Skin Irritation/Corrosion

Skin toxicity refers to the local toxic effects resulting from a topical exposure of the skin to a substance. Skin toxicities include skin irritation and skin corrosion. The Globally Harmonized System of Classification and Labeling of Chemicals (GHS) defines skin irritation as "the production of reversible damage to the skin following the application of a test substance for up to 4 hours" and defines skin corrosion as "the production of irreversible damage to the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to 4 hours [in at least one of three test animals]" (UNECE, 2004).

Systemic effects from the dermal absorption of a substance are covered in AltTox under Acute Systemic Toxicity and Repeated Dose/Organ Toxicity.

The skin is the largest human organ, but is more than just a protective covering for the body. The skin is composed of multiple layers and serves many functions important to the survival of the human/animal. The living skin is continuously regenerating and is metabolically active - phase 1 and phase 2 reactions can occur within the skin, although at significantly lower activity than in the liver (Williams, 2003). One of the functions of the skin is to protect the body from environmental hazards such as toxic or corrosive chemicals. When the skin is assaulted with a chemical, specific immunological and histological responses occur (Williams, 2003). A recent review describes the structure of the skin and the mechanisms of skin irritation (Weiss, et al., 2004).

The outermost layer of the skin is called the epidermis (Figure 1). The epidermis is made up of approximately four layers of epithelial cells called keratinocytes. The epidermal keratinocytes and junctions between these cells form the barrier of the skin, preventing substances from penetrating the skin, and water and electrolytes from leaking out of the body. Keratinocytes undergo a process of differentiation as they move from the bottom epidermal cell layer to the top (Williams, 2003). Terminal differentiation of the keratinocytes results in cornified (dead) keratinocytes on the surface of the skin forming the stratum corneum, another protective barrier for the skin. Langerhans cells, found in the epidermis, detect foreign antigens such as the molecules on the surface of invading microbes and travel to the lymph nodes where they present these antigens to cells of the immune system. Melanocyte cells, also in the epidermis, produce the melanin pigment that colors the skin.

The deeper layer of the skin is called the dermis. The high content of collagen and elastin in the dermis impart strength and elasticity to the skin. Blood vessels, nerves, sweat glands, and hair roots are also found within the dermis.

The innermost layer of the skin, called the subcutaneous layer, is relatively thick and primarily composed of fat cells. It is a source of insulation and physical protection for the body as well as a source of energy for the cells.

Cross section of skin - Visual Dictionary - Copyright © 2005-2008 - All rights reserved.

The Animal Test(s)

A test substance is applied to the shaved bare skin (about 6 cm²) of healthy young adult albino rabbits and the area is covered with gauze (OECD Test Guideline (TG) 404). The substance is removed after four hours and the rabbit's skin is observed at specific times for irritant responses for as many as 14 days. One animal is usually tested first. The GHS reports that animal skin irritation and corrosion responses are quite variable, so the document explains a range of responses for classification purposes. The grading of the skin responses by technicians is subjective, and is one source of the variability observed. Substance that are caustic (alkali) or of extreme pH are typically classified as corrosive without animal testing.

Regulatory Requirements & Test Guidelines

Skin irritation/corrosion is usually assessed by using a tiered testing scheme for hazard assessment. The Organisation for Economic Co-operation and Development (OECD) adopted a tiered approach for dermal testing in 2001, and the tired testing approach is described in the revised TG 404 (April 24, 2002), Acute Dermal Irritation/Corrosion. TG 404 recommends the use of validated in vitro or ex vivo methods when appropriate. TG 404 also describes a stepwise approach to rabbit testing. TG 404 notes that "in the interest of both sound science and animal welfare, in vivo testing should not be undertaken until all available data relevant to the potential dermal corrosivity/irritation of the substance have been evaluated in a weight-of-the-evidence analysis." The weight-of-evidence approach includes analysis of all existing human and animal data, pH extremes, and in vitro data.

Human data and experience, screening assays and pH extremes (to identify obvious corrosive materials), and data from structurally related materials are sometimes sufficient for classifying a substance. If this information is not sufficient, animal skin irritation tests and/or validated in vitro methods can then be used, as needed. The GHS guidance recommends "considering the totality of existing information and making an overall weight of evidence determination." The GHS provides a tiered testing and evaluation scheme for assessment of the information.

Five US agencies can require skin irritation/corrosion data when there is the potential for human skin exposure: Environmental Protection Agency (EPA); Food & Drug Administration (FDA); Consumer Product Safety Commission (CPSC); Department of Transportation (DOT); and Occupational Safety and Health Administration (OSHA).

Validated nonanimal methods to determine skin corrosion have been available for several years; however, in the US at least, confirmatory animal testing is generally still required for negative materials. Nonetheless, this still reduces the number of animals that would be exposed to corrosive materials.

