Draiz method

Table 6 Skin irritation data for F1BCIDE-K58 (using the Draize method)".

The extent of the skin irritation caused the sample was evaluated using the Draize method [7] on the skin of rabbits. Each animal was tested on a patch of unbroken skin. Each test area was treated with 0.5 g of the test substance. The test areas were covered for four hours. Inspections of the test areas for signs of erythema... 

The Draize method has generally erred on the side of safety in that it over predicts the severity of skin damage produced by chemicals, thus producing a safety factor for those exposed. Some investigators report repeatedly that the test is not sensitive enough to separate mild from moderate irritants. Although Draize-type tests will be replaced by in vitro assays some time in the future, we have no validated in vitro substitute at present. 

Table 3 Comparison of skin irritation test based on the Draize method...

Two similar experimental procedures have been used to quantify the skin contact effects of soluble silicates both are based on the Draize method(27). The first is the protocol adopted by the U.S. Food and Drug Administration and Consumer Product Safety Commission for determining the contact hazard of substances under the Federal Hazardous Substances Act, and is specified in 16 C.F.R. i1300.41 et seq. The second, is the protocol adopted by the U.S. Department of Transportation for determining the contact hazard of substances under the Federal Hazardous Materials Transporation Act, and is specified in 49 C.F.R. A173.240. 

The effects of eye contact with sodium silicates have been tested by industry and in government laboratories. The standard test for determining the hazard of eye contact is the FHSA Draize method specified in 16 C.F.R. t1500.42. The data in Table VII. indicates that at the ratios and concentrations tested, soluble silicates are irritating to the eyes, and severely irritating at high concentrations. A new test for assessment of eye contact effects is currently under development. 

Guinea pig 0.5 Draize method, 10 times per day 10 (daily) 15 Nonirritating... 

Rabbit (female. New Zealand) 0.5 Draize method 1 3 Nonirritating... 

In the U.S. market, three traditional amphoterics are used in most mild shampoos disodium cocoamphodiacetate, sodium lauroamphoacetate, and disodium lauroamphodiacetate. The eye irritation properties of these three surfactants were compared under identical conditions, with solutions of each surfactant prepared at four active levels 5, 10, 15, and 25%. After the pH was adjusted to 7.0, the solutions were evaluated according to the Draize method [12,13]. 

There are many in vivo and in vitro approaches to assessing the irritation potential of surfactants. For overall results, in vivo applications and observations are the most useful and realistic measures. The Draize method has been utilized for many years for this purpose and been found useful for assessing irritation potential. Recently, it has become conttoversial to utilize the Draize method with animals because of concerns for animal welfare, despite its purpose of isolating caustic materials in order to avoid human exposure. Many in vitro methods have been developed recently that are useful alternatives to the animal tests and even as functional tools for analyzing the cause of irritation [13,14],... 

Animal tests were commonly used in the past, also according to the Draize protocol [357,372]. However, such tests have been questioned for several reasons. In addition to the public concern about animal welfare, animal tests are conducted under conditions and at concentrations quite different from those that occur in practice, as in the case of eye irritancy. The effects of surfactants on the skin cannot be compared when the results have been obtained through different protocols and even results obtained by the same method are not comparable if conditions differ. 

Van Paassen [57,67] reported a synergistic decrease of the skin and eye irritation level of sodium lauryl ether sulfate by combination with lauryl ether carboxylates. The investigations have been carried out using the Draize eye irritation test and human patch test (Tables 13 and 14). Furthermore, measurements by in vitro methods, the Zein test, and the red blood cell test show low to no irritancy [251-253]. 

In a broad evaluation also the sulfosuccinate disodium laureth sulfosuccinate (DLSS) was a part of a variety of surfactants tested for their dermatological mildness, and some different test methods were applied [16]. Products were compared applying in vitro methods (Zein test, hemolysis) and in vivo methods (Duhring-Chamber test, skin mildness by intracutaneous test on mice and topical application on hairless mice, mucous membrane irritation according to the Draize procedure on rabbit eyes). In the Duhring-Chamber test the DLSS elicited no reactions in the animal tests it ranged in the least irritant third of the 15 products tested. 

In Fig. 11 results are given for sulfosuccinate surfactants of different molecular structure tested according to the Draize skin irritation procedure. The test method defines test scores verbally as described in Table 21. The data in Fig. 11 prove the nonirritant character of sulfosuccinates tested at 10% concentration. Similar results were found for another group of sulfosuccinates [103]... 

Fig. 12). The same holds true for the eye irritation scores. In this case the test method of Draize was again applied. 

Dermal Absorption. To determine the toxicity of parathion following dermal application, the method of Draize, Woodard, and Calvery (3) was followed. Variables considered in the design of these experiments were concentration as a factor of area, solvent, exposure time, and number of exposures. In some cases the wettable powder was applied in the dry form, while in other cases sufficient water was added to produce a viscid paste. All doses in the table are presented as milligrams per kilogram of parathion, regardless of the concentration or solvent. 

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-... 

Draize, J. H. Woodward, G., and Calvery, H. O., Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes, J. Pharm. Exp. Ther., 82, 377, 1944. 

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. 

The test guideline methods use the scoring system developed by Draize (1944), see Tables 4.8 and 4.9. The EU criteria for classification are based on the mean tissue scores obtained over the first 24—72h period after exposure and on the reversibUity or irreversibility of the effects observed. 

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. 

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. 

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. 

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