The Way Forward for In Vitro Skin Irritation and Corrosion Testing – 2010 Update

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The Way Forward for In Vitro Skin Irritation and Corrosion Testing – 2010 Update

Helena Kandárová, Yulia Kaluzhny, Mitchell Klausner & Patrick Hayden

Published: December 17, 2010

About the Author(s)
Dr. Kandárová’s main field of interest is the development of in vitro methods for reduction & replacement of in vivo tests for topical toxicity testing. Throughout her academic and professional career, she has been involved in projects aimed at the evaluation and validation of 3D reconstructed human tissue (RhT) models as replacements for animal tests.  In particular, she had been involved in successful validation and pre-validation studies for skin corrosion, skin and eye irritation testing.

Dr. Helena Kandárová studied chemistry at the Slovak University of Technology in Bratislava, where she graduated in 2001.  Dr. Kandárová performed the experimental part of her PhD work at ZEBET – The National Center for the Documentation and Evaluation of Alternatives to Animal Experiments, Federal Institute for Risk Assessment (BfR), Berlin, Germany – where she was involved in several research projects aiming in validation of alternative methods.  She defended her PhD thesis at the Freie Universität (FU) Berlin, Germany in 2006. In 2007, she joined MatTek Corporation, USA as a Senior Scientist and, in 2009, was appointed Executive Director of MatTek In Vitro Life Science Laboratories, Slovakia.

Dr. Kandárová is member of SOT, ESTIV, SETOX and several other professional organizations and expert panels. She (co)authored 17 publications and is a frequent speaker at national and international meetings and conferences. Dr. Kanďárová is co-founder of the In Vitro Toxicology Division of SETOX and member of the editorial board of Journal Interdisciplinary Toxicology.  She also serves as scientific reviewer for ATLA, Skin Pharmacology and Physiology, Regulatory Pharmacology and Toxicology.

Helena Kandárová
MatTek Corporation
200 Homer Avenue
Ashland, MA 01721
E-mail: hkandarova@mattek.com

Dr. Hayden received a B.S. in Chemistry from the University of MD at College Park in 1984 and a Ph.D. in Chemistry from Clarkson University, Potsdam, NY in 1991. His Ph.D. thesis work on biotransformation of halogenated ethylenes was conducted in the laboratory of Dr. James L. Stevens at the W. Alton Jones Cell Science Center in Lake Placid, NY. As a Post-Doctoral (PRAT) Fellow in the laboratory of Dr. Colin F. Chignell at the NIEHS, he studied ESR spectroscopy, spin-trapping of free radicals and free radical toxicity in skin cells.

Dr. Hayden first began working with in vitro skin models while employed by the Gillette Company (1995-1999), where he experimented with the Skin2™ and EpiDerm™ models in relation to irritation screening of consumer products.

Since 1999, Dr. Hayden is employed by MatTek Corporation as Vice President of Advanced In Vitro Systems, where he has led the development of MatTek’s full-thickness skin model (Epiderm-FT™) and manages the production and ongoing research related to the EpiDerm-FT™, EpiAirway™ and other in vitro human models.

Patrick J. Hayden
MatTek Corporation
200 Homer Avenue
Ashland, MA 01721
E-mail: phayden@mattek.com

Introduction

It has been more than 3 years since our first Way Forward essay on Skin Irritation Testing appeared on the AltTox website (1). A number of significant events and activities have occurred in the interim, and an update on the status and future direction of in vitro skin corrosion and irritation testing is now timely.

This update will focus on several recent developments and current issues including:

  • additional in vitro models that have been validated and fully accepted for skin irritation and corrosion testing,
  • harmonization of the EU and GHS systems for skin corrosion and irritation testing,
  • implementation of testing strategies (TS) combing validated in vitro/ex vivo methods,
  • need for additional work to address specialized skin corrosion and skin irritation classification and labeling systems (e.g. transport of dangerous goods, pesticides).

