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A Strategy for Skin Irritation Testing Michael K. Robinson and Mary A. Perkins Skin irritation safety testing and risk assessment for new products, and the ingredients they contain, is a critical requirement before market introduction. In the past, much of this skin testing required the use of experimental animals. However, new current best approaches for skin corrosion and skin irritation testing and risk assessment are being defined, obviating the need for animal test methods. Several in vitro skin corrosion test methods have been endorsed after successful validation and are gaining acceptance by regulatory authorities. In vitro test methods for acute, cumulative (repeat exposure), and chronic (prolonged exposure) skin irritation are under development. Though not yet validated, many are being used successfully for testing and risk assessment purposes as documented through an expanding literature. Likewise, a novel acute irritation patch test in human subjects is providing a valid and ethical alternative to animal testing for prediction of chemical skin irritation potential. An array of other human test methods also have been developed and used for the prediction of cumulative/chronic skin irritation and the general skin compatibility of finished products. The development of instrumental methods (e.g., transepidermal water loss, capacitance, and so on) has provided the means for analyzing various biophysical properties of human skin and changes in these properties caused by exposure to irritants. However, these methods do not directly measure skin inflammation. A recently introduced skin surface tape sampling procedure has been shown to detect changes in skin surface cytokine recovery that correlate with inflammatory skin changes associated with chemical irritant exposures or existing dermatitis. It holds promise for more objective quantification of skin irritation events, including subclinical (sensory) irritation, in the future. Copyright 2002, Elsevier Science (USA). All rights reserved.

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EFORE NEW PRODUCTS are introduced into the marketplace, the testing of these products and the ingredients they contain for potential adverse skin effects (irritation and allergy) is an essential element of the overall safety testing and risk assessment process.1-5 Procedures for the skin testing of new chemicals and finished products have been evolving in the face of technologic advancements and sociopolitical pressures. Traditionally, testing for skin corrosion and acute skin irritation (so-called hazard identification testing) has been conducted in animals. In 1944, Draize6 published a method for assessing skin corrosion and irritation hazard in rabbits which, although modified to varying degrees by regulatory authorities in different parts of the world7 became the world standard. To this day, different variations of this procedure (e.g., Organization of Economic Cooperation and Development [OECD] test guideline 404) form the basis for classification of skin corrosion and irritation hazard to man; however, the realization that animal tests for skin corrosion and irritation are rather poorly predictive for man,8,9 along with sociopolitical pressure to reduce or eliminate animal testing,10 has led to the emergence of new skin irritation testing and risk assessment strategies. This review summarizes a framework or strategy for skin irritation testing and risk assessment: highlighting in brief a stepwise approach that incorporates (1) the analysis of existing chemical data, (2) newly From the Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH. Address reprint requests to Michael K. Robinson, PhD, The Procter & Gamble Co, Miami Valley Laboratories, P.O. Box 538707, Cincinnati, OH 45253-8707. E-mail: [email protected] Copyright 2002, Elsevier Science (USA). All rights reserved. 1046-199X/02/1301-0006$35.00/0 doi:10.1053/ajcd.2002.30471

validated methods for in vitro skin corrosion testing, (3) developing methods for in vitro skin irritation testing, and (4) various human test methods for acute and cumulative/ chronic skin irritation and skin compatibility. Also described are newly emerging test procedures for the noninvasive assessment of skin inflammation.

The Testing Framework Figure 1 shows a schematic flow diagram of the steps in a skin irritation testing and risk assessment process. The steps mentioned above (i.e., existing data analysis, in vitro skin corrosion testing, in vitro skin irritation testing, and human testing) are listed in sequence along with decision points indicating either the continuation of the process or the decision to terminate or reformulate. A similar scheme has been published previously.11 However, the scheme shown in Figure 1 represents a unified, albeit generic, framework consistent with the strategies of multiple organizations.12

Steps in the Skin Irritation Testing and Risk Assessment Process Review Existing Data: Paper Toxicology

Prior to conducting any skin toxicity testing on a new ingredient, the toxicologist should first identify information that may exist already on the chemical. There are generally 3 types of data available. The first is pre-existing information from literature references, such as the skin irritation reference chemicals data bank,13 trade association archive data, national data archives (e.g., national hazardous substances database), or internal company databases.

