One component in the safety assessment of many types of products is the evaluation of their potential to cause eye injury. Eye irritation is defined as “the production of changes in the eye following the application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application” (UNECE, 2004). Eye corrosion (serious eye damage) is defined as “the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application” (UNECE, 2004).
The anterior surface of the eye is covered by the cornea and the conjunctiva. The cornea is the most exposed area and, therefore, the most likely part of the eye to be involved in a chemical exposure to the eye. Chemical injury to the cornea can result in the loss of vision. Accordingly, corneal injury in the animal test for eye irritation/corrosion accounts for 73% of the total ocular toxicity score. For these reasons, the cornea has been the primary tissue modeled in in vitro alternative models for accessing eye injury.
The cornea is composed of three cellular layers. The outermost layer, the corneal epithelium, is composed of 5-7 epithelial cell layers in the human cornea. The surface layers of cells are connected by tight junctions that modulate the permeation of molecules into the tissue. The stroma, beneath the corneal epithelium, is composed of keratocytes (fibroblast-like cells) interspersed in the stromal collagen matrix. A single cell layer of corneal endothelial cells forms the innermost cellular layer of the cornea. Nerve cells penetrate through the cornea into the corneal epithelium, making it one of the most highly innervated tissues in the body; these cells are readily perturbed when the corneal epithelial tight junctions are disrupted.
Many mild eye irritants act by disrupting or damaging only the surface cells of the corneal epithelium, and the corneal can repair this type of damage within a short time. The stronger the eye irritant, the deeper it penetrates into the next layer of the cornea, the stroma. Very damaging materials might penetrate deep enough to cause irreversible injury, including damage to the corneal endothelial cell layer, where tight junctions modulate the penetration of water and other substances from the cornea to the aqueous humor.
The conjunctiva covers the remaining surface of the eye and is also important in protecting the eye from environmental insults. Injury to the conjunctiva has been assigned about 18% of the in vivo eye injury score in the Draize test but has largely been ignored in in vitro assessments of chemical eye injury. Conjunctival injury may be irrelevant in moderate to severe eye injury, where the corneal effects are largely predictive of reversibility and outcome, but could be useful in assessing milder effects, especially for products used in and around the eye (Ward, et al., 2000). For example, the conjunctiva can exhibit different mechanisms of injury than the cornea due to its different physiology. The conjunctiva contains goblet cells, which secrete the mucin layer that protects the surface of the eye, as well as immune and vascular components important in the eye irritation response.
The iris is the third ocular tissue assessed for response to an irritant in the in vivo eye test. It is generally agreed that an in vitro iris assessment for most substances is not necessary, as iris responses occur only upon significant disruption to the ocular surface barrier of the cornea (Bagley, et al., 2006). Therefore, the degree of corneal injury should be predictive of potential iris effects.
Developed in 1944, the Draize rabbit eye irritation test remains the standard method for evaluating the ocular irritation/corrosion potential of a substance for regulatory purposes (Draize, et al., 1944; Friedenwald, et al., 1944; ILSI TCAAT, 1996). In this test, a material is instilled into one eye of albino rabbits (the other eye serving as the negative control), and the response of the animals is monitored using a standardized scoring system for injury to the cornea, conjunctiva, and iris. Ocular responses are scored at 1, 24, 48, and 72 hours. The animals are observed until the full magnitude and reversibility of the ocular injury can be evaluated—for up to 21 days. Reversibility of the ocular injury is an important component in the classification of a substance as an eye irritant versus an eye corrosive. Different modifications of the test require different numbers of animals, although no more than three animals is the current standard.
The Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 405 Acute Eye Irritation/Corrosion, the OECD Guidance Document No. 14, and Chapter 3.3 of the Globally Harmonized System (GHS) for Classification and Labeling of Chemicals (UNECE, 2004) describe internationally accepted guidelines for eye irritation/corrosion studies. Many national agencies will accept the GHS beginning in 2008 (EPA, 2006). Most test guidance documents, including the OECD and GHS, indicate that a chemical found to be strongly irritating or corrosive in skin studies does not need to be tested in eye studies because the response can be assumed to be at least that severe in the eye.
Regulatory authorities in most countries require ocular safety assessments and commonly have some version of the Draize rabbit eye test as part of their testing guidelines. The Introduction and Rationale… section of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) ocular background review documents provides an overview of the guidance and regulations for ocular testing and explains the classification systems that different authories use for eye irritation.