In addition, we will consider current and future needs for monitoring and assessing the impact that validated in vitro skin corrosion and irritation methods have for reduction of in vivo skin corrosion and irritation tests.

Specific areas of discussion include:

  • education, training and promotion of in vitro skin corrosion and irritation test methods,
  • mechanisms for monitoring in vitro skin corrosion and irritation test methods use and effectiveness for reduction of animal tests.
Newly Validated In Vitro Models for Skin Corrosion and Skin Irritation

At the time of our first Way Forward essay (2007), only one in vitro skin model (EPISKIN®, Skinethic Laboratories) was fully validated and endorsed by the European Centre for the Validation of Alternative Methods (ECVAM) as a standalone replacement for the in vivo skin irritation test (2). The EpiDerm model (MatTek Corporation) was at that time endorsed as a validated component of a tiered testing strategy (2). As anticipated, additional development and validations have now been performed. Re-development of the EpiDerm Skin Irritation Test (SIT) assay was conducted as a follow up activity to the original ECVAM validation study, with the aim to increase the sensitivity of the protocol to better match in vivo rabbit classifications (3,4,5). Additionally, a SkinEthic RHE assay validation study was conducted as a catch-up activity (6,7) based on Performance Standards derived from the EpiDerm and EPISKIN validations performed by ECVAM (8,9). Following this work, in 2008, the EpiDerm™ skin irritation test (SIT) and SkinEthic assays were fully validated and endorsed by ECVAM as standalone skin irritation tests (10). All three fully validated methods now provide similar performance while differing only in test chemical exposure time, ranging from 15 minutes (EPISKIN model), 42 minutes (SkinEthic model) to 60 minutes (EpiDerm model). Two additional reconstructed human epidermal (RhE) models, LabCyte (J-TEC, Japan) and EST-1000 (Cell Systems, Germany) are currently undergoing validation activities to catch-up with the methods described above. These studies are scheduled to be completed by mid-2011.

An important development that occurred in December, 2008 was the adoption of the Globally Harmonized System (GHS) of Classification and Labeling of Chemicals by the European Union (11). The EU system continues to use two categories to distinguish non irritating substances (no-category) from skin irritating substances (category 2, former EU R38). GHS category 3 (mild irritant classification) is optional in the EU and is used only for specialized national regulations. With the adoption of the GHS system, the former EU system cut-off score used to distinguish between no-category and skin irritating substances has been shifted from an in vivo score of 2.0 to 2.3. Consequently, substances with an in vivo score between 2.0 and 2.3 that were considered irritant under the former EU classification scheme are now considered non-irritants under the GHS system.

Since the ECVAM validated in vitro SIT protocols were designed to match in vivo classifications based on the former EU classification system, the change in cut-off necessitated a re-evaluation of the data from the validation studies. Retrospective statistical evaluation conducted by ECVAM and the German Federal Institute of Risk Assessment (BfR) showed that changing the cutoff increases the sensitivity of the validated methods for identification of skin irritants. Specificity decreased in all SITs, but was still within acceptable limits (12). Beginning in 2007, all validated in vitro SITs have been accepted for regulatory testing in the EU as Test Guideline B.46.

Further developments also occurred in the area of skin corrosion testing. In addition to EpiDerm, EPISKIN and SkinEthic skin corrosion assays, two additional skin corrosion methods obtained positive evaluations for compliance with OECD TG 431: Human skin corrosion test. These methods were EST-1000 (Cell Systems) which obtained ECVAM endorsement in 2009 (13) and Vitro-Life Skin (GUNZE Medical Division, Japan) which gained endorsement by the Japanese Center for the Validation of Alternative Methods (JaCVAM) in 2008 (14).

