Draize ocular irritation test

Borenfreund, E. and Borrero, O. (1984). In vitro cytotoxicity assays potential alternatives to the Draize ocular irritancy test. Cell Biol. Toxicol. 1 33-39. 

DNA binding of AP-1 and NFkB is concentration-dependent and corresponds to Draize scores for ocular irritancy. Test chemical exposure time and probelabeling efficiency may introduce variance to results. Despite the variance, detection of altered expression and/or activation of stress-response transcription factors like AP-1 and NFkB can serve as an early marker for subsequent deteriorative outcomes (Ramesh et al. 1999) of ocular toxicity. 

ILSI Health and Environmental Sciences Institute (1996) Replacing the Draize eye irritation test scientific background and research needs. J Toxicol Cutaneous Ocular Toxicol 15 211-234... 

As a part of the Federal Hazardous Substances Act (FHSA), a modified Draize test was adopted [63-65] as the official method for evaluation of acute ocular irritancy [66]. It is a pass/fail determination that remains in effect today. Two refinements have been accepted as alternatives (a) the test which uses a small volume more consistent with the capacity of the inferior con-... 

Thus far, a wide array of useful cell culture models of the corneal epithelium has been established. Many of these cell culture models focus on toxicity testing and ocular irritation, but some cell layer models for drug permeation studies are also available. Indispensable for successful drug penetration testing is a cell layer that exhibits a tight epithelial barrier. This latter requirement of tight barrier properties disqualifies some of the models that were established as substitutes for the Draize test. At least two cell lines are available for pharmaceutical studies and some newer models may qualify as a useful tool, once they are characterized for their barrier properties. 

Toxic responses in the eye can result from direct topical ocular exposure of drugs from direct installation into the eye and also from dermal products which patients may accidentally get into their eyes. Until recently, the Draize rabbit eye test using three rabbits has served as the major protocol to assess the irritancy potential of topically applied substances. 

Abraham MH, Hassanisadi M, Jalali-Heravi M et al. (2003) Draize rabbit eye test compatibility with eye irritation thresholds in humans a quantitative structure-activity relationship analysis. Toxicol Sci 76 384-391 Curren RD, Harbell JW (1998) In vitro alternatives for ocular irritation. Environ Health Perspect 106, Suppl 2 485M92 Draize JH, Woodard G, Calvery HO (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... 

PURPOSE AND RATIONALE This test uses an ex vivo model of corneal organ culture, preferentially porcine, to obtain information of the possible ocular toxicity of various chemicals. This test is used as an alternative to the Draize Test to minimize or replace the use of live animal testing of ocular irritancy (Symposium, Proceeding 1996). The test allows for determination of reversibility of corneal injury following exposure to chemicals, drugs or cosmetics (Xu etal. 2004). 

Comeal organ culture combined with objectively quantifiable assays for comeal epithelial barrier disruption reduces the high variability associated to the subjectively scored Draize Test. Although the surface biotinylation allows for an objective outcome measure, the scoring system is not yet quantitatively comparable for assessment of ocular irritancy to multiple test products. As it is utilized more extensively in varied laboratories with numerous test chemicals a standardized scoring system can be elicited similar to the familiar Draize Test. 

DNA binding of transcription factors is present for concentrations of test chemicals that elicit mild and moderate scores of ocular irritancy via the Draize. The binding is greatly reduced or absent for concentrations producing a severe ocular irritancy score by the Draize. The lack of binding indicates loss of viability and poor recovery. Concentration of test chemical used may be varied to reach a non-binding outcome to reveal stages of stress and recovery or lack there of. 

Various lecithin-based MEs were also characterized by Hasse and Keipert [131]. The formulations were tested in terms of their physicochemical parameters (pH, refractive index, osmolality, viscosity, and surface tension) and physiological compatibility (HET-CAM and Draize test). In addition, in vitro and in vivo evaluations were performed. The tested MEs showed favorable physicochemical parameters and no ocular irritation as well as a prolonged pilocarpine release in vitro and in vivo. 

Methods of testing for eye and skin irritation potential have been reviewed (163). The official FHSA procedure for evaluating ocular irritation potential of detergent products is a modified Draize rabbit eye test (164). Some controversy surrounds this method at present, and a search for a procedure less injurious to test animals is in progress. In general, the order of irritation is cationic > anionic > nonionic (165). 

As part of the optimisation programme, it is important to evaluate the ocular irritation potential of formulation prototypes. The Draize test, established in the 1940s, is the most widely used method for the identification of primary irritants. There have been modifications to the original test, but they all involve instilling a drop of the formulation into the conjunctival sac of one eye of an albino rabbit, the other eye acting as a control. The condition of both eyes is then evaluated after stipulated time periods and scored relative to the control eye. A high score indicates that the formulation is likely to be an irritant and would not be recommended for progression. 

Hutak, C. M., and R. B. Jacaruso. 1996. Evaluation of primary ocular irritation Alternatives to the Draize test. In Ocular therapeutics and drug delivery, edited by I. K. Reddy. Lancaster, Penn., USA Tech-nomic Publishing Company, pp. 489-525. 

