Animal Models Draize Rabbit Models

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The Draize model [2] and its modifications are commonly used to assay skin irritation using albino rabbits. Various governmental agencies have adopted these methods as standard test procedure. The procedure adopted in the U.S. Federal Hazardous Substance Act (FHSA) is described in Tables 2 and 3 [3,4,5]. Table 4 compares this method some other modifications of the Draize model.

Draize used this scoring system to calculate the primary irritation index (PII). This is calculated by averaging the erythema scores and the edema scores of all sites (abraded and nonabraded). These two averages are then added together to give the PII value. A value of less than 2 was considered nonirritating, 2 to 5 mildly irritating, and greater than 5 severely irritating. A value of 5 defines an irritant by Consumer Product Safety Commission (CPSC) standards. Subsequent laboratory and clinical experience has shown the value judgments (i.e., non-, mild, and severely irritating) proposed in 1944 requires clinical judgment and perspective, and should not be viewed in an absolute sense. Many materials irritating to the rabbit may be well tolerated by human skin.

Table 2 Draize-FHSA Model

Number of animals 6 albino rabbits (clipped)

2 X 1 inch2 sites on dorsum

One site intact, the other abraded, e.g., with hypodermic needle Applied undiluted to both test sites Liquids: 0.5 mL Solids/semisolids: 0.5g 1 inch2 surgical gauze over each test site Rubberized cloth over entire trunk 24 hours 24 and 72 hours Visual scoring system

Table 3 Draize-FHSA Scoring System


Erythema and eschar formation 0 No erythema

Very slight erythema (barely perceptible) 1

Well-defined erythema 2

Moderate to severe erythema 3

Severe erythema (beet redness) to slight eschar for- 4 mation (injuries in depth) Edema formation

No edema 0

Very slight edema (barely perceptible) 1

Slight edema (edges of area well defined by definite 2 raising)

Moderate edema (raised >1 mm) 3

Severe edema (raised >1 mm and extending beyond 4 the area of exposure)

Although the Draize scoring system does not include vesiculation, ulceration, and severe eschar formation, all of the Draize-type tests are used to evaluate corrosion as well as irritation. When severe and potentially irreversible reactions occur, the test sites are further observed on days 7 and 14, or later if necessary.

Modifications to the Draize assay have attempted to improve its prediction of human experience. The model is criticized for inadequately differentiating between mild and moderate irritants. However, it serves well in hazard identification, often overpredicting the severity of human skin reactions [5]. Therefore, Draize assays continue to be recommended by regulatory bodies for drugs and industrial chemicals.

Cumulative Irritation Assays

Several assays study the effects of cumulative exposure to a potential irritant. Justice et al. [6] administered seven applications of surfactant solutions at 10-minute intervals to the clipped dorsum of albino mice. The test site was occluded with a rubber dam to prevent evaporation and the skin was examined microscopically for epidermal erosion.

Frosch et al. [7] described the guinea pig repeat irritation test (RIT) to evaluate protective creams against the chemical irritants sodium lauryl sulfate (SLS), sodium hydroxide (NaOH), and toluene. The irritants were applied daily for 2 weeks to shaved back skin of young guinea pigs. Barrier creams were applied to the test animals 2 hours before and immediately after exposure to the irritant. Control animals were treated with the irritant only. Erythema was measured visually, and by bioengineering methods: laser doppler flowmetry and transepidermal water loss. One barrier cream was effective against SLS and toluene, whereas the other tested was not. In a follow-up study, another allegedly protective cream failed to inhibit irritation caused by SLS and toluene and exaggerated irritation to NaOH, contrary to its recommended use [8]. The RIT is proposed as an animal model to test the efficacy of barrier creams, and a human version, described below, has also been proposed.

Table 4 Examples of Modified Draize Irritation Method






Number of animals










2 of each


Dose liquids

0.5 mL undiluted

0.5 mL undiluted

0.5 mL

0.5 mL undiluted

0.5 mL

Dose solids in solvent

0.5 g

0.5 g moistened

0.5 g moistened

0.5 g

0.5 g

Exposure period (h)






Examination (h)

24, 72

24, 72

4, 48

0.5, 1, 24, 48, 72

0.5, 1, 24, 48, 72

Removal of test materials

Not specified

Not specified

Skin washed

Skin wiped

Skin washed

Excluded from testing

Toxic materials pH <2

Toxic materials pH <2

or >11.5

or >11.5

Abbreviations: FHSA, Federal Hazardous Substance Act; DOT, Department of Transportation; FIFRA, Federal Insecticide, Fungicide and Rodenticide Act; OECD, Organization for Economic Cooperation and Development. Source: Ref. 4.