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data sources on a chemical will be integrated into an initial risk assessment to determine whether it is acceptable to proceed directly to human exposure, whether the potential hazard is already known to be unacceptable, or whether additional test data are needed. Lastly, it is important to consider that skin safety testing and risk assessment is often a comparative toxicologic approach.1,3,4 That means that the assessment made for a new product and its ingredients can be compared with other similar products, those with a safe marketing pedigree as well as any product initiatives terminated because of unacceptable skin toxicity data.2 These comparisons can be invaluable in positioning the potential risk of new products and ingredients. Skin Corrosion Testing: A Shifting Paradigm

Figure 1. Flow diagram for a generic safety testing and risk assessment process for human skin corrosion and irritation.

The second source of information is in the form of physicochemical data on the substance of interest and any relevant quantitative structure-activity relationship (QSAR) information that might exist. Examples of physicochemical data are pH and acid/alkaline reserve, partition coefficient, melting point, and so on. Often these parameters are used to aid in the development of structure-activity relationships among related chemicals. Determination of potency profiles and specific relationships between structure and potency leads to development of quantitative structure-activity relationships or QSARs. An example is the comparison of skin corrosion data for fatty acids of differing alkyl chain lengths.14 The third source of information relevant to skin corrosion and irritation potential is that of exposure. Skin irritation potential is related not only to the inherent irritancy of the chemical (i.e., hazard), but also the nature of exposure to the chemical in the product context. Exposure assessment includes knowledge of concentration, duration, frequency, area of the body, exposure type (e.g., occluded or nonoccluded), and product type (e.g., rinse-off versus leave-on). For example, it may be possible to formulate corrosive chemicals into certain products at low, nonirritating concentrations, whereas even relatively mild irritants might be unacceptably irritating in high concentrations in products applied to and left on the skin. Each of the

Assuming that the evaluation of existing information does not permit human exposure without additional skin safety data, the first consideration is whether to conduct a skin corrosion test. As stated above, the Draize rabbit skin corrosion/irritation test has served as the basis for regulatory requirements pertaining to skin toxicology testing. First mandated in the United States by the Federal Hazardous Substance Act,7 multiple variations of the method exist worldwide. For skin corrosion testing, the speed of a corrosive response defines the appropriate packing group classification (I, II, or III) under regulation by the United States Department of Transportation and the appropriate hazard labeling requirements (i.e., risk phrase) under European Community directive. For such classification, lesions must penetrate into the dermis, must occur quickly (within 4 hours of exposure), and must be irreversible. Skin irritation (acute) testing involves the same duration or longer-term (24-hour) exposures over intact or preabraded skin. Resulting lesions are, by definition, less severe and reversible. The Draize test has come under increasing criticism for 2 main reasons, animal welfare concerns and predictive validity for the human response. Animal welfare organizations have targeted this and other tests as inhumane and unnecessary, leading to legislation (in Europe) that would ban the sale of products, the ingredients of which have been tested on animals.10 Also, the reproducibility and relevance of the Draize skin corrosion/irritation test results to human experience long have been questioned.15-18 This has led to a concerted effort to develop in vitro alternative test methods. The past 2 decades saw tremendous expenditure of effort on development of in vitro test methods for prediction of skin corrosion.14,19-28 This culminated in the successful validation of 2 methods, transcutaneous electrical resistance (TER) and viability assessment in the Episkin (Episkin SNC, Lyon, France) culture system, through an interlaboratory validation sponsored by the European Center for the Validation of Alternative Methods (ECVAM).29 More recently, a third test method, Corrositex (Corrositex,