Draize rabbit eye data has proved to be highly variable, generally overpredictive of human eye injury, and sometimes incorrect due to species differences in the ocular response to specific substances. However, there has been renewed interest in a variant of the traditional Draize test, the low-volume eye test (LVET). In this test, one-tenth the dosing volume of the traditional test is placed directly on the cornea, as opposed to the conjunctival sac. In addition to giving responses closer to those observed in humans, it has the potential utility of providing mechanistic data (Jester, et al., 1998; Maurer, et al., 2002; Jester, 2006). The LVET, used with confocal microscopy, has been shown to correlate with the degree of corneal injury for some classes of chemicals. A significant amount of new animal data (only a few of the publications are cited above) are being generated to demonstrate that the method is an improved in vivo model for use in the validation of in vitro eye irritation methods. In 2003, one of the new activities that the European Centre for the Validation of Alternative Methods (ECVAM) endorsed was “the evaluation of alternative methods aiming to refine the existing animal test, such as the Low-Volume-Eye-Test.”
A number of non-animal test methods have been developed in the search for a replacement for the Draize rabbit eye test. Protocols for many of the in vitro methods are available at the AnimAlt-Zebet Database and the ECVAM database service on alternative methods to animal experimentation (DB-ALM). Protocols for the four methods reviewed by ICCVAM for assessing severe eye irritants and corrosives are available in Background Review Documents (BRD). Many additional methods and ocular models used for ophthalmic and toxicological research and testing have been reported in the literature.
Alternative methods for assessing the eye irritation/corrosion potential of substances have been reviewed in recent symposia and publications (Curren & Harbell, 1998; ECVAM, 2002; Eskes, et al., 2005; ICCVAM, 2006; Salem & Katz, 2003). The ICCVAM/National Toxicology Program Interagency Center for Evaluation of Alternative Toxicological Methods (NICEATM) Ocular Toxicity Scientific Symposium I: Mechanisms of Chemically-Induced Ocular Injury and Recovery of May 2005 also reviewed current research and in vitro models, but the presentations and meeting summary are not yet available on ICCVAM’s website. A summary of at least some of the ocular models and assay endpoints that have been developed are summarized in Tables 1 and 2 [works in progress].
Table 1. Non-animal alternative methods for evaluating eye irritation and corrosion.*
|Human volunteer clinical eye testing||Makeup applied to outer eyelid for comparison to in vitro results||Debbasch, et al., 2005|
|Vapor exposures||Cometto-Muñiz, et al., 2007|
|Isolated eye assays||Bovine Corneal Opacity and Permeability (BCOP)||Gautheron, et al., 1992; Sina, et al., 1998; ICCVAM, 2006; Ubels, et al., 2004|
|Porcine Corneal Opacity and Permeability (PCOP)||Van den Berghe, et al., 2005; Xu, et al., 2000|
|Isolated Chicken Eye (ICE) assay||Prinsen & Koëter, 1993; Prinsen, 1996; ICCVAM, 2006|
|Isolated Rabbit Eye (IRE)||Cooper, et al., 2001; ICCVAM, 2006|
|Isolated Mouse Eye|
|Human Cornea (discarded from eyebanks)|
|Chicken egg membrane assays||Chorioallantoic Membrane Vascularization Assay (CAMVA)||Bagley, et al., 1994; 1999|
|Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM) assay)||Spielmann, et al., 1997; ICCVAM, 2006; Mehling, et al., 2007|
|Reconstituted human cornea models||Human corneal equivalent||Griffith, et al., 1999|
|Reconstituted rabbit cornea models||3D corneal tissue construct||Zieske, et al., 1994|
|Reconstituted bovine cornea models||Epithelium and stroma||Minami, et al., 1993; Parnigotto, et al., 1998|
|3D human corneal epithelial cell models||HCE-T human corneal epithelial cell model||Clothier, et al., 2000; Kahn & Walker, 1993; Kahn, et al., 1993; Kruszewski, et al., 1995; 1997; Ward, et al., 1997; 2003|
|SkinEthic HCE model; CEPI||Debbasch, et al., 2005; Van Goethem, et al., 2006; Mehling, et al., 2007|
|Coty corneal epithelial model||Doucet, et al., 2006|
|3D epithelial cell models||EpiOcular||Jones, et al., 2001|
|MDCK fluorescein leakage||Tchao, 1988; Shaw, et al., 1990; Jones, et al., 2001|
|Tissue equivalent assay||Osborne, et al., 1995; Curren, et al., 1997|
|3D human conjunctival epithelial cell models||Human conjunctival model||Ward, et al., 2000|
|Monolayer epithelial cell cultures||Human corneal epithelial cells||Ward, et al., 1997|
|Rabbit corneal cells; SIRC cell line||North-Root, et al., 1982; Grant, et al., 1992; Yang & Acosta, 1994|
|Various cultured cells||Harbell, et al., 1997|
|Epithelial and fibroblast cell lines||Pasternak & Miller, 1995|
|Red blood cells||Red cell hemolysis||Pape, et al., 1987; Lagarto, et al., 2006; Martinez, et al., 2006; Mehling, et al., 2007|
|Neural cell models (for detecting neurogenic ocular pain)||TRPV1-expressing neuroblastoma SH-SY5Y cells||Lilja & Forsby, 2004; Lilja, et al., 2007|
|Acellular models||EYTEX/Irritection||Curren, et al., 1997|
|Hemoglobin denaturation||Liao, et al., 2004|
|Computational models||SAR/(Q)SAR||Abraham, et al., 2003; Gerner, et al., 2005; Kulkarni & Hopfinger, 1999; Kulkarni, et al., 2001; Li, et al., 2005; Pospisil & Holzhütter, 2001; Tsakovska, et al., 2007|
*This table is a work in progress
A few of the non-animal methods for evaluating eye irritation and/or eye corrosion are described below.