Abbreviations: FHSA, Federal Hazardous Substance Act; DOT, Department of Transportation; FIFRA, Federal Insecticide, Fungicide and Rodenticide Act; OECD, Organization for Economic Cooperation and Development. Source: Ref. 4.

Copyright © Marcel Dekker, Inc. All rights reserved.

Repeat application patch tests have been developed to rank the irritant potential of products. Putative irritants are applied to the same site for 3 to 21 days, under occlusion. The degree of occlusion influences percutaneous penetration, which may in turn influence the sensitivity of the test. Patches used vary from Draize-type gauze dressings to metal chambers. Therefore, a reference irritant material is often included in the test to facilitate interpretation of the results. Various animal species have also been used, such as the guinea pig and the rabbit [9,10]. Wahlberg measured skinfold thickness with Harpenden calipers to assess the edema-producing capacity of chemicals in guinea pigs. This model showed clear dose-response relationships and discriminating power, except for acids and alkalis where no change in skinfold thickness was found.

Open application assays are also used for repeat irritation testing. Marzulli and Maibach [11] described a cumulative irritation assay in rabbits that uses open applications and control reference compounds. The test substances are applied 16 times over a 3-week period and the results are measured with a visual score for erythema and skin thickness measurements. These two parameters correlated highly. A significant correlation was also shown between the scores of 60 test substances in the rabbit and in man, suggesting that the rabbit assay is a powerful predictive model.

Anderson et al. [12] used an open application procedure in guinea pigs to rank weak irritants. A baseline response to SLS solution was obtained after 3 applications per day for 3 days to a 1 cm2 test area. This baseline is used to compare other irritants, of which trichloroethane was the most irritant, similar to 2% SLS. Histology showed a mononuclear dermal inflammatory response.

Immersion Assay

The guinea pig immersion assay was developed to assess the irritant potential of aqueous surfactant-based solutions, but might be extended to other occupational settings such as aqueous cutting fluids. Restrained guinea pigs are immersed in the test solution while maintaining their head above water. The possibility of systemic absorption of a lethal dose restricts the study to products of limited toxic potential. Therefore, the test concentration is usually limited to 10%.

Ten guinea pigs are placed immersed in a 40°C solution for 4 hours daily for three days. A comparison group is immersed in a reference solution. Twenty-four hours after the final immersion, the animals' flanks are shaved and evaluated for erythema, edema, and fissures [13,14,15,16]. Gupta et al. [17] concomitantly tested the dermatotoxic effects of detergents in guinea pigs and humans, using the immersion test and the patch test, respectively. Epidermal erosion and a 40 to 60% increase in the histamine content of the guinea pig skin was found, in addition to a positive patch test reaction in seven of eight subjects.

Mouse Ear Model

Uttley and Van Abbe [18] applied undiluted shampoos to one ear of mice daily for four days, visually quantifying the degree of inflammation as vessel dilatation, erythema, and edema. Patrick and Maibach [19] measured ear thickness to quantify the inflammatory response to surfactant-based products and other chemicals. This allowed quantification of dose-response relationships and comparison of chemicals. Inoue et al. [20] used this model to compare the mechanism of mustard oil-induced skin inflammation to the mechanism of capsaicin-induced inflammation. Mice were pretreated with various receptor an-

tagonists, such as 5-HT2, H1; and tachykinin antagonists, showing that the tachykinin NK1 receptor was an important mediator of inflammation induced by mustard oil. The mouse models provide simplicity and objective measurements. Relevance for man requires elucidation.

Other Methods

Several other assays of skin irritation have been suggested. Humphrey [21] quantified the amount of Evans blue dye recovered from rat skin after exposure to skin irritants. Trush et al. [22] used myeloperoxidase in polymorphonuclear leukocytes as a biomarker for cutaneous inflammation.


Human models for skin irritation testing are species relevant, thereby eliminating the precarious extrapolation of animal and in vitro data to the human setting. As the required test area is small, several products or concentrations can be tested simultaneously and compared. Inclusion of a reference irritant substance facilitates interpretation of the irritant potential of the test substances. Prior animal or in vitro studies, depending on model relevance and regulatory issue, can be used to exclude particularly toxic substances or concentrations before human exposure.

Single-Application Patch Testing

The National Academy of Sciences (NAS) [23] outlined a single-application patch test procedure determining skin irritation in humans. Occlusive patches may be applied to the intrascapular region of the back or the volar surface of the forearms, using a relatively nonocclusive tape for new or volatile materials. More occlusive tapes or chambers generally increase the severity of the responses. A reference material is included in each battery of patches.

The exposure time may vary to suit the study. NAS suggests a 4-hour exposure period, although it may be desirable to test new or volatile materials for 30 minutes to 1 hour. Studies longer than 24 hours have been performed. Skin responses are evaluated 30 minutes to 1 hour after removal of the patch, using the animal Draize scale (Table 2) or similar. Kligman and Wooding [24] described statistical analysis on test data to calculate the IT50 (time to produce imitation in 50% of the subjects) and the ID50 (dose required to produce irritation in 50% of the subjects after a 24-hour exposure).