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In Vitro International, Irvine, TX), was accepted by the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) as a valid test method for certain classes of chemicals.30 An additional skin culture method, EpiDerm (Mattek, Ashland, MA), successfully completed a catch-up validation process31,32 and also was endorsed by ECVAM33 as an alternative skin corrosion test method. Recently, the European Union adopted a new test method for skin corrosion assessment, incorporating the TER and generic human skin model assays under the dangerous substances directive.34 The human skin model was left as generic so as to avoid governmental endorsement of a commercial vendor; however, any human skin model used must be qualified as valid through a process equivalent to the ECVAM-sponsored validation effort. A draft guideline on in vitro methods for skin corrosion testing also is under review by member countries of the OECD. An example of the use of the EpiDerm culture system to discriminate between corrosive and noncorrosive test chemicals is shown in Figure 2A. Options for Skin Irritation Testing: In Vitro Systems

Assuming a test material clears corrosivity screening, various skin irritation test methods may be considered as alternatives to the Draize irritation test. In vitro methods for skin irritation testing are currently in active development but none are validated to date. There is an expanding list of publications highlighting the development of human skin culture methods for prediction of both acute and cumulative/chronic skin irritation potential.35-46 Recently, several test methods were enrolled in an ECVAM-sponsored prevalidation test for prediction of the acute irritation potential of chemicals.47 None of 5 test methods proved to be well developed enough to progress into a full validation study. Work was needed to improve interlaboratory reproducibility and/or predictiveness for all of the test methods.48 However, the lack of a formal validation for prediction of acute chemical irritation does not preclude the utility of different methods for internal testing and risk assessment for both chemicals and finished products.39,44-46 An example of the use of the EpiDerm culture system for prediction of the skin irritation of antiperspirant and deodorant products is shown in Figure 2B. In spite of the fact that the human data used for comparison was a relatively subjective ranking of irritancy based on diary records of reported skin complaints from a consumer test, the rank correlation of the in vitro and human data was quite good. This type of in vitro irritation testing can be valuable for screening of multiple chemicals or formulations to make initial decisions on materials to advance to more time consuming and costly human tests. Options for Skin Irritation Testing: Human Test Methods

Often overlooked in the push to develop in vitro methods for skin irritation testing has been the value of conducting

Figure 2. (A) Test substances were rank ordered based on the percent viability after a 3-minute exposure in the EpiDerm cultures. Any test material with cell viability ⬍ 50% after the 3-minute exposure is considered to be corrosive. Test materials marked with the asterisk (*) denote already classified (DOT) corrosive chemicals (From Perkins, et al99 with permission). (B) The percent of human subjects reporting either subjective or objective skin irritation for 11 antiperspirant/deodorant products in a consumer use study were plotted against the in vitro release of IL-1␣ from EpiDerm cultures exposed to the same products for 20-hours. Symbols represent the mean (⫾ SE) for 4 replicate cultures. Linear regression analysis was used to compare responses in the two studies. (From Perkins, et al45 with permission.)

ethical tests in volunteer human subjects. Human subjects long have been used for many types of skin compatibility tests. Examples of common direct chemical/formulation application procedures include extended duration or repeat application patch tests,49-54 repeat open application tests,55 cumulative irritation patch tests,56-58 and scarification tests.59 Also well documented are a variety of laboratory-controlled or in-home tests, such as forearm wash tests,60 hand immersion tests,61 exaggerated fabric rubbing tests,62 and extended duration product home use tests.63-65 Various methods applicable to compatibility testing of

