-in-oil emulsion–solvent evaporation method. The BH loaded into ion-exchange Mt was 47.45%±0.54%. After the encapsulation of Mt-BH into Eudragit microspheres, the encapsulation efficiency of BH into Eudragit microspheres was 94.35%±1.01% and BH loaded into Eudragit microspheres was 14.31%±0.47%.ResultsBoth Fourier transform infrared spectra and X-ray diffraction patterns indicated that BH was successfully intercalated into acid-Mt to form Mt-BH and then Mt-BH was encapsulated into Eudragit microspheres to obtain BMEMs. Interestingly, in vitro release duration of the prepared BMEMs was extended to 12 hours, which is longer than both of the BH solution (2.5 hours) and the conventional BH microspheres (5 hours). Moreover, BMEM exhibited lower toxicity than that of BH solution as shown by the results of cytotoxicity tests, chorioallantoic membrane-trypan blue staining, and Draize rabbit eye test. In addition, both in vivo and in vitro preocular retention capacity study of BMEMs showed a prolonged retention time. The pharmacodynamics showed that BMEMs could extend the drug duration of action.ConclusionThe developed BMEMs have the potential to be further applied as ocular drug delivery systems for the treatment of glaucoma.

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.

Assessment of the eye irritation potential of chemicals: A comparison study between two test methods based on human 3D hemi-cornea models

We have recently developed two hemi-cornea models (Bartok et al., Toxicol in Vitro 29, 72, 2015; Zorn-Kruppa et al. PLoS One 9, e114181, 2014), which allow the correct prediction of eye irritation potential of chemicals according to the United Nations globally harmonized system of classification and labeling of chemicals (UN GHS). Both models comprise a multilayered epithelium and a stroma with embedded keratocytes in a collagenous matrix. These two models were compared, using a set of fourteen test chemicals. Their effects after 10 and 60minutes (min) exposure were assessed from the quantification of cell viability using the MTT reduction assay. The first approach separately quantifies the damage inflicted to the epithelium and the stroma. The second approach quantifies the depth of injury by recording cell death as a function of depth. The classification obtained by the two models was compared to the Draize rabbit eye test and an ex vivo model using rabbit cornea (Jester et al. Toxicol in Vitro. 24, 597–604, 2010). With a 60min exposure, both of our models are able to clearly differentiate UN GHS Category 1 and UN GHS Category 2 test chemicals.

The EpiOcular™ Eye Irritation Test is the Method of Choice for the In Vitro Eye Irritation Testing of Agrochemical Formulations: Correlation Analysis of EpiOcular Eye Irritation Test and BCOP Test Data According to the UN GHS, US EPA and Brazil ANVISA Classification Schemes.

The Bovine Corneal Opacity and Permeability (BCOP) test is commonly used for the identification of severe ocular irritants (GHS Category 1), but it is not recommended for the identification of ocular irritants (GHS Category 2). The incorporation of human reconstructed tissue model-based tests into a tiered test strategy to identify ocular non-irritants and replace the Draize rabbit eye irritation test has been suggested (OECD TG 405). The value of the EpiOcular™ Eye Irritation Test (EIT) for the prediction of ocular non-irritants (GHS No Category) has been demonstrated, and an OECD Test Guideline (TG) was drafted in 2014. The purpose of this study was to evaluate whether the BCOP test, in conjunction with corneal histopathology (as suggested for the evaluation of the depth of the injury( and/or the EpiOcular-EIT, could be used to predict the eye irritation potential of agrochemical formulations according to the UN GHS, US EPA and Brazil ANVISA classification schemes. We have assessed opacity, permeability and histopathology in the BCOP assay, and relative tissue viability in the EpiOcular-EIT, for 97 agrochemical formulations with available in vivo eye irritation data. By using the OECD TG 437 protocol for liquids, the BCOP test did not result in sufficient correct predictions of severe ocular irritants for any of the three classification schemes. The lack of sensitivity could be improved somewhat by the inclusion of corneal histopathology, but the relative viability in the EpiOcular-EIT clearly outperformed the BCOP test for all three classification schemes. The predictive capacity of the EpiOcular-EIT for ocular non-irritants (UN GHS No Category) for the 97 agrochemical formulations tested (91% sensitivity, 72% specificity and 82% accuracy for UN GHS classification) was comparable to that obtained in the formal validation exercise underlying the OECD draft TG. We therefore conclude that the EpiOcular-EIT is currently the best in vitro method for the prediction of the eye irritation potential of liquid agrochemical formulations.