BCOP Assay: The BCOP assay uses enucleated cow eyes that would otherwise be discarded at slaughterhouses. The cornea is isolated from the rest of the eye and maintained in a holder. A test substance is applied to this isolated cornea for a specified time then removed, and the effect of the substance on the permeability of the cornea to fluorescein (a colored dye) and the increase in corneal opacity (transmission of light through the cornea) are determined. As stated previously, corneal opacity is the primary component of the Draize rabbit eye test—hence, the BCOP assay has a direct mechanistic link to the rabbit test.
The BCOP assay has undergone a number of interlaboratory studies, and some companieshave used it for internal product decisions for many years. It has been found to be most predictive of the rabbit test for moderate to severe/corrosive substances. Additional endpoints are sometimes evaluated, and there are variations in the protocols used by different labs. The assay has been used to test a wide range of product types including liquids, powders, and creams. Standardized protocols for the BCOP assay are available, and contract labs conduct the assay for companies needing data for regulatory submissions.
Table 2. Endpoints used for assessing eye irritation and corrosion in ocular models.*
|Cytotoxicity/viability/proliferation||MTT (tetrazolium salt; mitochondrial dehydrogenase dye reduction)||Clothier, et al., 2000; Ward, et al., 1997|
|Lactate dehydrogenase (LDH)|
|Neutral red release (NRR)|
|Neutral red uptake (NRU)||Pasternak & Miller, 1995|
|Lactate release||Clothier, et al., 2000; Ward, et al., 2000|
|Akamar blue™||Clothier, et al., 2000|
|WST-1 (tetrazolium salt that yields water soluble cleavage products)||Huhtala, et al., 2003|
|Histology/ultrastructure||Histology in BCOP model||Cater & Harbell, 2006; Cooper, et al., 2001; Ubels, et al., 2004|
|Transmission electron microscopy (TEM) in cell models||Ward, et al., 1997|
|Barrier function||Fluorescein permeability in isolated eye assays (BCOP, etc.)||Gautheron, et al.,1992; Sina, et al., 1998; Ubels, et al. 2004; Van den Berghe, et al., 2005; Xu, et al., 2000|
|Fluorescein leakage/MDCK cells (FL)||Tchao, 1988; Shaw, et al., 1990; Jones, et al., 2001|
|Transepithelial fluorescein permeability (TEP)||Kahn & Walker, 1993; Kahn, et al., 1993; Kruszewski, et al., 1995; 1997; Ward, et al., 1997; 2000; 2003|
|Transepithelial electrical resistance (TER or TEER) and other measures of tight junctions||Wang, et al., 2004; Ward, et al., 1997; 2000|
|Inflammatory mediator release or expression||Cytokines||Burbach, et al., 2001; Smit, et al., 2003; Debbasch, et al., 2005|
|Arachadonic acid metabolites|
|Adhesion and other receptor molecule expression||ICAM-1||Yannariello-Brown, et al., 1998|
|CD14 and toll-like receptor 4||Song, et al., 2001|
|Stress gene/protein expression||HO-1|
|Toxicogenomics||Boulton & Wride, 2006|
|Transcription factor modulation||NF?-b||Xu, et al., 2000|
|Cellular metabolism||Lactate release/glucose uptake|
|Other||Total glutathione content||Pasternak & Miller, 1995|
|ATP content||Pasternak & Miller, 1995|
|Methionine incorporation||Pasternak & Miller, 1995|
*This table is a work in progress
Isolated chicken eye: The ICE assay uses enucleated chicken eyes obtained from slaughterhouses. The eyes are placed in an apparatus, kept moist, and treated with the test substance. Three responses of the cornea are evaluated: corneal swelling, corneal opacity, and fluorescein retention. The irritation potential of a substance is calculated from the mean values of these measurements.
EpiOcular assay: “MatTek’s EpiOcular™ corneal model consists of normal, human-derived epidermal keratinocytes that have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. The epidermal cells, which are cultured on specially prepared cell culture inserts using serum free medium, differentiate to form a multi-layered structure that closely parallels the corneal epithelium…. EpiOcular has been utilized with several common tests of cytotoxicity and irritancy, including MTT, IL-1a, PGE2, LDH, and sodium fluorescein permeability.”
