Rosemarie Osborne

A proposed eye irritation testing strategy to reduce and replace in vivo studies using Bottom–Up and Top–Down approaches

In spite of over 20 years of effort, no single in vitro assay has been developed and validated as a full regulatory replacement for the Draize Eye Irritation test. However, companies have been using in vitro methods to screen new formulations and in some cases as their primary assessment of eye irritation potential for many years. The present report shows the outcome of an Expert Meeting convened by the European Centre for the Validation of Alternative Methods in February 2005 to identify test strategies for eye irritation. In this workshop test developers/users were requested to nominate methods to be considered as a basis for the identification of such testing strategies. Assays were evaluated and categorized based on their proposed applicability domains (e.g., categories of irritation severity, modes of action, chemical class, physicochemical compatibility). The analyses were based on the data developed from current practice and published studies, the ability to predict depth of injury (within the applicable range of severity), modes of action that could be addressed and compatibility with different physiochemical forms. The difficulty in predicting the middle category of irritancy (e.g. R36, GHS Categories 2A and 2B) was recognized. The testing scheme proposes using a Bottom-Up (begin with using test methods that can accurately identify non-irritants) or Top-Down (begin with using test methods that can accurately identify severe irritants) progression of in vitro tests (based on expected irritancy). Irrespective of the starting point, the approach would identify non-irritants and severe irritants, leaving all others to the (mild/moderate) irritant GHS 2/R36 categories.

Xenobiotic metabolism gene expression in the EpiDerm™ in vitro 3D human epidermis model compared to human skin

There is an urgent need to validate in vitro human skin models for use in safety testing. An important component of validation is characterizing the metabolizing capacity of these models. We report comparison of the expression of 139 genes encoding xenobiotic metabolizing enzymes in the EpiDerm model and human skin. In microarray analysis, the expression of 87% of the genes was consistent between the EpiDerm model and human skin indicating the presence of similar metabolic pathways suggesting commonality in function. Analysis of EpiDerm models constructed from four donors showed highly comparable expression of xenobiotic metabolizing genes demonstrating reproducibility of the model. Overall, the expression of Phase II enzymes appeared to be more pronounced in human skin and the EpiDerm model than that of Phase I enzymes, consistent with the role of skin in detoxification of xenobiotics. Though the basal expression of CYPs in particular was low in EpiDerm, significant induction of CYP1A1/1B1 activity was observed following treatment with 3-methylcholanthrene. These results indicate that the xenobiotic metabolizing capacity of the EpiDerm model appears to be representative of human skin. Models such as EpiDerm provide a valuable in vitro approach for evaluation of metabolism and toxicity of cutaneous exposures to xenobiotics.

Development and Intralaboratory Evaluation of an in Vitro Human Cell-Based Test to Aid Ocular Irritancy Assessments

A human cell-based in vitro method was developed to screen for ocular irritancy potential of aqueous compatible and incompatible test agents, such as liquids, insoluble solids, powders, granulars, emulsions, and acids/alkalis. Methods were developed for topical application (an exposure that mimics in vivo testing) and wash-off of test substances on the epithelial surface of human skin derived epithelial-fibroblast cocultures (Skin2 Model ZK1200 from Advanced Tissue Sciences). These cultures contain noncornified stratified squamous epithelium, providing a three-dimensional in vitro model that resembles noncornified mucosal epithelium, such as cornea and conjunctiva. The hypothesis tested and confirmed in this work was that the rate of cytotoxicity induced by topical application of test substances to the stratified epithelial cell cultures would correlate with ocular irritancy. Test substances were applied to the cell system for up to 30 min, and cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) vital dye metabolism. The time (in minutes) of exposure to test agent that reduced MTT metabolism to 50% of control levels (the t50 value) was calculated for each test substance, and these values showed a good correlation (r = 0.87) with historic rabbit low-volume eye test maximum average scores (MAS) for a range of liquid, solid, granular, powder, and other test materials, including consumer product formulations and ingredient chemicals. Additionally, t50 values confirmed the mild nature of selected cosmetic formulations. Taken together, these data indicate that this in vitro test, termed the tissue equivalent assay, is a valuable tool to screen for irritancy of test substances to mucosal tissues, such as cornea and conjunctiva.