All the official recommended methods for determining the ocular Irritation potential of a chemical derive from the Draize rabbit eye test (Draize et al., 1944). Despite of its historical impact, to day most scientists as well as the general public are disapproved of the Draize test for ethical and scientific reasons (Sharpe, 1985, Swanston, 1985). A number of in vitro test systems were described, e.g. the isolated eyes (Price and Anders, 1985), the embryonated hen s egg-choriallantoic membrane (HET-CAM) test (Luepke, 1985), the neutral red/kenacid blue method (Borenfreund and Puermer, 1985 Knox et al., 1986 Riddell et al., 1986), or the EYTEXR method (Martin, 1993 Regnier, 1994). 

The LjD ratio is the relationship between the HC50 and the hemoglobin denaturation index (DI). This ratio is used to predict the potential ocular irritation of these surfactants relating to the SDS compound (L/Z>sds 0.44 irritant). According to the results of the LjD ratio first and by the in vivo eye irritation Draize test later (see Table 2), these linear arginine-based surfactants have no irritant effect in the eyes (non-eye irritants, LjD > 100). 

There are some other acute toxicity tests in which non-lethal outcome are sought. These include studies of the amount of chemical needed to cause skin or eye irritation or more serious damage. Test systems developed by J. H. Draize and his associates at the Food and Drug Administration in the early 1940s were used to study ocular effects. Warning labels on consumer products were typically based on the outcome of the Draize test. 

Ocular Effects. No studies were located regarding ocular effects in humans after exposure to 3,3 -dichlorobenzidine by any route. No adverse effects on the eye were noted when dichlorobenzidine (isomer unspecified, free base) was directly placed in the conjunctival sac of the eye of rabbits (Gerarde and Gerarde 1974). However, 0.1 mL 3,3 -dichlorobenzidine dihydrochloride (dihydro salt of 3,3 -dichloro-benzidine) in a 20% com oil suspension produced erythema, pus, and comeal opacity, giving a 76% score in the Draize test within an hour when placed in the conjunctival sac of the eye of the rabbit (Gerarde and Gerarde 1974). Apparently, the irritant effects of hydrochloric acid Ifom the salt-compoimd contributed... 

Ocular damaging and irritant agents can be identified and evaluated by the Draize rabbit test [114]. However, more recently this test has been criticized on the basis of ethical considerations and unreliable prognosis of human response. Alternative methods such as the evaluation of toxicity on ocular cell cultures have been recommended and are being indicated as promising prognostic tools [115-120]. Direct confocal microscopic analysis [121], hydration level of isolated corneas [122], and various other tests on isolated corneas or animal eyes have also been proposed for evaluation of ocular toxic effects. 

Eye Irritation. Because of the prospect of permanent blindness, ocular toxicity has long been a subject of both interest and concern. Although all regions of the eye are subject to systemic toxicity, usually chronic but sometimes acute, the tests of concern in this section are tests for irritancy of compounds applied topically to the eye. The tests used are all variations of the Draize test, and the preferred experimental animal is the albino rabbit. 

Due to the fact that the Draize test procedures can be very painful and result in visible suffering of the animals and trauma and reactions in the rabbit eyes, animal activists have often used this assay as a symbol for cruelty. In addition, the Draize test which was originally developed to predict the irritation potential of materials that come into contact with the eye and ocular adnexa such as ophthalmological preparations and cosmetics, has often been reported to present several drawbacks such as [18] ... 

Ocular administration of 1,1,1-trichloroethane caused only mild eye irritation in rabbits (Duprat et al. 1976 Krantz et al. 1959 Marzulli and Ruggles 1973 Torkelson et al. 1958). The study by Marzulli and Ruggles (1973) was a survey in which 10 laboratories conducted the Draize eye test in rabbits using 1,1,1-trichloroethane and reported little or no eye irritation. 

The last type of acute test we will discuss is the (in)famous Draize test. This is an acute test that evaluates potential eye irritation, and was developed for the cosmetic industry to ensure that products used in eye makeup would not irritate the eyes. In this test, the chemical is placed directly on the cornea of the test animal using an eyedropper. The scientist then observes the signs and describes any lesions seen in the cornea, iris, or elsewhere in the eye and surrounding tissue. The test is considered cruel by many because the effects of the chemical often include pain and swelling of the eyes. However, this is one situation where in vitro tests would not provide similar information because ocular inflammation and other signs would not be reproduced in a beaker. 

The conventional test for the irritant and corrosive potential of chemicals is the rabbit eye test, which was developed by Draize et al. (1944), and has become the international standard assay for acute eye irritation and corrosion (EC B.5, Directive 2004/73/EC OECD TG 405, 2002). The test material is applied to the conjunctival sac of the animal s eye and subsequent grading of ocular lesion is established cornea opacity, iris lesion, redness of conjunctivae, and oedema of conjunctivae (chemosis). 

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