HCE-T TEP assay: The HCE-T TEP assay measures the dose-dependent effect of a test material on fluorescein transepithelial permeability (TEP) across stratified cultures of human corneal epithelial cells (HCE-T cell line). HCE-T cells are grown at the air-liquid interface on a collagen membrane where the cells stratify and differentiate to form an epithelial barrier similar to the corneal surface. The concentration of a test material that causes fluorescein retention by the HCE-T cultures to decrease to 85% relative to the control cultures (FR85) is the assay endpoint. TEP and transepithelial electrical resistance (TER) are not equally altered by a treatment, indicating that fluorescein permeability in the HCE-T model is regulated by tissue properties in addition to tight junction integrity, including the multiple cell layers, cell viability, and desmosomal junction integrity. Other endpoints that have been evaluated using the HCE-T model include lactate release, PGE2 release, various cytokines, and MTT dye uptake.
MDCK FL assay: The Fluorescein Leakage (FL) assay measures the dose-dependent effect of a test material on the amount of fluorescein that penetrates across a monolayer of MDCK cells cultured on permeable membrane culture inserts. MDCK cells form tight junctions that prevent passage of the fluorescein unless damaged by an applied chemical.
In vitro ocular methods have a long and disappointing history regarding test method validation (Balls, et al., 1999; Spielmann & Liebsch, 2001). A recent approach to validation of ocular alternative test methods involves the separate assessment of methods for eye irritation and eye corrosion. ICCVAM/NICEATM has already evaluated four methods for their ability to assess substances that are severe/corrosive to the eye, and ECVAM is conducting a retrospective assessment of the validity of additional in vitro methods for their ability to assess substances that are mild to moderately irritating or nonirritating to the eye.
In 2003, four alternative test methods were nominated to ICCVAM/NICEATM for review as screening methods for severe eye irritation/corrosion: the Isolated Chicken Eye (ICE), Isolated Rabbit Eye (IRE), Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM), and Bovine Corneal Opacity and Permeability (BCOP) assays. Several EU authorities had already accepted these methods for the classification of severe/corrosive eye irritants. The ICCVAM review of these methods concluded with an expert panel report in March 2005 and with ICCVAM’s endorsement of the BCOP and ICE methods as valid screens for corrosive and severe eye irritants. However, ICCVAM has determined that negative results still require in vivo testing before a conclusion of nonsevere/corrosive can be made.
The ICCVAM endorsement was stated as follows: “In 2007, BCOP and ICE recommended as screening tests for identifying corrosives and severe irritants, with certain limitations; HET-CAM and IRE not recommended for regulatory hazard classification purposes until further developed and evaluated.” The specific limitations on the uses of these in vitro methods are described in the documents on the ICCVAM website. The US Food and Drug Administration (FDA) and US Environmental Protection Agendy (EPA) reported previous acceptance of BCOP data in specific circumstances and are expected to continue to do so considering ICCVAM’s recommendations.
The ECVAM Scientific Advisory Committee (ESAC) statement on the ICCVAM retrospective study of the four in vitro screening assays for ocular corrosives/severe irritants endorsed the validity of the BCOP and ICE methods for use in a tiered strategy as part of the weight of evidence approach. ESAC indicated further work is needed for the IRE and HET-CAM methods (ESAC Statement, April 27, 2007).
Current non-animal methods considered valid for regulatory testing purposes (for limited applications) are listed in Table 3.
Table 3. In vitro ocular test methods considered valid for limited regulatory testing applications.
|Bovine Corneal Opacity and Permeability (BCOP) assay||Eye corrosion/severe irritation||
|Isolated Chicken Eye (ICE) assay||Eye corrosion/severe irritation||
|Isolated Rabbit Eye (IRE) assay||Eye corrosion||
|Hen’s Egg Test—Chorio-Allantoic Membrane assay (HET-CAM)||Eye corrosion||
(a) Although not formally endorsed as valid, positive outcomes can be used for classifying and labeling substances as severe eye irritants (R41) in the EU
Validation studies for two cell-based in vitro test methods for assessing ocular irritants, the Gillette HCE-T TEP assay and the MatTek EpiOcular assay, were completed in 2001. The prevalidation study results for the HCE-T TEP assay were presented at the 2000 Alternative Toxicological Methods for the New Millennium meeting in Bethesda, MD, US (Ward, et al., 2003) and were submitted to the ICCVAM Ocular Toxicity Working Group (OTWG) in January 2001. The validation study was completed in January 2001, and the results were prepared for submission to the OTWG in 2001. Validation study results from the first phase of the EpiOcular study were also submitted to ICCVAM and reviewed by the OTWG. Additional studies were conducted and submitted to ECVAM for review. ICCVAM’s nonsevere ocular irritant page now indicates only that ICCVAM is reviewing the validation status of in vitro methods for identifying nonsevere ocular irritants and nonirritants. NICEATM is still requesting the submission of in vivo and/or in vitro ocular data that can be added to its database of alternative methods for assessing nonsevere irritants.