In vitro skin irritation testing with human skin cell cultures

Cultured human skin cells are a potentially useful model for skin irritancy testing. We have evaluated the effects of chemical irritants on human epidermal keratinocytes (NHEK) and on keratinocyte-dermal fibroblast (NHEK/DF) co-cultures. Cell viability in NHEK cultures, measured as incorporation of the vital dye neutral red (NR), was reduced in a dose-dependent manner in response to the chemical irritants tested. The half-maximal effective concentration (NR(50)) values correlated with irritation scores in human patch tests with these materials. Certain materials were found to be incompatible with this test system. NHEK/DF cultures were treated with ten prototype surfactants, and were evaluated for cell viability (MTT incorporation), cytotoxicity (release of the enzymes lactate dehydrogenase and N-acetyl glucosaminidase), metabolism (glucose utilization), and inflammatory mediator (prostaglandin E(2)) release. There was a close correlation of the dose-response characteristics for all the endpoints tested, and between the in vitro responses and human patch test scores for the surfactants tested. These results demonstrate the usefulness of human skin cell cultures and of cell viability, cytotoxicity, and inflammatory mediator release as endpoints, for the in vitro assessment of skin irritancy.

In Vitro and Human Testing Strategies for Skin Irritation

Abstract: Prior to the manufacture, transport, and marketing of chemicals or products, it is critical to assess their potential for skin toxicity (corrosion or irritation), thereby protecting the worker and consumer from adverse skin effects due to intended or accidental skin exposure. Traditionally, animal testing procedures have provided the data needed to assess the more severe forms of skin toxicity, and current regulations may require animal test data before permission can be obtained to manufacture, transport, or market chemicals or the products that contain them. In recent years, the use of animals to assess skin safety has been opposed by some as inhumane and unnecessary. The conflicting needs of the industrial toxicologist to (1) protect human safety, (2) comply with regulations, and (3) reduce animal testing have led to major efforts to develop alternative, yet predictive, test methods. A variety of in vitro skin corrosion test methods have been developed and several have successfully passed initial international validation. These have included skin or epidermal equivalent assays that have been shown to distinguish corrosive from noncorrosive chemicals. These skin/epidermal equivalent assays have also been modified and used to assess skin irritation potential relative to existing human exposure test data. The data show a good correlation between in vitro assay data and different types of human skin irritation data for both chemicals and consumer products. The effort to eliminate animal tests has also led to the development of a novel human patch test for assessment of acute skin irritation potential. A case study shows the benefits of in vitro and human skin irritation tests compared to the animal tests they seek to replace, and strategies now exist to adequately assess human skin irritation potential without the need to rely on animal test methods.

Beyond the blot: cutting edge tools for genomics, proteomics and metabolomics analyses and previous successes

The skin has an amazing array of complex interacting biological processes. Recent advances in investigational techniques now allow evaluation of these processes at the level of the gene, protein and metabolite. Sometimes collectively known as the omics, these fields of inquiry, known as genomics, proteomics and metabolomics, respectively, are yielding new and important insights into skin structure and processes, its responses to injury and age, and the mechanisms by which new interventions and compounds may work to improve the health and integrity of this crucial organ.

Application of genomics to breakthroughs in the cosmetic treatment of skin ageing and discoloration.

The use of global gene expression profiling, also known as transcriptomics or genomics, provides a means to identify key pathways affected in ageing skin that can be improved with appropriate cosmetic compounds. Aspects of skin ageing that can be addressed include matrix production, barrier, lipid synthesis, antioxidant capacity and hyperpigmentation. Gene expression profiling together with in vitro human skin cell cultures for compound screening and verification have led to the identification of cosmetic compounds and an understanding of the biological effects of compounds such as niacinamide, Pal‐KTTKS, hexamidine, retinyl propionate and sodium dehydroacetate. In addition, understanding of the decreased antioxidant capacity of aged skin has led to the identification of new antiageing ingredients, olive‐derived fatty acid ethoxylates, which have been shown to restore antioxidant enzymes in skin keratinocytes and fibroblasts. Gene expression profiling of age spots has also provided an understanding of the role of undecylenoyl phenylalanine in reducing melanin production by an adrenergic receptor mechanism in melanocytes. The use of these compounds in cosmetic formulations for skin care can aid improvements in the appearance of aged skin, including the improved appearance of fine lines, wrinkles and age spots.