Robinson et al. [25] suggested a 4-hour patch test as an alternative to animal testing. Assessing erythema by visual scoring, they tested a variety of irritants on Caucasians and Asians. A relative ranking of irritancy was obtained using 20% SLS as a benchmark. |

Taking this model further, McFadden et al. [26] investigated the threshold of skin irritation §

in the six different skin types. Again using SLS as a benchmark, they defined the skin

irritant threshold as the lowest concentration of SLS that would produce skin irritation under the 4-hour occluded patch conditions. They found no significant difference in irritation between the skin types.

Cumulative Irritation Testing

Lanman et al. [27] and Phillips et al. [9] described a cumulative irritation assay, which s has become known as the ''21-day'' cumulative irritation assay. The purpose of the test 2

was to screen new formulas before marketing. A 1 inch square of Webril was saturated with liquid of 0.5 g of viscous substances and applied to the surface of the pad to be applied to the skin. The patch was applied to the upper back and sealed with occlusive tape. The patch is removed after 24 hours, and then reapplied after examination of the test site. This is repeated for 21 days and the IT50 can then be calculated. Note that the interpretation of the data is best done by comparing the data to an internal standard for which human clinical experience exists.

Modifications have been made to this method. The chamber scarification test (see the following) was developed to predict the effect of repeated applications of a potential irritant to damaged skin, rather than healthy skin. The cumulative patch test described above had failed to predict adverse reactions to skin damaged by acne or shaving, or sensitive areas such as the face [28].

Wigger-Alberti et al. [29] compared two cumulative models by testing skin reaction to metalworking fluids (MWF). Irritation was assessed by visual scoring, transepidermal water loss, and chromametry. In the first method, MWF were applied with Finn Chambers on the volunteers' midback, removed after 1 day of exposure, and reapplied for a further 2 days. In the second method, cumulative irritant contact dermatitis was induced using a repetitive irritation test for 2 weeks (omitting weekends) for 6 hours per day. The 3-day model was preferred because of its shorter duration and better discrimination of irritancy. For low-irritancy materials in which discrimination is not defined with visual and palpatory scores, bioengineering methods (i.e., transepidermal water loss) may be helpful.

The Chamber Scarification Test

This test was developed [30,31] to test the irritant potential of products on damaged skin. Six to eight 1 mm sites on the volar forearm were scratched eight times with a 30-gauge needle without causing bleeding. Four scratches were parallel and the other four are perpendicular to these. Duhring chambers, containing 0.1 g of test material (ointments, creams, or powders), were then placed over the test sites. For liquids, a fitted pad saturated (0.1 mL) may be used. Chambers containing fresh materials are reapplied daily for 3 days. the sites are evaluated by visual scoring 30 minutes after removal of the final set of chambers. A scarification index may be calculated if both normal and scarified skin are tested to reflect the relative degree of irritation between compromised and intact skin; this is the score of scarified sites divided by the score of intact sites. However, the relationship of this assay to routine use of substances on damaged skin remains to be established. Another compromised skin model, the arm immersion model of compromised skin, is described in the following immersion tests section.

The Soap Chamber Test

Frosch & Kligman [32] proposed a model to compare the potential of bar soaps to cause ''chapping.'' Standard patch testing was able to predict erythema, but unable to predict the dryness, flaking, and fissuring seen clinically. In this method, Duhring chambers fitted with Webril pads were used to apply 0.1 mL of an 8% soap solution to the human forearm. The chambers were secured with porous tape, and applied for 24 hours on day 1. On days 2 to 5, fresh patches were applied for 6 hours. The skin is examined daily before patch application and on day 8, the final study day. No patches are applied after day 5. Applica

tions were discontinued if severe erythema was noted at any point. Reactions were scored on a visual scale of erythema, scaling, and fissures. This test correlated well with skin-washing procedures, but tended to overpredict the irritancy of some substances [33].

Immersion Tests

These tests of soaps and detergents were developed in order to improve irritancy prediction by mimicking consumer use. Kooyman & Snyder [34] describe a method in which soap solutions of up to 3% are prepared in troughs. The temperature was maintained at 105°F while subjects immersed one hand and forearm in each trough, comparing different products (or concentrations). The exposure period ranged from 10 to 15 minutes, three times each day for 5 days, or until irritation was observed in both arms. The antecubital fossa was the first site to show irritation, followed by the hands [6,34]. Therefore, antecubital wash tests (see the following) and hand immersion assays were developed [5].