ECVAM is also reviewing the validation status of several additional in vitro methods (Fluorescein leakage, Red blood cell lysis, Neutral red release, and Cytosensor microphysiometer). A prevalidation study has been completed for the SkinEthic HCE model (Van Goethem, et al., 2006), which may be included in the ECVAM assessment.
Ahmad, S., Stewart, R., Yung, S., et al. (2007). Differentiation of human embryonic stem cells into corneal epithelial-like cells by in vitro replication of the corneal epithelial stem cell niche. Stem Cells. 25, 1145-1155.
Bagley, D.M., Waters, D. & Kong, B.M. (1994). Development of a 10-day chorioallantoic membrane vascular assay as an alternative to the Draize rabbit eye irritation test. Food Chem. Toxicol. 32, 1155-1160.
Bagley, D.M., Cerven, D. & Harbell, J.W. (1999). Assessment of the chorioallantoic membrane vascular assay (CAMVA) in the COLIPA in vitro eye irritation validation study. Toxicol. in Vitro. 13, 285-293.
Bagley, D.M., Casterton, P.L., Dressler, W.E., et al. (2006). Proposed new classification scheme for chemical injury to the human eye. Regul. Toxicol. Pharm. 45, 206-213.
Balls, M., Berg, N., Bruner, L.H., et al. (1999). Eye irritation testing: The way forward. The report and recommendations of ECVAM workshop 34. Altern. Lab. Anim. 27, 53-77.
Boulton, M. & Wride, M. (2006). Can toxicogenomics be used to identify chemicals that cause ocular injury? ALTEX. 23, Suppl., 318-320.
Buehler, E.V. & Newman, E.A. (1964). A comparison of eye irritation in monkeys and rabbits. Toxicol. Appl. Pharmacol. 6, 701-710.
Burbach, G.J., Naik, S.M., Harten, B.J., et al. (2001). Interleukin-18 expression and modulation in human corneal epithelial cells. Curr. Eye Res. 23, 64-68.
Cater, K.C. & Harbell, J.W. (2006). Prediction of eye irritation potential of surfactant-based rinse-off personal care formulations by the bovine corneal opacity and permeability (BCOP) assay. Cutan. Ocul. Toxicol. 25, 217-233.
Clothier, R., Orme, A., Walker, T.L., et al. (2000). Comparison of three cytotoxicity assays using the corneal HCE-T model. Altern. Lab. Anim. 28, 293-302.
Cometto-Muñiz, J.E., Cain, W.S., Abraham, M.H. & Sánchez-Moreno, R. (2007). Cutoff in detection of eye irritation from vapors of homologous carboxylic acids and aliphatic aldehydes. Neuroscience. 145, 1130-1137.
Cooper, K.J., Earl, L.K., Harbell, J.W. & Raabe, H. (2001). Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay. Toxicol. In Vitro. 15, 95-103.
Curren, R.D., Sina, J.F., Feder, P., et al. (1997). IRAG working group 5. Other assays. Interagency Regulatory Alternatives Group. Food Chem. Toxicol. 35, 127-158.
Curren, R.D. & Harbell, J.W. (1998). In vitro alternatives for ocular irritation. Environ. Health Perspect. 106, Suppl. 2, 485-492.
de Silva, O. & European Federation of the Cosmetics Industry Steering Committee on Alternatives to Animal Testing (2002). The contributions of the European cosmetics industry to the development of alternatives to animal testing: Dialogue with ECVAM and future challenges. Altern. Lab. Anim. 30, Suppl. 2, 189-193.
Debbasch, C., Ebenhahn, C., Dami, N., et al. (2005). Eye irritation of low-irritant cosmetic formulations: Correlation of in vitro results with clinical data and product composition. Food Chem. Toxicol. 43, 155-165.
Doillon, C.J., Watsky, M.A., Hakim, M., et al. (2003). A collagen-based scaffold for a tissue engineered human cornea: Physical and physiological properties. Int. J. Artif. Organs. 26, 764-773.
Doucet, O., Lanvin, M., Thillou, C., et al. (2006). Reconstituted human corneal epithelium: A new alternative to the Draize eye test for the assessment of the eye irritation potential of chemicals and cosmetic products. Toxicol. In Vitro. 20, 499-512.
Draize, J.H., Woodward, G., Calvery, H.O. (1944). Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 82, 377-390.
Duan, X., McLaughlin, C., Griffith, M. & Sheardown, H. (2007). Biofunctionalization of collagen for improved biological response: Scaffolds for corneal tissue engineering. Biomaterials. 28, 78-88.