Developments at the OECD Level

Successful validation of the EPISKIN®, EpiDerm™ and SkinEthic RHE skin irritation tests has led to the development and adoption of a new regulatory Test Guideline (TG) 439 In Vitro Skin Irritation: “Reconstructed Human Epidermis Test Method”. The guideline was adopted on July 22, 2010 by the Organization for Economic Co-operation and Development (OECD). The TG references all three SITs as full replacement methods for the in vivo skin irritation test (15). The OECD has thus adopted a total of 4 in vitro TGs for dermal toxicity testing: 1) OECD TG 430 “Rat Skin Trans-Cutaneous Electrical Resistance (TER) Test”; 2) OECD TG 431 “Skin Corrosion Test using RhE models”; 3) OECD TG 435 “Corrositex” and 4) OECD TG 439 “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.”

It should be noted that OECD TG 430 uses ex vivo rat skin treated with antibiotics for skin corrosion testing, and is therefore considered to be an animal test in some countries. Therefore, TG 430 may not be the method of choice for testing cosmetic ingredients. OECD TG 435 (Corrositex) has a limited applicability domain since it is restricted only to extreme pH ranges. The RhE tests are therefore expected to play a major role in future testing. Amongst many other activities of the OECD Test Guidelines program, revision of the OECD TG 404 “Acute Dermal Irritation/Corrosion” (the in vivo method) is planned to reflect the adoption of the in vitro skin corrosion and irritation tests. Combining these methods into a testing strategy is expected to minimize if not completely eliminate the use of animals needed for most dermal toxicity testing. Notable exceptions discussed below include corrosion testing for transport of dangerous goods, and US EPA pesticide testing.

in vitro skin corrosion/irritation testing

The extensive amount of work and rapid progress accomplished during the past 3 years has demonstrated a remarkable level of co-operation and a worldwide commitment to the 3Rs principles by numerous stakeholders including chemical, household product and cosmetic industry manufacturers, in vitro model developers/producers, validation authorities and government regulatory agencies. However, as the currently validated protocols begin to be implemented, several noteworthy situations where they will not adequately satisfy regulatory requirements are brought more sharply into focus.

Implementation of procedures for handling MTT reducing agents and colored test materials

All currently validated RhE corrosion and irritation assays utilize cellular reduction of MTT to a purple formazan compound as the accepted assay endpoint. However, some test chemicals may be capable of directly reducing MTT, which can lead to a false negative corrosion or irritation result. Fortunately, methods to account for direct MTT reduction have been developed (16,17). When properly implemented, these methods can identify and eliminate false negative results in the vast majority of cases. Addition of these procedures for dealing with direct MTT reducers into existing skin corrosion and irritation protocols is needed. In addition, test agents that are highly colored and have absorption spectra that significantly overlap with that of formazan may interfere with the MTT assay and lead to false negative results. Utilization of additional (viable) control tissues that do not undergo the MTT assay step has been proposed to allow for determination of the amount of a colored test article that is bound to the tissue (18). Different viability endpoints, such as ATP release from damaged cells, (19,20) may also be workable alternatives for these situations. Other endpoints such as cytokine and chemokine synthesis/secretion may also find utility in these situations. Further work on standardization of the protocols and development of reliable prediction models, followed by formal validation, is still required before implementation of these promising endpoints into testing strategies for regulatory purposes.

Development and validation of assay protocols for more complicated classification schemes
Testing requirements for transport of dangerous goods

One important testing requirement that is not adequately satisfied by current validated in vitro skin testing protocols relates to national and international guidelines for transport of dangerous goods. Current legislation is designed to regulate transport of dangerous goods via ground, rail, air, and sea to ensure that transport is done in a manner that protects the safety of transport personnel as well as the general public.

Current GHS package labeling guidelines utilize a classification scheme consisting of 3 corrosion sub-categories (1A – very dangerous, 1B medium danger, and 1C minor danger). A similar United Nations (UN) classification scheme for package labeling utilizes skin corrosion sub-categories designated as Group 1 (very dangerous), Group II (medium danger) and Group III (minor danger). Some countries have adopted skin corrosion classification and labelling systems without a requirement to distinguish between corrosivity sub-categories. However, industry may experience problems using this approach. Due to the lack of subdivisions, corrosion sub-category IA (very dangerous) might have to be applied by default to many moderately corrosive chemicals or chemicals with pH extremes having only mild or moderate skin irritation potential. Although respecting precautionary principles, assigning a chemical or formulation to sub-category 1A will have important consequences for most products in this class. These include very small volume package limits for air transport, prohibition from passenger aircraft, protective storage conditions, costly containers and low acceptance of the product on the market.