Assessment of the Cytosensor Microphysiometer Assay in the COLIPA In Vitro Eye Irritation Validation Study

The Cytosensor(TM) microphysiometer assay and its associated prediction model were evaluated in the COLIPA ocular irritation validation study for cosmetic ingredients and formulations. Test materials were prepared in low-buffer medium and exposed to L929 cells grown in transwells. The metabolic rate of the cell population was measured after each dose and the dose inducing a 50% decrease in the rate (MRD(50)) was determined and used to predict the ocular irritation potential. Only 29 of the 55 materials could be tested because of solubility limitations. The irritancy potential of many chemical classes was underpredicted by the assay, particularly acids, bases and organics. The use of the assay for surfactants and surfactant-based formulations showed promise, confirming the use of the method for these types of materials, although some revision of the prediction model would be necessary. Excellent interlaboratory reproducibility for the MRD(50) values across all test materials was observed.

The Performance of the Tissue Equivalent Assay using the Skin2TM ZK1200 Model in the COLIPA International Validation Study on Alternatives to the Draize Eye Irritation Test

The tissue equivalent assay (TEA) (Osborne et al., 1995) was used to evaluate 55 mixed ingredients and formulations in the COLIPA International Validation Study on Alternatives to the Draize Rabbit Eye Irritation Test (Brantom et al., 1997). The TEA can be used to test all types of materials since it uses a topical application approach and is not limited to only testing liquid or soluble materials. A prediction model (PM) for the test was developed using historical eye irritation data from a total of 132 materials on which in vivo and in vitro data were available. A regression model was derived from these data and used to relate the in vitro endpoint (t(50)) obtained in the study to a Draize MMAS (modified maximum average score). This provided a measure of the predicted in vivo eye irritation scores. In the current study, two separate laboratories used the same protocol to test the same set of coded materials and the results of both laboratories were compared to the initial PM. The TEA met the reliability criteria of the validation study in reproducing the predefined PM in both laboratories, and a good relationship between predicted and observed Draize MMAS values was obtained (r=0.906 and r=0.850). Good correlations were maintained when separate analyses were made of the formulations and ingredients included in the test set. Good relationships between the in vitro endpoint and individual Draize tissue scores (r>0.8) were also exhibited. Although insufficient data were available to make an assessment of interlaboratory variation, some difference in the reproducibility of the assay was noted between the two laboratories, particularly for the highly irritating materials. However, the consistency of data was encouraging and the discrepancies seen between the laboratories suggested a sensitivity of the model to subtle differences in application techniques, and in handling and timing. Taken together, these results indicate the utility of the TEA test for these types of substances and the need to more fully address the issue of interlaboratory reproducibility.

Examination of the Reversibility of Corneal Opacity Using an Historical Eye Irritation Database

AbstractThe appearance of corneal opacity in a rabbit eye irritation test is of concern due to the importance of corneal clarity to vision. An important consideration, both for human safety assessments and regulatory classifications, is whether the corneal opacity is readily and completely reversible, or is irreversible. In fact, new harmonized regulatory guidelines for classification of eye irritants and corrosives assume that the most severe of corneal opacities (grade 4) are irreversible. The purpose of the current work was to use an extensive historical database to examine this assumption and to identify factors that are important in reversibility. Eye test scores from historic rabbit eye irritation tests were analyzed to determine the reversibility within 21 days of corneal opacity responses graded as 4, on a scale of 0-4, at 1, 2, or 3 days after instillation of test material, and to define factors influencing reversibility. Overall, the historic data indicated that 45% of corneal opacities graded a...