Characterization of new eye drops with choline salicylate and assessment of their irritancy by in vitro short time exposure tests

The aim of our study was to examine the irritation potential of new eye drops containing 2% choline salicylate (CS) as an active pharmaceutical ingredient (API) and various polymers increasing eye drop viscosity (hydroxyethylcellulose, hydroxypropyl methylcellulose, methylcellulose, polyvinyl alcohol, polyvinylpyrrolidone). The standard method for assessing the potential of irritating substances has been the Draize rabbit eye test. However the European Centre for Validation of Alternative Methods and the Coordinating Committee for Validation of Alternative Methods recommend, short time exposure (STE) in vitro tests as an alternative method for assessing eye irritation. The eye irritation potential was determined using cytotoxicity test methods for rabbit corneal cell line (SIRC) after 5min exposure. The viability of cells was determined using two cytotoxicity assays: MTT and Neutral Red Uptake. According to the irritation rankings for the short time exposure test, all tested eye drops are classified as non-irritating (cell viability >70%).

Identification of cornifelin and early growth response-1 gene as novel biomarkers for in vitro eye irritation using a 3D reconstructed human cornea model MCTT HCE™.

Evaluation of the eye irritation is essential in the development of new cosmetic products. Draize rabbit eye irritation test has been widely used in which chemicals are directly applied to rabbit eye, and the symptoms and signs of eyes are scored. However, due to the invasive procedure, it causes substantial pain and discomfort to animals. Recently, we reported in vitro eye irritation test method using a 3D human corneal epithelial model (MCTT HCE™) which is reconstructed from remaining human tissues after a corneal transplantation. This model exhibited an excellent predictive capacity for 25 reference chemicals (sensitivity 100%, specificity 77% and accuracy 88% vs. GHS). To improve the test performance, we explored new biomarkers for the eye irritation through transcriptomic approach. Three surfactants were selected as model eye irritants that include sodium lauryl sulfate, benzalkonium chloride and triton X-100. After test chemicals were treated, we investigated differentially expressed genes through a whole-gene microarray (Affymetrix GeneChip(®) Human Gene 2.0 ST Array, 48,000 probes). As a result, we identified that mRNAs of cornifelin (CNFN), a constituent of the insoluble cornified cell envelope of stratified squamous epithelia, and early growth response-1 (EGR1), a nuclear transcriptional regulator, were significantly up-regulated by all three irritants. Up-regulation of CNFN and EGR1 was further confirmed by Q-RT-PCR, and immunohistochemistry revealed increased level of CNFN in irritant-treated tissues, supporting the relevance of CNFN and EGR1 as new biomarkers for eye irritation.

Improvement of the Bovine Corneal Opacity and Permeability (BCOP) assay as an in vitro alternative to the Draize rabbit eye irritation test