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cosmetic ingredients and products have recently been summarized.66,67 Given the difficulty, noted above, in validating in vitro tests for acute skin irritation, it is important to recognize the recent development and application of a human patch test method for prediction of acute chemical skin irritation potential. The human 4-hour patch test for chemical skin irritation classification was first developed in the mid 1990s.68 The method was based on 3 basic premises (1) that, by definition, an irritation reaction produced in human skin is a valid response; (2) comparison of irritation responses by a test chemical versus a reference irritant would enable test chemical classification as irritating to skin (R38) or nonclassified; and (3) acceptability of the method would require that it be shown to be reliable in chemical irritation classification and that the procedure adhere to the highest ethical standards for the protection of volunteer human test subjects.69,70 The test method involves application of generally undiluted test chemicals under occluded patch conditions for up to 4 hours.68 Exposure time is ratcheted from 30 minutes or 1 hour through 2, 3, and 4 hours. Skin reactions are graded 24, 48, and 72 hours after patch removal using a simple scale (1⫹, 2⫹, 3⫹) of increasing erythema. Exposure to any test material is stopped after any indication (⫹ or greater) of a positive response. In practice, most responses observed are mild (1⫹). It is the incidence (not severity) of positive responses in a test population of approximately 30 subjects that determines irritation classification. Test chemical response incidences are compared with a positive control chemical (20% sodium dodecyl sulfate [SDS]). The concentration of 20% was selected as the concentration of SDS classified as R38 under the European Union dangerous substances directive. Test chemicals showing statistically greater or equal irritation compared with 20% SDS (using Fisher’s exact test), are classified as R38, whereas chemicals showing statistically less irritation are nonclassified. An example of such a comparative test is shown in figure 3. Figures 3A and 3B (data from 2 separate laboratories) provide the statistical comparison between the collective incidences of positive responses to each chemical. Figures 3C and 3D show the same data in a time-response curve format. The calculated TR50 values (time of exposure required for 50% of the subject population to respond positively) are not used for irritation classification but facilitate cross-study comparisons. The 4-hour patch test has undergone considerable intralaboratory and interlaboratory evaluation, showing excellent consistency.71-74 The small-scale interlaboratory study shown in Figure 3, compares the irritation potential of fatty acids of differing alkyl chain lengths.74 Not only were the statistical comparisons between the 2 laboratories consistent (Fig 3A and 3B), there was remarkable consistency in the comparative TR50 values (Fig 3C and 3D). As noted earlier, one of the criticisms of the Draize skin corrosion/irritation test has been the discrepancy seen

between the animal and human tests conducted on the same set of chemicals.15-18 This was confirmed in comparisons between human 4-hour patch test results on a set of 65 chemicals and their existing European Commission classifications.8 A breakdown of these results (Table 1) indicates the relatively poor predictive potential of the animal test. In addition to its use for chemical irritation classification, the human 4-hour patch test has shown utility in investigating basic parameters of the acute skin irritation response, including the effects of atopy,75 differences in skin reactivity based on skin type76 and race73,77 and interindividual78 and intra-individual79 variation in skin reactivity. Because this test was initially developed for irritation classification (so-called hazard identification) purposes, it has been criticized by some on ethical grounds and has not been formally accepted into testing guidelines. However, because the method relies on time of response (not severity of response) for irritation comparisons, the skin reactions produced are typically less severe than those commonly produced by other tests (including so-called confirmatory safety tests). The ethical considerations surrounding this test method have been thoroughly reviewed.9 Given the useful results generated to date from studies employing this method, both chemical classification data and basic skin reactivity data and the high ethical standards under which the procedure is conducted,9 there is much to recommend the human 4-hour patch test for continued development and use in skin irritation testing and risk assessment.

Future Advances in Skin Irritation Test Methodologies The advances in skin corrosion and irritation test methods described previously in this article today are enabling the dermatotoxicologist to conduct thorough testing and risk assessment programs without the need to rely on animal test procedures (at least when not required by regulation). However, the field of skin toxicology research is not standing pat. Further advances are on the horizon to expand our testing capabilities even more. Development and application of genomics and proteomics tools is affecting all research disciplines in the life sciences. One might reasonably consider the use of such expensive methods for simple skin corrosion or irritation screening to be analogous to swatting a fly with a sledgehammer, when simpler and cheaper methods are readily available; however, these tools are well suited for basic mechanistic skin research that could reveal novel targets for dermatotoxicology screening in the future. The discipline of neurosensory skin reactivity, or chemesthesis, is critically important in the overall safety assessment of skin products. Often consumer satisfaction with the products they buy and use is undermined, not by development of visible skin reactions, but by disagreeable