Thus, it is desirable that in vitro methods for skin corrosion should be able to discriminate at least between 1A (UN group I) and 1B/1C (UN group II/III) classess. Successful validation of such an in vitro test would have a substantial impact on reducing animal tests for packing group determinations. Analysis of existing in vitro data from previous validation studies provides some answers regarding the ability of current RhE models and protocols to partially satisfy corrosion packing group classification requirements. For example, in the EPISKIN validation study conducted in 1998, the sensitivity for identifying skin corrosion category 1A was 39% and sensitivity for combined category 1B+1C was 75% (21,22). Skin corrosion sub-categorization was not assessed during the EpiDerm protocol validation study. However, retrospective analysis of the 3 minute timepoint data from the Phase I EpiDerm validation study, where 50 chemicals were tested, shows a sensitivity of 100% and specificity of 72.2% for identification of corrosion class 1A (16). Additional protocol development and new prospective evaluation studies may be required to fully satisfy sub-categorization requirements for transport purposes.

US EPA testing requirements for pesticides

Another testing requirement that is not adequately satisfied by currently validated protocols is the United States Environmental Protection Agency (US EPA) Office of Prevention, Pesticides and Toxic Substances (OPPTS) 870.2500 Guideline on Acute Dermal Irritation. This guideline is one of a series of test guidelines that have been developed by the EPA for use in the testing of pesticides and toxic substance. This guideline utilizes a 4 hour rabbit test with a scheme that categorizes chemicals into four classes: Category I – corrosion, Category II – severe irritation at 72 hours after exposure, Category III – Moderate irritation at 72 hours after exposure and category IV – Mild or slight irritation. This scheme does not correlate with the GHS system due to the different approach to scoring and the number of classes utilized. It will therefore be difficult to address EPA guideline requirements in vitro without a harmonization of EPA and GHS systems for classification and labeling principles.

The GHS system for skin irritation classification utilizes 3 skin irritation categories: no-label (in vivo score < 1.5), slight skin irritant (SI) (in vivo score ≥1.5 – in vivo score >2.3) (23). Harmonization of the currently validated in vitro SITs with the GHS scoring system cutoff for skin irritation (i.e. corresponding to in vivo score ≥ 2.3) as described above was a helpful step. However, validated protocols that define the slight irritant category remain to be developed.

One approach towards developing an in vitro GHS skin irritation protocol is currently being explored with the EpiDerm model by MatTek Corporation. This approach involves incorporation of an additional treatment time-point into the currently validated skin irritation protocol in order to match the 2 cutoff scores (corresponding to in vivo skin irritation scores of 1.5 and 2.3) utilized by the GHS system (11). One difficulty associated with developing this concept is a lack of mildly irritating reference substances with reliable in vivo data. Another problem is that the in vivo data are highly variable, and therefore no clear cutoffs exist between the three classes. Only few repeated in vivo experiments were reported in publicly available databases, and especially with mildly irritating chemicals, discordant classifications can be obtained (24,25). Additionally, the final classification of many chemicals has been found to be very much influenced by purity, supplier and age of the chemicals. Finally, some chemicals that are irritating to rabbit skin, and many chemicals that are mildly irritating to rabbit skin, are not found to cause the corresponding effects in human skin (26,27).

To-date, the EpiDerm GHS SIT protocol that is currently under development has produced promising results for a set of 36 reference chemicals including 12 chemicals from each GHS classification category (28). However, due to the narrow range of the SI category (in vivo score ≥1.5 – in vivo or in vitro) to attain a high concordance with the SI category. Nevertheless, protocols with three GHS skin irritation categories may be useful for certain applications and circumstances. Transferability of the EpiDerm™ GHS-SIT protocols will be further evaluated prior to proceeding to formal validation studies.