Measurement of ocular irritancy is a necessary step in the safety evaluation of both industrial and consumer products. Assessment of the acute eye irritation potential is therefore part of the international regulatory requirements for testing of chemicals.The Bovine Corneal Opacity and Permeability (BCOP) assay is generally accepted as a valid in vitro alternative method to the Draize eye irritation test to detect corrosive and severe eye irritants (category 1), but has not proven sensitive enough to discriminate accurately moderate (category 2A/2B) to mild and non-irritating compounds. In the currently accepted BCOP assay, opacity is determined by the amount of light transmission through the cornea, and permeability is determined by the amount of sodium fluorescein dye that passes through all corneal cell layers. Both measurements are used to assign an In Vitro Irritancy Score (IVIS) for prediction of the in vivo ocular irritation potential of a test substance. Nowadays, opacity is measured by an OP-KIT opacitometer providing a center-weighted reading of light transmission by measuring changes in voltage when the transmission of white light passes through the cornea alters. As a consequence, this may underestimate opacity that develops as spots or heterogeneous opaque areas on the periphery of an isolated cornea.A prototype of a laser light-based opacitometer (PLLBO) allowing better measurement of opacities was developed by Van Goethem et al. (2010). This new device showed improved sensitivity to detect subtle changes in corneal transparency. Furthermore, the new opacitometer allowed the analysis of the complete corneal surface and was able to detect more efficiently opaque spots located along the sides of the excised corneas.A further improved prototype of the PLLBO was constructed in combination with a camera and a speckle noise reducer. Treatment conditions of the corneas in the cornea holders were optimized in order to mimic more the real in vivo situation. A set of test compounds with irritancy potencies especially in the mild and moderate range was tested. The improved LLBO showed some promising features which potentially could improve the usefulness of the BCOP test. Adaptation of cornea holders showed to be of limited value and only restricted to concentrations up to 15% which mimics more test conditions in industry.This 3-year research project was sponsored by the Stavros Niarchos Foundation (Greece).

A new cell-based method for assessing the eye irritation potential of chemicals: An alternative to the Draize test

Using a human corneal cell line (HCE-T cells) and 2 evaluation criteria, we developed a new alternative method to assess the eye irritation potential of chemicals. We exposed HCE-T cells to different concentrations of 38 chemicals for 1h and measured relative cell viability (RCV) as an endpoint at each concentration. Using the RCV values, we calculated the RCV50. We also exposed HCE-T cells to 3 fixed concentrations of the 38 chemicals (5%, 0.5%, and 0.05%) for 1h and measured the RCV at each concentration. Using the RCV values at 5%, 0.5%, and 0.05%, we developed a new criterion for eye irritation potential (total eye irritation score, TEIS) and estimated the ocular irritancy. We then assessed the correlation of the results of RCV50 and TEIS with those of the Draize rabbit eye irritation. Both the RCV50 and TEIS results exhibited good positive correlations (sensitivity: 80.77%, specificity: 83.33%, and accuracy: 81.58% for TEIS; sensitivity: 73.08–76.92%, specificity: 75.00%, and accuracy: 73.68–76.32% for RCV50). We conclude that the new in vitro model using HCE-T cells is a good alternative evaluation model for the prediction of the eye irritation potential of chemicals.

In-house Validation of the EpiOcular™ Eye Irritation Test and its Combination with the Bovine Corneal Opacity and Permeability Test for the Assessment of Ocular Irritation

In 2009, the Bovine Corneal Opacity and Permeability (BCOP) test was accepted by the regulatory bodies for the identification of corrosive and severe ocular irritants (Global Harmonised System [GHS] Category 1). However, no in vitro test is currently accepted for the differentiation of ocular irritants (GHS Category 2) and non-irritants (GHS No Category). Human reconstructed tissue models have been suggested for incorporation into a tiered testing strategy to ultimately replace the Draize rabbit eye irritation test (OECD TG 405). The purpose of this study was to evaluate whether the EpiOcular(TM) reconstructed cornea-like tissue model and the COLIPA pre-validated EpiOcular Eye Irritation Test (EpiOcular-EIT) could be used as suitable components of this testing strategy. The in-house validation of the EpiOcular-EIT was performed by using 60 test substances, including a broad variety of chemicals and formulations for which in vivo data (from the Draize rabbit eye irritation test) were available. The test substances fell into the following categories: 18 severe irritants/corrosives (Category 1), 21 irritants (Category 2), and 21 non-irritants (No Category). Test substances that decreased tissue viability to ≤ 60% (compared to the negative control tissue) were considered to be eye irritants (Category 1/2). Test substances resulting in tissue viability of > 60% were considered to be non-irritants (No Category). For the assessed dataset and the classification cut-off of 60% viability, the EpiOcular-EIT provided 98% and 84% sensitivity, 64% and 90% specificity, and 85% and 86% overall accuracy for the literature reference and BASF proprietary substances, respectively. Applying a 50% tissue viability cut-off to distinguish between irritants and non-irritants resulted in 93% and 82% sensitivity, 68% and 100% specificity, and 84% and 88% accuracy for the literature reference and BASF proprietary substances, respectively. Further, in the EpiOcular-EIT (60% cut-off), 100% of severely irritating substances under-predicted by the BCOP assay were classified as Category 1/2. The results obtained in this study, based on 60 test substances, indicate that the EpiOcular-EIT and the BCOP assay can be combined in a testing strategy to identify strong/severe eye irritants (Category 1), moderate and mild eye irritants (Category 2), and non-irritants (No Category) in routine testing. In particular, when the bottom-up strategy with the 60% viability cut-off was employed, none of the severely irritating substances (Category 1) were under-predicted to be non-irritant. Sensitivity for Category 1/2 substances was 100% for literature reference substances and 89% for BASF SE proprietary substances.

