Skin irritation and skin corrosion refer to localized toxic effects resulting from a topical exposure of the skin to a substance. The systemic effects from the dermal absorption of a substance are covered in AltTox under Acute Systemic Toxicity and Repeated Dose/Organ Toxicity.
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" (UNECE, 2009).
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 survival. 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 exposed to a chemical, specific immunological and histological responses can occur (Williams, 2003). The review article by Weiss, et al., (2004) also describes the structure of the skin and the mechanisms of skin irritation.
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.
Figure 1. 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; Acute Dermal Irritation/Corrosion). 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. Substances that are caustic (alkali) or of extreme pH are typically classified as corrosive without animal testing.
Regulatory Requirements & Test Guidelines
The Organisation for Economic Co-operation and Development (OECD) adopted a tiered approach for dermal testing in 2001, and this approach is described in the revised TG 404 (April 24, 2002). 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 the skin irritation/corrosion potential of a substance. If this information is not sufficient, validated in vitro methods can then be used. 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.
Updated approaches to the use of tiered and integrated testing strategies for predicting skin irritation potential without the use of animals are discussed in the Emerging Research, Methods, & Policies section.
Five US agencies may consider 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).
A number of validated, non-animal methods to determine skin corrosion are available (see section below). Some regulatory agencies may still be requesting confirmatory animal testing on a case-by-case basis. However, the use of existing data and a tiered in vitro approach (in conjunction with an intelligent selection of tests) should be sufficient.
The recent validation of several types of in vitro tests for skin irritation (see section below) 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 non-animal methods requires their use. The European Commission (EC) has accepted several reconstructed human epidermis models (EpiSkin, SkinEthic and EpiDerm) for skin irritation testing into their regulations (see draft TG B 46); this test guideline is based on the recently accepted draft OECD TG for skin irritation. The US validation authority, ICCVAM, has not reviewed or endorsed in vitro skin irritation test methods yet. However, the pending acceptance of the OECD TG for skin irritation will establish the basis for its use in OECD member states for appropriate test applications.
See the Validation and Acceptance of Non-animal Alternative Methods section (below) for details on protocols and OECD TGs for in vitro skin irritation and skin corrosion testing.
Non-animal 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). Research is also underway into the use of stem cells to create or regenerate in vitro skin cell models (discussed in the Emerging Research, Methods, & Policies section).
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.
The 3D skin models, or reconstructed human epidermis (RHE) models, 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. Skin models that are commercially available include the following: EpiDerm Skin Irritation Test (SIT) (MatTek Corporation, Ashland, MA, US), EpiSkin SIT (SkinEthic, Nice, France), SkinEthic RHE (SkinEthic, Nice, France), LabCyte EPI-MODEL (J-TEC, Japan), EST-1000 RHE (Advanced CellSystems, Germany), and Vitrolife-Skin RHE (GUNZE Medical Division, Japan).
The endpoints typically evaluated for skin irritation testing are cytotoxicity (MTT assay), or cytotoxicity (MTT assay) plus IL-1a (cytokine release). The endpoint evaluated for skin corrosion is cytotoxicity (MTT assay).
A video that demonstrates the protocol used with the EpiDerm SIT has been published.
Figure 2. EpiDerm human skin tissue equivalent - histological cross section magnified 400 times.
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. The Corrositex assay is based on the time it takes for a chemical to penetrate an artificial biobarrier.
Human volunteer skin testing (patch testing) can be ethically conducted on products and ingredients where the hazard of the substance is already substantially understood (Basketter, et al., 2004; Grindon, et al., 2007; Jírová, et al., 2010; Robinson, et al., 2000; Robinson & Perkins, 2002). Generally only 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 useful in the validation of human cell-based methods (Basketter, et al., 2004).
Jírová, et al. (2010) compared human patch test data with in vitro and animal data and found that only 5 substances were human skin irritants out of 16 materials classified as skin irritants in the rabbit test. The authors concluded the "results confirm observations that rabbits overpredict skin effects in humans," and that human data should therefore be taken into consideration when validating in vitro methods and making conclusions about their predictive capacity.