ECVAM. (2002). Local toxicity. Acute dermal and ocular effects. Altern. Lab. Anim. 30, Suppl. 1, 35-47.
Environmental Protection Agency (EPA). (2006). Globally Harmonized System (GHS) for Classification and Labeling of Chemicals. Available here.
Eskes, C., Bessou, S., Bruner, L., et al. (2005). Eye irritation. Altern. Lab. Anim. 33, Suppl. 1, 47-81.
Freeberg, F.E., Griffith, J.F., Bruce, R.D. & Bay, P.H.S. (1984). Correlation of animal test methods with human experience for household products. J. Toxicol. Cutaneous Ocul. Toxicol. 3, 53-64.
Friedenwald, J. S., Hughes, W. F. & Herrmann, H. (1944). Acid-base tolerance of the cornea. Arch. Ophthalmol. 31, 279-283.
Gad, S.C. & Chengelis, C.P. (1991). Acute Toxicology Testing. Academic Press, San Diego, 57-83.
Gautheron, P., Dukic, M., Alix, D. & Sina, J.F. (1992). Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam. Appl. Toxicol. 18, 442-449.
Gerner, I., Liebsch, M. & Spielmann, H. (2005). Assessment of the eye irritating properties of chemicals by applying alternatives to the Draize rabbit eye test: The use of QSARs and in vitro tests for the classification of eye irritation. Altern. Lab. Anim. 33, 215-237.
Grant, W.M. & Schuman, J.S. (1993). Toxicology of the Eye. 4th Ed. Charles C. Thomas, Springfield, IL.
Grant, R.L., Yao, C., Gabaldon, D. & Acosta, D. (1992). Evaluation of surfactant cytotoxicity potential by primary cultures of ocular tissues: I. Characterization of rabbit corneal epithelial cells and initial injury and delayed toxicity studies. Toxicol. 76, 153-176.
Griffith, M., Osborne, R., Munger, R., et al. (1999). Functional human corneal equivalents constructed from cell lines. Science. 286, 2169-2172.
Harbell, J., Koontz, S.W., Lewis, R.W., et al. (1997). IRAG working group 4: Cell cytotoxicity assays. Food Chem. Toxicol. 35, 79-126.
Huhtala, A., Alajuuma, P., Burgalassi, S., et al. (2003). A collaborative evaluation of the cytotoxicity of two surfactants by using the human corneal epithelial cell line and the WST-1 test. J. Ocul. Pharmacol. Ther. 19, 11-21.
ICCVAM. (2006). In vitro test methods for detecting ocular corrosives and severe irritants. Background Review Documents. Available here.
ILSI Technical Committee on Alternatives to Animal Testing (TCAAT). (1996). Replacing the Draize eye irritation test: Scientific background and research needs. J. Toxicol. Cutaneous Ocul. Toxicol. 15, 211-234.
Jester, J.V. (2006). Extent of corneal injury as a biomarker for hazard assessment and the development of alternative models to the Draize rabbit eye test. Cutan. Ocul. Toxicol. 25, 41-54.
Jester, J.V., Petroll, W.M., Bean, J., et al. (1998). Area and depth of surfactant-induced corneal injury predicts extent of subsequent ocular responses. Invest. Ophthalmol. Vis. Sci. 39, 2610-2625.
Jones, P.A., Budynsky, E., Cooper, K.J., et al. (2001). Comparative evaluation of five in vitro tests for assessing the eye irritation potential of hair-care products. Altern. Lab. Anim. 29, 669-692.
Kahn, C.R. & Walker, T.L. (1993). Human corneal epithelial cell lines with extended lifespan: Transepithelial permeability across membranes formed in vitro. Invest. Ophthalmol. Vis. Sci. 34, 1010.
Kahn, C.R., Young, E., Lee, I.H. & Rhim, J.S. (1993). Human corneal epithelial primary cultures and cell lines with extended life span: In vitro model for ocular studies. Invest. Ophthalmol. Vis. Sci. 34, 3429-3441.
Kruszewski, F.H., Walker, T.L., Ward, S.L. & DiPasquale, L.C. (1995). Progress in the use of human ocular tissues for in vitro alternative methods. Comments on Toxicol. 5, 203-224.
Kruszewski, F.H., Walker, T.L. & Dipasquale, L.C. (1997). Evaluation of a human corneal epithelial cell line as an in vitro model for assessing ocular irritation. Fundam. Appl. Toxicol. 36, 130-140.
Kulkarni, A.S. & Hopfinger, A.J. (1999). Membrane-interaction QSAR analysis: Application to the estimation of eye irritation by organic compounds. Pharm. Res. 16, 1245-1253.
Kulkarni, A., Hopfinger, A.J., Osborne, R., et al. (2001). Prediction of eye irritation from organic chemicals using membrane-interaction QSAR analysis. Toxicol. Sci. 59, 335-345.