Formulation of Indomethacin Eye Drops via Complexation with Cyclodextrins

Purpose: Topically administered indomethacin is used in the management and prevention of ocular inflammation and cystoid macular edema related to cataract surgery and in the maintenance of mydriasis during cataract surgery. Pharmaceutically, the main obstacle in formulating indomethacin as eye drops is its low solubility and aqueous instability. Inclusion complexation of indomethacin with cyclodextrins (CDs) was our interest in this research to obtain stable and effective aqueous indomethacin eye drops.Materials and Methods: The influence of β-CD, hydroxypropyl-β-CD (HP-β-CD), and sulfobutyl ether-β-CD (SBE-β-CD) on indomethacin solubility was investigated. Indomethacin-HP-β-CD complex was prepared and characterized by infrared spectroscopy, differential scanning calorimetry, and X-ray diffractometry and was subjected to in vitro release and stability studies. The anti-inflammatory effect of formulated indomethacin-HP-β-CD eye drops on chemically burned albino rabbit eyes was compared to those of Indocollyre® and Voltaren® eye drops.Results: HP-β-CD was found to have a high solubilizing effect towards indomethacin and was thus selected for its formulation. Formulated indomethacin-HP-β-CD eye drops exhibited delayed release and high drug stability compared to the drug solution. Draize rabbit eye irritation test and histological examination on albino rabbit eyes treated with indomethacin-HP-β-CD eye drops revealed that these eye drops were non-irritant. The anti-inflammatory studies indicated that formulating indomethacin eye drops via complexation with HP-β-CD significantly improves the therapeutic efficacy of the indomethacin compared to the investigated eye drops.Conclusion: An aqueous ocular delivery system for the poor water soluble anti-inflammatory indomethacin was prepared using HP-β-CD as a complexing agent. Besides a sufficient solubility for the drug, many factors were studied in the development of this system, such as stability and safety. In addition, indomethacin-HP-β-CD eye drops showed promising management to corneal inflammation.

Survey of ocular irritation predictive capacity using Chorioallantoic Membrane Vascular Assay (CAMVA) and Bovine Corneal Opacity and Permeability (BCOP) test historical data for 319 personal care products over fourteen years

The Chorioallantoic Membrane Vascular Assay (CAMVA) and Bovine Corneal Opacity and Permeability (BCOP) test are widely used to predict ocular irritation potential for consumer-use products. These in vitro assays do not require live animals, produce reliable predictive data for defined applicability domains compared to the Draize rabbit eye test, and are rapid and inexpensive. Data from 304 CAMVA and/or BCOP studies (319 formulations) were surveyed to determine the feasibility of predicting ocular irritation potential for various formulations. Hair shampoos, skin cleansers, and ethanol-based hair styling sprays were repeatedly predicted to be ocular irritants (accuracy rate=0.90–1.00), with skin cleanser and hair shampoo irritation largely dependent on surfactant species and concentration. Conversely, skin lotions/moisturizers and hair styling gels/lotions were repeatedly predicted to be non-irritants (accuracy rate=0.92 and 0.82, respectively). For hair shampoos, ethanol-based hair stylers, skin cleansers, and skin lotions/moisturizers, future ocular irritation testing (i.e., CAMVA/BCOP) can be nearly eliminated if new formulations are systematically compared to those previously tested using a defined decision tree. For other tested product categories, new formulations should continue to be evaluated in CAMVA/BCOP for ocular irritation potential because either the historical data exhibit significant variability (hair conditioners and mousses) or the historical sample size is too small to permit definitive conclusions (deodorants, make-up removers, massage oils, facial masks, body sprays, and other hair styling products). All decision tree conclusions should be made within a conservative weight-of-evidence context, considering the reported limitations of the BCOP test for alcohols, ketones, and solids.