(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 Non-animal Alternative Methods
In 1999, an ICCVAM review of Corrositex recommended its use as a stand-alone assay for evaluating acids, bases, and acid derivatives for the US Department of Transportation, and otherwise, as part of a tiered testing strategy. A reciprocal statement of validity was issued by ESAC in 2000. That same year, Corrositex achieved formal acceptance by US regulators, and in 2006, the method achieved international acceptance as OECD TG 435.
EpiSkin, EpiDerm and the rat TER tests were validated by ECVAM and endorsed by the ESAC in 1998 as "scientifically validated for use as a replacement for the animal test, and … ready to be considered for regulatory acceptance." These tests underwent a subsequent validation review by ICCVAM, which in 2002 recommended their use as part of a tiered testing strategy. They achieved formal EU acceptance for regulatory purposes in 2000, followed by international acceptance as OECD TGs 430 and 431 in 2004. A Draft Revised TG 431 is currently under consideration, which includes a technical update on performance standards and recommends substitution of two reference chemicals.
The following RHE models have subsequently been endorsed as valid for predicting skin corrosion based on their meeting the performance standards in OECD TG 431: SkinEthic (ESAC, 2006), VitroLife-Skin (JaCVAM, 2008), and EST-1000 (ESAC, 2009; Hoffmann, et al., 2005).
Variants of the EpiSkin and EpiDerm corrosivity tests were evaluated for their ability to predict skin irritation (Spielmann, et al., 2007). On the basis of this validation study, ESAC (2007) concluded that the EpiSkin Skin Irritation Test (SIT) "showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation...and for being used as a replacement...for the Draize Skin Irritation Test...for the purposes of distinguishing between R38 skin irritating and no-label (non-skin irritating) test substances." With the protocol used in this study, the EpiDerm SIT test was endorsed as a component of a tiered testing strategy, in which negative results required further confirmation.
ECVAM Performance Standards (PS) were established for validating skin irritation testing methods based on RHE methods in comparison to the validated EpiSkin SIT (ECVAM, 2007). The ESAC endorsed revised Performance Standards in 2009 due to the adoption of the United Nations Globally Harmonized System (UN GHS) system by the EU in December 2008.
The following year, the ESAC (2008) endorsed the Modified EpiDerm SIT and the SkinEthic Reconstructed Human Epidermis (RHE) models for assessing skin irritation potential (Alépée, et al., 2010; Kandárová, et al., 2009). Both showed results similar to the validated reference method, EpiSkin SIT, and are considered as valid replacement methods for the hazard identification and labeling of chemicals within EU regulations. This second study of the EpiDerm SIT involved modifications of the exposure conditions of the tissues to the test substances that resulted in increased agreement of the test results with the in vivo data.
In 2009, the ESAC endorsed the three ECVAM-validated methods (EpiSkin; Modified EpiDerm SIT; SkinEthic RHE) for testing under the UN GHS.
The in vitro skin irritation test methods endorsed by the ESAC have achieved regulatory acceptance in the EU (see draft TG B.46), and have also been proposed for international acceptance as a draft OECD TG. On March 25, 2010 the OECD National Coordinators Meeting WNT22 approved the draft Test Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method. The TG will be submitted to the Joint Meeting and then Council for final adoption.
A peer review of the skin irritation test method, LabCyte EPI-MODEL 24, a reconstructed human epidermis model produced in Japan "has been validated in accordance with the principles and criteria documented in the OECD Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment, No. 34. The test method is a 'me-too' submission following the Performance Standards of the draft Test Guideline for 'In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method'.... The Peer Review is anticipated to be finalised by the end of March 2010."
Additional information on in vitro methods for skin irritation/corrosion toxicity testing can be found on AltTox at Existing Alternatives: Skin Irritation/Corrosion.
Table 1. Non-animal test methods for skin irritation/corrosion.
* Specific strengths and limitations on the use of these in vitro methods are described in the documents available on the ECVAM and ICCVAM websites.
** The Draft TG was approved at the OECD National Coordinators Meeting in March 2010, and is awaiting final adoption.
*** A "me-too" submission following the Performance Standards of the draft OECD Test Guideline for In Vitro Skin Irritation; anticipated to be finalized by end of March 2010.