The recent validation of several types of in vitro tests for skin irritation has the potential to further reduce animal testing for this toxicity endpoint. However, the degree of regulatory acceptance by the many authorities requiring skin irritancy data will impact the degree of reduction in animal testing for skin irritation. For certain substances in certain geographic areas, such as cosmetic ingredients in the EU, the availability of validated nonanimal methods requires their use.

Nonanimal Alternative Methods

A variety of cell-based methods have been developed and used for the assessment and/or ranking of skin irritants. Cell models include monolayer cultures of human and animal skin cells (keratinocytes); multilayered (3-dimensional (3D)) cultures of skin cells that provide a barrier function like the surface of the skin; and co-culture models where two or more of the types of cells found in the skin are represented. A recent review article describes the features and evolution of the 3D skin models composed of human keratinocytes cultured at the air-liquid interface to induce stratification and development of a barrier function (Poumay & Coquette, 2007). Commercial in vitro skin models are now available for conducting reproducible in vitro assessments, and several of the models have been endorsed as scientifically valid for certain testing applications (discussed in the following section).

The 3D skin models usually consist of human cells grown on a membrane at the air-liquid interface (Figure 2). This method of culture induces the cells to grow in multilayers and to form junctions between the cells so that the cultures are similar to mini pieces of human skin in the wells of a petri plate. Research labs have produced these types of models, and several are commercially available, for example: EpiDerm™ (MatTek Corporation, Ashland, MA, US), EPISKIN™ (Skinethic, Nice, France), and RealSkin™ (Skinethic, Nice, France). These models are used to assess skin irritation and corrosion.

Ex vivo models, or skin explants, consist of pieces of skin from humans or animals for in vitro testing applications. These have been used in screening for skin irritants but are more useful for testing skin corrosion or dermal absorption (skin penetration). Examples of this type of model used for skin irritation testing are the mouse skin integrity function test (SIFT), human cadaver skin, human skin from surgery, and the pig ear test (Zuang, et al., 2005). The rat skin transcutaneous electrical resistance (TER) method has been validated for skin corrosion testing.

An acellular (no living tissues involved) barrier model called CORROSITEX® has also been validated for skin corrosion testing.

Human volunteer skin testing (patch testing) can be ethically conducted on products and ingredients where the hazard of the substance is already substantially understood (Grindon, et al., 2007; Robinson, et al., 2000; Robinson & Perkins, 2002). Generally only very mild substances would be tested for irritation in this way, and no materials thought to be corrosive could be assessed on human volunteers. A database of human skin irritation data from 4-hour patch tests for 65 substances has been published, which could be used in the validation of human cell-based methods (Basketter, et al., 2004).

(Quantitative) structure-activity relationship ((Q)SAR) models have been developed for skin irritation studies but are not commonly used. Summaries of these models can be found in several ECVAM reports (ECVAM, 2002; Zuang, et al., 2005), and the review of Grindon, et al. (2007).

Validation and Acceptance of Nonanimal Alternative Methods

Validated alternatives for skin irritation and corrosion testing are summarized below, and are described in more detail in a recent report (Grindon, et al., 2007).

EPISKIN and EpiDerm were recently evaluated for their ability to predict skin irritants (Spielmann, et al., 2007). ECVAM endorsed EPISKIN as scientifically valid for a specific type of skin irritation testing. It is the first in vitro test to be found valid for replacing the rabbit test for determining R38 hazard labeling in the EU (using the MTT endpoint). The EpiDerm method was endorsed by ECVAM for use in a tiered assessment - it can be used to identify skin irritants, but negative results require additional assessment. ICCVAM has not yet endorsed in vitro methods for assessing skin irritation.

Four methods are considered as scientifically valid by ICCVAM for skin corrosion testing: EpiDerm, EPISKIN, rat skin TER, and CORROSITEX. EpiDerm, EPISKIN, rat skin TER, CORROSITEX and the SkinEthic method are considered scientifically valid by ECVAM for determining skin corrosives. CORROSITEX is endorsed for testing only specific classes of chemicals. Other limitations on the use of these in vitro methods are described in the documents available on the ECVAM and ICCVAM websites.

OECD Test Guidelines are available for the in vitro skin corrosion test methods: OECD TG 430, In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER); TG 431, In Vitro Skin Corrosion: Human Skin Model Test; and TG 435, In Vitro Membrane Barrier Test Method for Skin Corrosion.

EU regulatory agencies have accepted EpiDerm, EPISKIN, and the rat skin TER for skin corrosion testing. US regulatory agencies have accepted EpiDerm, EPISKIN, and CORROSITEX for skin corrosion testing.

Nonanimal methods for assessing skin irritation or skin corrosion, which are considered scientifically valid by ECVAM or ICCVAM, are summarized in Table 1.

Table 1. Nonanimal test methods for skin irritation/corrosion.