Assessment of the dermal and ocular irritation potential of alcohol hand sanitizers containing aloe vera with in vitro and in vivo methods

Alcohol-based hand sanitizers have been proven effective in reducing the spread of microbial infections. However, frequent use of alcohol-based formulations of hand sanitizers can cause adverse skin effects, such as allergic and irritant contact dermatitis. Thus, we evaluated the irritant potency of alcoholbased hand sanitizers containing aloe vera and clove extract with four irritation studies and compared in vivo and in vitro results. The Draize rabbit eye and skin irritancy tests were conducted to evaluate the in vivo ocular and dermal irritation potential. For in vitro evaluation of skin irritation, tissue damage was assessed with the MTT assay and by examining the release of interleukin-1α (IL-1α) using engineered epidermal equivalents (KeraSkin™). A 3T3-neutral red uptake (NRU) assay was performed as a potential alternative to the Draize eye irritation test. Sanitizers containing aloe vera and clove extract were non-irritating to skin, and the results were comparable with both the in vivo and in vitro MTT viability assay. However, an increase in IL-1α release was observed. The results of the in vivo eye irritation toxicity studies in rabbits demonstrated reversible conjunctival redness and edema, whereas the test article was non-irritating in the in vitro 3T3-NRU study. These data indicate that the eye is more sensitive to alcohol-based hand sanitizerinduced damage than the skin. Furthermore, in vitro tests are useful for screening purposes, but not for the elimination of in vivo testing.

HET-CAM test for determining the possible eye irritancy of pesticides

Agrochemicals and veterinary products must undergo numerous toxicological tests before registration. The use of animals in these studies is a controversial issue. The Draize eye irritation test is one of the most criticised methods because of the injuries inflicted on the test animals. Several in vitro methods have been used to investigate the toxicity of potential eye irritants with a view to replacing in vivo eye irritation testing. One of these alternative methods is the Hen's Egg Test--Chorioallantoic Membrane (HET-CAM) test. In the present studies comparative screening was performed with a set of agrochemicals to establish parallel data on in vitro (HET-CAM) and in vivo (Draize) results. The examined materials were: Totril (ioxynil), Omite 57 E (propargit), Actellic 50 EC (pyrimiphos-methyl), Stomp 330 EC (pendimethalin), Mospilan 3 EC (acetamiprid), Alirox 80 EC (EPTC), Match 050 EC (lufenuron), Nonit (dioctyl sulphosuccinate sodium), Perenal (haloxyfop-R methyl ester), Pyrinex 48 EC (chlorpyrifos). These experiments showed good correlation between results obtained by the HET-CAM test and those of the Draize rabbit eye test in most cases. The present form of the HET-CAM test can be proposed as a pre-screening method for the determination of eye irritative potential, therefore the number of test animals can be reduced and/or experimental animals can be replaced.

