Dermal toxicity: alternative methods for risk assessment.

Conceptually, irritant contact dermatitis (irritation) and allergic contact dermatitis (ACD) in man should provide the ideal platforms to launch in vitro toxicology into the pantheon of in vitro testing assays. In theory, irritant dermatitis has been considered by most a simple area of cutaneous biology, whereas ACD is a complex area of biology. However, both result in responses that are reasonably stereotypical and well characterized. The biology of the underlying mechanisms is becoming characterized and will thus allow development of mechanistically based in vitro assays that will be scientifically validated and thus acceptable to regulatory agencies.


Irritant Dermatitis Syndrome: Contemporary (Tentative) Definition
Dermatopathologists and toxicologists generally consider cutaneous irritation a homogeneous and monomorphous biologic event, having been lulled by its mundane morphology. However, current knowledge suggests the contrary-a relatively homogeneous appearance but a complex, variegated sequence of mechanisms. Our current clinical and mechanistic classification (Table 1) undoubtedly represents a vast undersimplification, as we are only beginning to understand this common and heterogeneous syndrome. Irritant Dermatitis Syndrome: Localized or Systemic? Conventional dogma suggests that irritant dermatitis is a localized (site of contact) phenomenon; surely the reasoning appears impeccable. Yet, current knowledge suggests that, although the point of contact phenomena must be primal, other systemic factors may be decisive. Some possible systemic factors influencing irritant dermatitis are a) age, b) race, c) preexisting and/or previous skin diseases, and d) atopic dermatitis. Irritation in Vtro Methods to evaluate potential irritation have been well described in In Vitro Skin Toxicology (1).
For irritancy testing, physical-chemical measurements, quantitative structureactivity relationship (QSAR), and historical data can provide significant data. In vitro methods that measure cytotoxic interleukins (ILs)la, arachidonic acid, and the prostaglandins (1,2) should provide adequate information on acute mild irritants through and including corrosivity. Additionally, reconstituted tissue equivalents (RTE) and skin explants may be useful in other situations.
All these systems are in development or in use in research laboratories. They have not gone through adequate optimization yet to be ready for validation, but one can expect that this will begin to happen in the near future. Table 2 is not meant to be indusive, but to identify current, best-guess approaches to specific end points. There is a This paper was prepared as background for the 13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): Alternative Testing Methodologies held 26-31 January 1997 in Ispra, Italy. Manuscript received at EHP 9 May 1997; accepted 21  substantial need to a) more clearly define relationships between interleukins (both time relationship and biologic interactions); b) understand the biology of adhesion molecules; c) improve and define the conditions of the biologic systems; and d) establish relationships between these biochemical systems, molecules, and exogenous chemicals.
In evaluating acute toxicants (including dermal), it has been suggested that once data from in vitro testing are evaluated, including what is known about the chemicals and evaluation of these chemicals [e.g., QSAR (3,4), literature, physical-chemical measures], it may then be appropriate to establish safety of these materials directly in the human ( Figure 1).
Corrosivity, a physical destruction of the skin, is the extreme case of irritancy. It is likely that in the near future we will be able to predict corrosive ability using QSAR, physical and chemical assays, and historical information to fully assess the hazard (3-6). It is inappropriate for us to assess the degree of severe corrosives using either whole animal or human clinical studies.

Phototoxicity
Dermal phototoxicity results from photo-activation of chemicals that cause either a photoirritant or a photoallergic response. A method to examine phototoxicity has recently been described (7,8). The developers of this assay suggest that it is validated, but it has not yet been submitted for fully independent, anonymous peer   Figure 1. System for using in vitro assays as part of a tiered testing structure. The diagram illustrates a sequential process for making the safety assessment of a hypothetical chemical or product. It can be seen that in vitro methods (as well as traditional animal tests) supply only a portion of the information needed to make the safety assessment and that this information is integrated with other data so that a weight-of-evidence decision is finally made. From Curren et al. (36).
review. A guideline document is being prepared for submission to the Organisation for Economic Co-operation and Development (OECD).

Allergic Contact Dermatitis
Allergic contact dermatitis (ACD) is a complex set of biologic reactions that is not yet fully understood. However, significant advances in defining an approach to predict the phenomenon is rapidly developing. ACD is the result of cutaneous T-lymphocyte-mediated reaction to exogenous chemicals. The biology of ACD has been well described by Elmets (9) and relevant sections are quoted here for easy reference. Once complete antigens have been formed, they are "taken up" by Langerhans cells within the epidermis and other antigen presenting cells, such as macrophages and dendritic cells, within the dermis (10-12). Epidermal Langerhans cells are a specialized type of antigen-presenting cell (13). They comprise 2-4% of the entire epidermal cell population. These cells are derived from the bone marrow, and migrate to the suprabasilar layer of the epidermis where they reside for long periods of time. In that location they form a reticulo-endothelial network that traps: a) exogenous xenobiotics that may have deleterious effects on the skin, b) pathogenic microorganisms that may cause a local or systemic disease and c) neoantigens on cells that without eradication would develop into dinically apparent tumors (10). The major function of epidermal Langerhans cells and other antigen-presenting cells is to activate subpopulations of antigen reactive T-lymphocytes (11). As their part of their role as antigen-presenting cells, Langerhans cells "take-up" antigen and degrade it within lysosomal granules or in the endoplasmic reticulum and then reexpress the degraded molecule on the cell surface complexed with class I or class II major histocompatibility determinants. While this is occurring, Langerhans cells migrate to regional lymph nodes, a site where they actually present the antigen to T-lymphocytes (14). Interestingly, while they migrate Langerhans cells undergo a series of differentiation steps that prepare them to more efficiently present the antigen to T cells (15,16). This includes an increase in the expression of class II major histocompatibility determinants (17) and of the adhesion molecules ICAM-1 [CD54] (18,19), B7-1 [CD80] (20,21), and B7-2 [CD86] (20). The costimulatory molecules ICAM-1, B7-1 and B7-2 are important adhesion molecules, which are expressed on antigen-presenting cells and are required for optimal activation of T cells. During this differentiation process, Langerhans cells also lose their ability to process antigens (15,22) and their capacity to express E-cadherin, an adhesion molecule that allows Langerhans cells to attach to keratinocytes (23). The cytokines GM-CSF (20,24) and, to a lesser extent, IL-1 (24)  Interleukin-8 (IL-8) is an 8-kDa heparin-binding basic polypeptide that is chemotactic for T cells (26). There is evidence that it acts in that capacity to bring in T cells into cutaneous sites in urushiol allergic contact dermatitis (27). IL-8 mRNA can be induced in cultured keratinocytes in response to IL-1oa (26).
The potential systems and end points are summarized in Table 2.
If one uses the schematic in Figure 1 then QSAR, historical data, and literature may provide adequate data to classify a compound or will identify which specific in vitro tests will be appropriate. The next sequential step will be to use cell culture and RTE and measure appropriate cytokines, adhesion molecules, and/or histochemistry.

Needs and Future Direction
Many methods have been evaluated by different laboratories. There is a clear need for additional studies to more completely define and identify the underlying biology of the cytokines, adhesion molecules, and other inflammatory molecules. This knowledge will provide the rationale for specific batteries of in vitro tests to provide measures of irritancy, corrosivity, and allergic potential.
What remains to be done is not only validating the assays for man. This is a needed step, but only after appropriate methods are fully developed to generally accepted standards of scientific rigor using in principle the criteria described by the Interagency Coordinating Committee for the Validation of Alternative Methods (34) and the OECD (35) for validation and regulatory acceptance. Then an understanding of how to use the information appropriately for risk assessment will be the next challenge.