Clarys et al. [35] used a 30-minute/4-day immersion protocol to investigate the effects of temperature as well as anionic character on the degree of irritation caused by detergents. The irritation was quantified by assessment of the stratum corneum barrier function (transepidermal water loss), skin redness (a* color parameter), and skin dryness (capacitance method). Although both detergents tested significantly affected the integrity of the skin, higher anionic content and temperature, respectively, increased the irritant response.

Allenby et al. [36] describe the arm immersion model of compromised skin, which is designed to test the irritant or allergic potential of substances on damaged skin. Such skin may show an increased response, which may be negligible or undetectable in normal skin. The test subject immersed one forearm in a solution of 0.5% sodium dodecyl sulfate for 10 minutes, twice daily until the degree of erythema reached 1 to 1+ on visual scale. This degree of damage corresponded to a morning's wet domestic work. Patch tests of various irritants were applied to the dorsal and volar aspects of both the pretreated and untreated forearms, and also to the back. Each irritant produced a greater degree of reaction on the compromised skin.

Wash Tests

Hannuksela and Hannuksela [37] compared the irritant effects of a detergent in use testing and patch testing. In this study of atopic and nonatopic medical students, each subject washed the outer aspect of the one forearm with liquid detergent for 1 minute, twice daily for 1 week. Concurrently, a 48-hour chamber patch test of five concentrations of the same detergent was performed on the upper back. The irritant response was quantified by bioengineering techniques: transepidermal water loss, electrical capacitance, and skin blood flow. In the wash test, atopics and nonatopics developed irritant contact dermatitis equally, whereas atopics reacted more readily to the detergent in chamber tests. The disadvantage of the chamber test is that, under occlusion, the detergent can cause stronger irritation than it would in normal use [38]. Although the wash test simulates normal use of the product being tested, its drawback is a lack of standard guidelines for performing the test. Charbonnier et al. [39] included squamometry in their analysis of a hand-washing model of subclinical irritant dermatitis with SLS solutions. Squamometry showed a significant

difference between 0.1 and 0.75% SLS solutions whereas visual, subjective, capacitance, transepidermal water loss, and chromametry methods were unable to make the distinction. Charbonnier suggests squamometry as an adjunct to the other bioengineering methods.

Frosch [33] describes an antecubital washing test to evaluate toilet soaps, using two washing procedures per day. Simple visual scoring of the reaction (erythema and edema) allows products to be compared. This comparison can be in terms of average score, or number of washes required to produce an effect.

Assessing Protective Barriers

Zhai et al. [40] proposed a model to evaluate skin protective materials. Ten subjects were exposed to the irritants SLS and ammonium hydroxide (in urea), and Rhus allergen. The occluded test sites were on each forearm, with one control site on each. The irritant response was assessed visually using a 10-point scale, which included vesiculation and maceration unlike standard Draize scales. The scores were statistically analyzed for non-parametric data. Of the barrier creams studied, paraffin wax in cetyl alcohol was found to be the most effective in preventing irritation.

Wigger-Alberti and Elsner [41] investigated the potential of petrolatum to prevent epidermal barrier disruption induced by various irritants in a repetitive irritation test. White petrolatum was applied to the backs of 20 human subjects who were exposed to SLS, NaOH, toluene, and lactic acid. Irritation was assessed by transepidermal water loss and colorimetry in addition to visual scoring. It was concluded that petrolatum was an effective barrier cream against SLS, NaOH, and lactic acid, and moderately effective against toluene.

Frosch et al. [42] adapted the guinea pig RIT previously described for use in humans. Two barrier creams were evaluated for their ability to prevent irritation to SLS. In this repetitive model, the irritant was applied to the ventral forearm, using a glass cup, for 30 minutes daily for 2 weeks. One arm of each subject was pretreated with a barrier cream. As in the animal model, erythema was assessed by visual scoring, laser doppler flow, and transepidermal water loss. Skin color was also measured by colorimetry (La* value). The barrier cream decreased skin irritation to SLS, the most differentiating parameter being transepidermal water loss and the least differentiating being colorimetry.

Bioengineering Methods in Model Development

Many of the models previously described do not use the modern bioengineering techniques available, and therefore data based on these models may be imprecise. Despite the investigations skill, subjective assessment of erythema, edema, and other visual parameters may lead to confounding by inter and intraobserver variation. Although the eye may be more sensitive than current spectroscopy and chromametric techniques, the reproducibility and increased statistical power of such data may provide greater benefit. A combination of techniques, such as transepidermal water loss, capacitance, ultrasound, laser doppler flowmetry, spectroscopy, and chromametric analysis, in addition to skilled observation may increase the precision of the test. Andersen and Maibach [43] compared various bioengineering techniques, finding that clinically indistinguishable reactions induced significantly different changes in barrier function and vascular status. An outline of many of these techniques is provided by Patil et al. [5].

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