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Figure 3. (A,B) Patches containing the indicated test materials were applied for 0.5 to 4h, increasing the duration of exposure until a positive response was observed. The asterisk (*) or pound (#) symbols indicate a statistically significant differences (the Fisher exact test) at P ⬍ .05 relative to the response to 20% SDS at the same time point. Graph A represents a study conducted by P&G study and graph B represents a study conducted by Unilever. (C,D) The data shown in A and B were graphed using logistic curve fitting procedures and a TR50 value (time for 50% of test subjects to respond) was derived for each test material. Graph C is the P&G study and graph D is the Unilever study. Reprinted from Robinson, et al74 with permission.

Table 1. Correspondence Between European Classification of 65 Chemicals and Human Skin Irritation Potential Human 4h Patch Test Classification† Existing EC Classification* R34 R38 Nonclassified

R38

Nonclassified

3‡ 7 2§

1‡ 23‡ 29

*Based upon EC, Supplier, and Trade Association Data. †Based on results of human 4h patch test. ‡Bold values indicate over classification (EC) versus human 4h patch test. §Italicized value indicates under classification (EC) versus human 4h patch test. Reprinted from Robinson, et al9 with permission.

sensations such as stinging, burning, or itching. Various subjective methods have been introduced to try and study the phenomenon of sensory irritation.80-83 Application of the tools of neurophysiology has improved the ability to more objectively quantify this symptomatic data.84,85 The identification of biomarkers in skin as truly objective correlates of sensory skin irritation also is under investigation.86 Also, there have been steady advances in the development of instrumental methods for the noninvasive evaluation of skin biology.87 Most of the techniques developed in this discipline assess biophysical parameters of skin, such as barrier properties, water content, structural dynamics, and so on. What also has been needed is the ability to study inflammatory properties of skin by noninvasive means. Techniques such as skin biopsy, suction blister, and exten-

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Figure 4. Photographs of Sebutape application to various skin and mucosal sites.

sive tape stripping have been fruitful for basic studies of skin inflammation in small numbers of subjects.88-91 However, they are not particularly amenable to large-scale screening or studies in sensitive populations, such as infants. To meet this need, we have recently developed a truly noninvasive skin surface tape sampling procedure to quantify adsorbed inflammatory molecules from normal versus compromised skin.92 The method relies on the adsorption of skin surface inflammatory molecules onto the dermatologic tape, Sebutape (CuDerm, Dallas, TX), a material commonly used for sebum collection and quantification.93 This tape is minimally adhesive, removes few actual corneocytes (compared with cellophane tape) and can be applied to virtually any skin or mucosal surface (Fig 4). The tapes are left in place for only 1 minute, then removed and stored frozen until analysis. Molecules of interest are extracted in buffer and assayed with various methods of analysis. Inflammatory cytokines are assayed using commercial enzyme immunoassay kits. Results from the initial development and application of the method have recently been published.92 Two examples of the utility of the method for the study of diaper dermatitis in infants are shown in Figure 5. The analysis of interleukin-1␣ (IL-1␣) recovered from normal and compromised skin of the diaper area of infants is shown in Figure 5A. IL-1␣ is constitutively present in the stratum corneum,94 hence the recoverable levels from normal skin sites. However, 3 different types of compromised diaper area skin (diaper rash, erythema, and heat rash) all showed significantly increased recovery of IL-1␣ normalized to total recovered protein. In contrast to IL-1␣, interleukin-8 (IL-8) is an inducible chemokine not typically

detected in normal skin.89 When we compared inflamed diaper area skin with normal skin, only the inflamed skin showed detectable IL-8 levels. Though sporadic among the test subjects (Fig 5B), the difference versus normal skin was significant (Fig 5C). The variability was likely caused by a single sampling time point in this study relative to the individualized onset and resolution of the diaper rash among the test subjects. Though much remains to be learned about this method, we also have successfully applied the basic technique to the study of surfactant-induced irritation,92 gingivitis,95 and scalp disorders.96 As noted above, we also are examining its potential utility in detecting molecular markers that might correlate with neurosensory skin irritation.86 This and similar methods91,97,98 hold great promise for improving our ability to more objectively quantify various types of skin inflammation.

