Draize test—

TABLE 12 Influence of the Ethoxylation Degree on the Eye Irritation (Draize test) of Sodium Lauryl Polyglycol Ether Carboxylates [R0(CH2CH20)xCH2C00Na]... 

TABLE 15 Eye Irritation According to the Draize Test of Alkyl Ether Carboxylates Compared to Other Mild Cosurfactants ... 

The potential of diisopropyl methylphosphonate to cause eye irritation was evaluated with the Draize Test (Hart 1976). The compound was directly applied to the conjunctival sac of one eye in each of nine New Zealand White rabbits. Significant irritation of the conjunctivae was observed in all rabbits, and the comeal surface was characterized by a diffuse opacity. The opacity was temporary and cleared within 8 days. Irrigation with lukewarm water following application of diisopropyl methylphosphonate reduced but did not prevent irritation (Hart 1976). 

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-... 

The Irritation module estimates the potential of selected compound to cause eye or skin irritation in standard rabbit Draize test [64]. 

Booman, K.A., DeProspo, J., Demetrulias, J., Diedger, A., Griffith, J.F., Grochosky, G., Kong, B., McCormick, W.C., North-Root, H., Rozen, M.G., and Sedlak, R. I. (1989). The SDA alternatives program Comparison of in vitro data with Draize test data. J. Toxicol.—Cut. Ocular Toxicol. 8 35 19. 

Opacity. Corneal opacity is the most heavily weighted of the components of the Draize eye score (80 out of 110 possible points) (Conquet et al., 1977). Thus, an in vitro system that provides an accurate measure of opacification should contribute substantially toward in vitro modeling of the classical Draize test. Two assays attempting to model this process are discussed. 

If animal testing is required, a full-scale Draize test may not be necessary given the background established in the beginning of the tier approach. For instance, the compound could be tested in a single sentinel animal to obtain confirmation of in vitro data. In addition, other modifications could be used, such as the administration of appropriate anesthetics to the test animals, or the use of the low-volume Draize modification (Falahee et al., 1982 Freeberg et al., 1984 Griffith, 1987). 

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. 

In another approach, Parnigotto and coworkers reconstructed corneal structures in vitro by using corneal stroma containing keratocytes to which corneal epithelial cells from bovine primary cultures were overlaid [73], However, this particular corneal model did not contain an endothelial layer. This model was histochemically characterized and the toxicity of different surfactants was tested using MTT methods. This stroma-epithelium model has been reported to show a cornea-like morphology, where a multilayered epithelial barrier composed of basal cells (of a cuboidal shape) and superficial cells (of a flattened shape) is noted. Furthermore, the formation of a basement membrane equivalent and expression of the 64-kDa keratin were reported, indicating the presence of differentiated epithelial cells. The toxicity data for various surfactants obtained with this model correlate well with those seen by the Draize test [73], However, this corneal equivalent was not further validated or used as a model for permeation studies. 

As can be seen above, it has been shown that cultivation of 3-D in vitro models of animal and human cornea is possible and the model resembles the in vivo cornea of the animal and human. In order to use these cornea constructs as models for in vitro drug absorption studies and alternative to the Draize test, further... 

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. 

No effeets were reported in rabbits when 100 mg of dichlorobenzidine (free base) was plaeed in the conjunctival sac of the eye (Gerarde and Gerarde 1974). It should be noted that the authors did not report the duration of exposure or the vehicle used. However, 0.1 mL of 3,3 -dichlorobenzidine dihydrochloride in a 20% com oil suspension produced eiythema, pus, and comeal opaeity, 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). This response is very likely associated with the release of hydrochloric acid following the salt s contact with the moist surface of the eye. 

In the Draize test, a single dose of 0.1 mL or 0.1 g is introduced into the conjunctival sac of the right eye, the left eye acting as a control. The reactions of the conjunctivae, iris and cornea are scored for irritancy at approximately 1, 3, 8, 24, 48 and 72 h and again at 7 days after dosing. Test materials shown to be severe skin irritants or that are below pH 2 or above pH 11 are not tested but are assumed to be eye irritants. 

The use of the Draize tests has been receiving attention for a number of years because of animal welfare considerations. Consequently, the modifications of the existing protocol and the development of alternative methods have been extensively examined by the cosmetic and chemical industry to reduce animal usage and the occurrence of severe reactions. One modification of this model uses reduced volumes of 0.01 mL and 0.01 g, which reduces severe reactions but does not compromise the predictive value of the test. 

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. 

Attempts to solve the dilemma have taken two forms to find substitute in vitro tests and to modify the Draize test so that it becomes not only more humane but also more predictive for humans. Substitute tests consist of attempts to use cultured cells or eyes from slaughtered food animals, but neither method is yet acceptable as a routine test. Modifications consist primarily of using fewer animals. Usually one animal is tested first and, if the material is severely irritating no further eye testing is conducted. EPA has reduced the required number of animals from 6 to 3. In addition eye irritation should never be carried out on materials with a pH of less than 2 or more than 10 as these materials can be assumed to be potential eye irritants. 

Objective irritation is defined as nonimmunologically mediated, localized inflammation of the skin, usually resulting from contact with a substance that chemically damages the skin.2,9 The exact mechanism is unknown, and it is likely that both endogenous and exogenous factors are involved. In vivo predictive testing in animals (e.g., modified Draize test, repeated application patch tests,... 

A number of simulations were performed to assess the effects of applying the different combinations of the three alternative tests before the Draize skin corrosion test. Each combination of alternative tests is referred to hereafter as a different sequence. Specifically, assessments were made of the sequences applied before the Draize test ... 

Chemical Step 1 Step 2 Step 3 Draize Test... 

The possible outcomes obtained when the 4 sequences of alternative tests and the Draize test are applied to the data for the 51 chemicals are summarized in Table 18.8, along with the outcome of applying just one in vitro test (the EPISKIN test) before the Draize test. For each sequence, Table 18.8 gives the number of chemicals that enter each step, the distribution of these chemicals in terms of their known corrosion potential (C or NC), the number of positive predictions (i.e., chemicals for which no further assessment is made), and the numbers of true and false positives. 

The numbers of false negatives should also equal the number of chemicals identified as corrosive by the Draize test (Table 18.8). 

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