Lagarto, A., Vega, R., Vega, Y., et al. (2006). Comparative study of red blood cell method in rat and calves blood as alternatives of Draize eye irritation test. Toxicol. In Vitro. 20, 529-533.
Li, Y., Liu, J., Pan, D. & Hopfinger, A.J. (2005). A study of the relationship between cornea permeability and eye irritation using membrane-interaction QSAR analysis. Toxicol. Sci. 88, 434-446.
Liao, Y., Wang, X., Zhang, L.S., et al. (2004). Study on the use of haemoglobin denaturation test as an alternative to Draize eye irritation test. Sichuan Da Xue Xue Bao Yi Xue Ban. 35, 654-657.
Lilja, J. & Forsby, A. (2004). Development of a sensory neuronal cell model for the estimation of mild eye irritation. Altern. Lab. Anim. 32, 339-343.
Lilja, J., Lindegren, H. & Forsby, A. (2007). Surfactant-induced TRPV1 activity: A novel mechanism for eye irritation? Toxicol. Sci. 99, 174-180.
Martinez, V., Corsini, E., Mitjans, M., et al. (2006). Evaluation of eye and skin irritation of arginine-derivative surfactants using different in vitro endpoints as alternatives to the in vivo assays. Toxicol. Lett. 164, 259-267.
Maurer, J.K., Parker, R.D. & Jester, J.V. (2002). Extent of initial corneal injury as the mechanistic basis for ocular irritation: Key findings and recommendations for the development of alternative assays. Regul. Toxicol. Pharmacol. 36, 106-117.
Mehling, A., Kleber, M. & Hensen, H. (2007). Comparative studies on the ocular and dermal irritation potential of surfactants. Food Chem. Toxicol. 45, 747-758.
Minami, Y., Sugihara, H. & Oono, S. (1993). Reconstruction of cornea in three-dimensional collagen gel matrix culture. Invest. Ophthalmol. Vis. Sci. 34, 2316-2324.
North-Root, H., Yackovich, F., Demetrulias, J., et al. (1982). Evaluation of an in vitro cell toxicity test using rabbit corneal cells to predict the eye irritation potential of surfactants. Toxicol. Lett. 14, 207-212.
Nussenblatt, R.B., Bron, A., Chambers, W., et al. (1998). Ophthalmologic perspectives on eye irritation testing. J. Toxicol. Cutaneous Ocul. Toxicol. 17, 103-109.
Osborne, R., Perkins, M.A. & Roberts, D.A. (1995). Development and intralaboratory evaluation of an in vitro human cell-based test to aid ocular irritancy assessments. Fundam. Appl. Toxicol. 28, 139-153.
Pape, W.J.W., Pfannenbecker, U. & Hoppe, U. (1987). Validation of the red blood cell test system as an in vitro assay for the rapid screening of irritation potential of surfactants. Mol. Toxicol. 1, 525-536.
Parnigotto, P.P., Bassani, V., Montesi, F. & Conconi, M.T. (1998). Bovine corneal stroma and epithelium reconstructed in vitro: Characterization and response to surfactants. Eye. 12, 304–310.
Pasternak, A.S. & Miller, W.M. (1995). First-order toxicity assays for eye irritation using cell lines: Parameters that affect in vitro evaluation. Fundam. Appl. Toxicol. 25, 253-263.
Pospisil, H. & Holzhütter, H.G. (2001). A compartment model to calculate time-dependent concentration profiles of topically applied chemical compounds in the anterior compartments of the rabbit eye. Altern. Lab. Anim. 29, 347-365.
Prinsen, M.K. (1996). The chicken enucleated eye test (CEET): a practical (pre)screen for the assessment of eye irritation/corrosion potential of test materials. Food Chem. Toxicol. 34, 291-296.
Prinsen, M.K. & Koëter, H.B.W.M. (1993). Justification of the enucleated eye test with eyes of slaughterhouse animals as an alternative to the Draize eye irritation test with rabbits. Food Chem. Toxicol. 31, 69–76.
Salem, H. & Katz, S.A. (2003). Part II. Development of predictive methods based on mechanisms of eye irritation at the ocular surface: Meeting industry and regulatory needs. Alternative Toxicological Methods for the New Millennium. CRC Press, Boca Raton, FL. 75-186.
Shaw, A.J., Clothier, R.H. & Balls, M. (1990). Loss of trans-epithelial impermeability of a confluent monolayer of Madin-Darby canine kidney(MDCK) cells as a determinant of ocular irritancy potential. Altern. Lab. Anim. 18, 145-151.
Shortt, A.J., Secker, G.A., Munro, P.M., et al. (2007). Characterization of the limbal epithelial stem cell niche: Novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells. Stem Cells. 25, 1402-1409.