Summary Skin irritation testing and risk assessment procedures are rapidly evolving in the wake of methodologic advances as well as societal pressures to eliminate animal testing. Through a stepwise skin testing and risk assessment strategy (e.g., Fig 1), experienced dermatotoxicologists can today provide robust and defensible skin irritation assessments in support of new products and the ingredients contained therein5,11,12 without the need to conduct animal tests. Some of the procedures used are common sense (e.g., review of physicochemical properties and existing test data), some have been validated and are being accepted into testing guidelines (e.g., in vitro skin corrosion tests), and still others are in development (e.g., QSAR and in vitro

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Figure 5. (A) Sebutape samples were collected from infants (n ⫽ 13) participating in a clinical study. The IL-1␣ levels (mean ⫾ SE) were significantly increased (*P ⱕ .05; paired Student’s ttest) in compromised skin sites (⫹) over control sites (⫺). (B,C) Sebutape samples were collected from infant’s skin sites with different rash severity and from normal control leg sites. Individual levels of IL-8 for each subject (n ⫽ 28) for control and diaper rash sites are shown in (A). The normalized IL-8 values (B) were significantly higher (P ⱕ .05) in rash versus control sites. Reprinted from Perkins, et al92 with permission.

skin irritation tests). Regardless, most or all are used today in some fashion,12 and, with other newly emerging technologies84,92 and methods,9 will only enhance our testing and risk assessment capabilities in the years to come.

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4.

Acknowledgments The authors thank Dr. G. Frank Gerberick and Ms. Cindy A. Ryan for their thoughtful review and critique of the manuscript.

5. 6.

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References 1. Robinson MK, Stotts J, Danneman PJ et al: A risk assessment process for allergic contact sensitization. Food Chem Toxicol 27:479-489, 1989 2. Robinson MK, Parsell KW, Breneman DL, et al: Evaluation of the primary

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skin irritation and allergic contact sensitization potential of transdermal triprolidine. Fundam Appl Toxicol 17:103-119, 1991 Gerberick GF, Robinson MK, Stotts J: An approach to allergic contact sensitization risk assessment of new chemicals and product ingredients. Am J Contact Dermat 4:205-211, 1993 Gerberick GF, Robinson MK: A skin sensitization risk assessment approach for evaluation of new ingredients and products. Am J Contact Dermat 11:65-73, 2000 Robinson MK, Osborne R, Perkins MA: In vitro and human testing strategies for skin irritation. Ann NY Acad Sci 919:192-204, 2000 Draize JH, Woodard G, Calvery HO: Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J Pharm Exp Therap 82:377-390, 1944 Patil SM, Patrick E, Maibach HI: Animal, human, and in vitro test methods for predicting skin irritation, in Marzulli FN, Mailbach HI (eds): Dermatotoxicology Methods. Washington, DC, Taylor & Francis, 1998, pp 89-113 Basketter D, Gerberick F, Kimber I, et al: Toxicology of Contact Dermatitis: Allergy, Irritancy, and Urticaria. Chichester, UK, John Wiley and Sons, 1999, pp 1-180