Novel cultured porcine corneal irritancy assay with reversibility endpoint

Several alternative assays exist to assess ocular irritancy without the use of live animals. However, these assays cannot address ocular injury reversibility. Reversibility is an issue critical to regulatory authorities and manufactures of commercial products, as ocular irritation caused by misuse or accidental exposure to a product may cause irreversible eye damage. Here we report the development and initial characterization of a novel ocular irritation assay that addresses ocular injury reversibility. This assay, the Porcine Corneal Ocular Reversibility Assay (PorCORA), uses an air-interface porcine corneal culture system to sustain ex vivo porcine corneas as a model system. These corneas are maintained in culture for 21 days to determine if cornea injury, once inflicted, will reverse. Corneal injury reversibility is measured using Sodium Fluorescein (NaFl) stain to detect compromised epithelial barrier function. In this study, we examined the effects of five compounds on the cultured corneas: phosphate-buffered saline (PBS), 100% Ethanol (EtOH), 3% Sodium Dodecyl Sulfate (SDS), 1% Benzalkonium Chloride (BAK), and 10% Sodium Hydroxide (NaOH). Overall, the persistence of corneal effects between historical Draize rabbit eye data and PorCORA indicates a correlation coefficient of 0.98 (for the five compounds tested) and a correlation coefficient of 0.97 with the Draize modified maximal average score (MMAS). Finally, both fluorescence confocal microscopy and histopathology evidence demonstrates that the PorCORA and NaFl measurements are indicative of actual cellular and tissue damage. PorCORA shows promise as a potential non-animal replacement assay capable of predicting ocular damage reversibility.

Nanoemulsion as a Potential Ophthalmic Delivery System for Dorzolamide Hydrochloride

Dilutable nanoemulsions are potent drug delivery vehicles for ophthalmic use due to their numerous advantages as sustained effect and high ability of drug penetration into the deeper layers of the ocular structure and the aqueous humor. The aim of this article was to formulate the antiglaucoma drug dorzolamide hydrochloride as ocular nanoemulsion of high therapeutic efficacy and prolonged effect. Thirty-six systems consisting of different oils, surfactants, and cosurfactants were prepared and their pseudoternary-phase diagrams were constructed by water titration method. Seventeen dorzolamide hydrochloride nanoemulsions were prepared and evaluated for their physicochemical and drug release properties. These nanoemulsions showed acceptable physicochemical properties and exhibited slow drug release. Draize rabbit eye irritation test and histological examination were carried out for those preparations exhibiting superior properties and revealed that they were nonirritant. Biological evaluation of dorzolamide hydrochloride nanoemulsions on normotensive albino rabbits indicated that these products had higher therapeutic efficacy, faster onset of action, and prolonged effect relative to either drug solution or the market product. Formulation of dorzolamide hydrochloride in a nanoemulsion form offers, thus, a more intensive treatment of glaucoma, a decrease in the number of applications per day, and a better patient compliance compared to conventional eye drops.

Assessing ocular irritation potential using a modified ex vivo rabbit eye test

We have evaluated the ocular irritancy potential of an unknown environmental contaminant, para-toluene sulfonic acid (pTSA), compared with that of known irritants, 5% sodium dodecyl sulfate (SDS) and 10% acetic acid (AA), using a simplified, ex vivo rabbit eye test modified to measure cytotoxicity as a mechanistic correlate to the Draize rabbit eye test. Rabbit eyes were obtained fresh within 24 hours from an abattoir and then exposed to 50 μL of test material. Eyes were then incubated intact for 3 hours or 1 day, and the corneas were removed, stained with calcein acetoxymethylester (AM)/ethidium homodimer (live/dead assay, Invitrogen Corp., Carlsbad, CA, USA), and evaluated by laser scanning confocal microscopy. The number of dead cells was then quantified and the difference was statistically compared. For corneas exposed to 1 ppm and 1% pTSA, there was no significant difference in the number of dead cells compared with water-exposed, control corneas at either 3 hours or 1 day after exposure. However, corneas exposed to 10% and 50% pTSA showed significantly increased (p < .0001) numbers of dead cells, averaging (mean ± standard deviation) 486 ± 133 and 1,052 ± 101 cells/field of view (460 × 460 μm). The level of cytotoxicity was comparable with that observed for 10% AA, which averaged 409 ± 142 cells/field of view. The data suggest that pTSA is an innocuous irritant at exposure levels environmentally encountered, but that higher concentrations (10% and above) might be considered a slight to mild irritant. We conclude that this modified ex vivo rabbit eye test using the live/dead assay may be a useful model for developing ocular irritation assays.