Sina, J.F., Gautheron, P., Casterton, P., et al. (1998). Report from the bovine corneal opacity and permeability technical workshop. In Vitro Mol. Toxicol. 11, 315–351.
Smit, E.E., Sra, S.K., Grabowski, L.R., et al. (2003). Modulation of IL-8 and RANTES release in human conjunctival epithelial cells: Primary cells and cell line compared and contrasted. Cornea. 22, 332-337.
Song, P.I., Abraham, T., Park, Y., et al. (2001). The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells. Invest. Ophthalmol. Vis. Sci. 42, 2867-2877.
Spielmann, H. & Liebsch, M. (2001). Lessons learned from validation of in vitro toxicity test: From failure to acceptance into regulatory practice. Toxicol. In Vitro. 15, 585-590.
Spielmann, H., Liebsch, M., Moldenhauer, F., et al. (1997). IRAG working group 4: CAMbased assays. Food Chem. Toxicol. 35, 39-66.
Stepp, M.A. & Zieske, J.D. (2005). The corneal epithelial stem cell niche. Ocul. Surf. 3, 15-26.
Stramer, B.M., Zieske, J.D., Jung, J.C., et al. (2003). Molecular mechanisms controlling the fibrotic repair phenotype in cornea: Implications for surgical outcomes. Invest. Ophthalmol. Vis. Sci. 44, 4237-4246.
Tchao, R. (1988). Trans-epithelial permeability of fluorescein in vitro as an assay to determine eye irritants. Alternative Methods in Toxicology. Vol 6 (Ed. A.M. Goldberg). Mary Ann Liebert, New York.
Tsakovska, I., Saliner, A.G., Netzeva, T., et al. (2007). Evaluation of SARs for the prediction of eye irritation/corrosion potential: Structural inclusion rules in the BfR decision support system. SAR QSAR Environ. Res. 18, 221-235.
Ubels, J.L., Ditlev, J.A., Clousing, D.P. & Casterton, P.L. (2004). Corneal permeability in a redesigned corneal holder for the bovine cornea opacity and permeability assay. Toxicol. In Vitro. 18, 853-857.
United Nations Economic Commission for Europe (UNECE). (2004). Globally Harmonized System of Classification and Labeling of Chemicals (GHS). Part 3, Health and environmental hazards. Chapter 3.3. Serious eye damage/ irritation. Available here.
Van den Berghe, C., Guillet, M.C. & Compan, D. (2005). Performance of porcine corneal opacity and permeability assay to predict eye irritation for water-soluble cosmetic ingredients. Toxicol. In Vitro. 19, 823-830.
Van Goethem, F., Adriaens, E., Alépée, N., et al. (2006). Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals. Toxicol. In Vitro. 20, 1-17.
Wang, Y., Zhang, J., Yi, X.J. & Yu, F.S. (2004). Activation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells. Exp. Eye Res. 78, 125-136.
Ward, S.L., Walker, T.L. & Dimitrijevich, S.D. (1997). Evaluation of chemically-induced toxicity using an in vitro model of human corneal epithelium. Toxicol. In Vitro. 11, 121-139.
Ward, S., Walker, T., Trocme, S., et al. (2000). A human conjunctival model for the evaluation of eye irritants. Progress in the Reduction, Refinement and Replacement of Animal Experimentation. (Eds: M. Balls, A.M. van Zeller & M.E. Halder). Elsevier Science B.V., Amsterdam. 305-317.
Ward, S.L., Gacula, Jr., M. & Edelhauser, H.F. (2003). The Human Corneal Epithelial HCE-T TEP assay for eye irritation: Scientific relevance and summary of prevalidation study results. Alternative Toxicological Methods for the New Millennium. (Eds. H. Salem & S.A. Katz). CRC Press, Boca Raton, FL. 161-186.
Weil, C.S. & Scala, R.A. (1971). Study of intra- and inter-laboratory variability in the results of rabbit eye and skin irritation test. Toxicol. Appl. Pharmacol. 19, 276-360.
Xu, K.P., Li, X.F. & Yu, F.S. (2000). Corneal organ culture model for assessing epithelial responses to surfactants. Toxicol. Sci. 58, 306-314.
Yang, W. & Acosta, D. (1994). Cytotoxicity potential of surfactant mixtures evaluated by primary cultures of rabbit corneal epithelial cells. Toxicol. Lett. 70, 309–318.
Yannariello-Brown, J., Hallberg, C.K., Häberle, H., et al. (1998). Cytokine modulation of human corneal epithelial cell ICAM-1 (CD54) expression. Exp. Eye Res. 67, 383-393.
Zieske, J.D., Mason, V.S., Wasson, M.E., et al. (1994). Basement membrane assembly and differentiation of cultured corneal cells: Importance of culture environment and endothelial cell interaction. Exp. Cell Res. 214, 621-633.