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9. Robinson MK, McFadden JP, Basketter DA: Validity and ethics of the human 4-h patch test as an alternative method to assess acute skin irritation potential. Contact Dermatitis 45:1-12, 2001 10. EEC: Council Directive 93/35/EEC of 14 June 1993 amending for the 6th time Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products. Off J Eur Comm L15:32, 1993 11. Robinson MK, Osborne R, Perkins MA: Strategies for the assessment of acute skin irritation potential. J Pharmacol Toxicol 42:1-9, 1999 12. Robinson MK, Cohen C, de Brugerolle de Fraissinette A, et al: Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finish products. Food Chem Toxicol (in press) 13. Bagley DM, Gardner JR, Holland G, et al: Skin irritation: Reference chemicals data bank. Toxicol In Vitro 10:1-6, 1996 14. Whittle E, Barratt MD, Carter JA, et al: Skin corrosivity potential of fatty acids: In vitro rat and human skin testing and QSAR studies. Toxicol In Vitro 10:95-100, 1996 15. Phillips L, Steinberg M, Maibach HI, et al: A comparison of rabbit and human skin response to certain irritants. Toxicol Appl Pharmacol 21:369382, 1972 16. Nixon GA, Tyson CA, Wertz WC: Interspecies comparisons of skin irritancy. Toxicol Appl Pharmacol 31:481-490, 1975 17. Campbell RL, Bruce RD: Comparative dermatotoxicology. I. Direct comparison of rabbit and human primary skin irritation responses to isopropylmyristate. Toxicol Appl Pharmacol 59:555-563, 1981 18. Nixon GA, Bannan EA, Gaynor TW, et al: Evaluation of modified methods for determining skin irritation. Regul Toxicol Pharmacol 12:127-136, 1990 19. Oliver GJ, Pemberton MA: An in vitro epidermal slice technique for identifying chemicals with potential for severe cutaneous effects. Food Chem Toxicol 23:229-232, 1985 20. Oliver GJ, Pemberton MA, Rhodes C: An in vitro skin corrosivity test— modifications and validation. Food Chem Toxicol 24:507-512, 1986 21. Oliver GJA, Pemberton MA, Rhodes C: An in vitro model for identifying skin-corrosive chemicals: I. Initial validation. Toxicol In Vitro 2:7-18, 1988 22. Barlow A, Hirst R, Pemberton MA, et al: Refinement of an in vitro test for the identification of skin corrosive chemicals. Toxicol Methods 1:106-115, 1991 23. Botham PA, Hall TJ, Dennett R, et al: The skin corrosivity test in vitro: Results of an inter-laboratory trial. Toxicol In Vitro 6:191-194, 1992 24. Lewis RW, Botham PA: Measurement of transcutaneous elecrical resistance to assess the skin corrosivity potential of chemicals, in Rougier A, et al (eds): In Vitro Skin Toxicology. New York, NY, Mary Ann Liebert, 1994, pp 161-169 25. Basketter DA, Whittle E, Chamberlain M: Identification of irritation and corrosion hazards to skin: An alternative strategy to animal testing. Food Chem Toxicol 32:539-542, 1994 26. Gordon VC, Harvell JD, Maibach HI: Dermal corrosion, the CORROSITEX system: A DOT accepted method to predict corrosivity potential of test materials, in Rougier A, et al (eds): In Vitro Skin Toxicology. New York, NY, Mary Ann Liebert, 1994, pp 37-45 27. Cassidy SL, Stanton ES: In vitro skin irritation and corrosivity studies on organosilicon compounds. J Toxicol-Cutan Ocular Toxicol 15:355-367, 1996 28. Perkins MA, Osborne R, Johnson GR: Development of an in vitro method for skin corrosion testing. Fundam Appl Toxicol 31:9-18, 1996 29. Fentem JH, Archer GEB, Balls M, et al: The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the management team. Toxicol In Vitro 12:483-524, 1998 30. Scala R, Fentem JH, Chen J et al: Corrositex: An In Vitro Test Method for Assessing Dermal Corrosivity Potential of Chemicals. Available at: http:// iccvam.niehs.nih.gov/corprep.htm 1999. 31. Balls M, Fentem JH: The validation and acceptance of alternatives to animal testing. Toxicol In Vitro 13:837-846, 1999 32. Liebsch M, Traue D, Barrabas C, et al: The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity testing. ATLA-Altern Lab Anim 28:371-401, 2000

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