Testing of mixtures and formulations

Currently validated skin corrosion and irritation assays were designed to identify acute hazard potential of chemicals, and most validation testing for this purpose was conducted with individual neat chemical compounds, with only a few simple chemical mixtures included in the validation studies. It is unclear how well the current assays will perform with more complex mixtures and formulations. Testing of formulations, especially those that are designed to stick to surfaces (e.g. pesticides), may be challenging for the currently validated in vitro assays. For prediction of moderate to mild effects, rank ordering and risk assessment needs, various time-to-toxicity protocols may be more suitable (29). When developing new in vitro test protocols, it will be important to consider the needs for different testing purposes such as accidental exposure conditions (chemicals, pesticides), intended “wash-off” or “leave-on” conditions (cosmetic ingredients and products), or applications on impaired skin (pharmaceuticals).

Alternate skin irritation assay endpoints

All of the currently validated RHE SITs are based on tissue viability endpoints. Other endpoints, such as cytokine (e.g. IL-1, IL-8 and others) or lipid derived mediator (e.g. PGE2) secretion, have also been evaluated. There have been some suggestions and reports that use of multiple endpoints (e.g. MTT viability and IL-1α) may improve SIT performance (30,31). As mentioned above, alternate endpoints might prove useful for situations where the MTT assay is not possible (i.e. with highly colored test agents that interfere with the MTT reading). However, so far, MTT viability has proven to be the most robust and reliable endpoint (29,32). In general, for chemicals whose mechanism or mode of action involves perturbation of cellular functions in the viable epidermal cells, cytokine release assays have tended to simply mirror viability results and have proven to be more variable as well. Thus, to-date, cytokine assays have not provided significant improvements when combined with MTT viability results (29,32).

There are some specific exceptions and situations, however, where cytokine assays may be expected to offer advantages over viability endpoints. For example, disruption of epidermal barrier function is a well established mechanism for production of skin irritation (33). Epidermal stratum corneum barrier disruption can occur without overt toxic effects to the underlying keratinocytes. Certain aliphatic hydrocarbon components of jet fuel are known skin irritants. In recent studies it has been shown that jet fuel components with a specific lipophilicity and volatility range induce cytokine release without significant MTT viability loss (34) while histological assessment shows stratum corneum disruption without signs of toxicity to the viable epidermal layers. Release of IL-6, and IL-8 was correlated with the in vivo skin irritation potential in these cases (34). Thus, for skin irritants that have a specific mode of action primarily involving stratum corneum disruption, cytokine release appears to be a useful endpoint. Additional work on cytokine and other multiple endpoint assays may ultimately yield improved performance of RHE SITs, as well as additional information regarding mode of action. Additional experimentation and close cooperation between model developers, industry users and experts from dermatology, biochemistry and biology will likely facilitate advancements in this area.

Education, training, promotion and usage monitoring of in vitro skin corrosion and irritation tests

Now that OECD TGs are available for in vitro skin corrosion and irritation tests, it is important that efforts and resources are allocated towards education, training and promotion of the tests. Workshops, hands-on demonstrations and dedicated training in the use of alternative methods are effective tools for outreach to young scientists and general public education. It will be important for these activities to be supported by agencies or organizations such as ECVAM, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the German Centre for Documentation and Evaluation of Alternatives to Animal Experiments (ZEBET), non-profit organizations such as the Institute for In Vitro Sciences (IIVS), national toxicology societies, universities and industry stakeholders concerned about animal welfare.

Due to recently enacted or upcoming legislative changes, it will be important for industry to implement the existing in vitro OECD test methods, and to submit their data to regulatory agencies for review. It will also be important for regulatory agencies to have transparent mechanisms in place for tracking and evaluating in vitro test method usage. Data should be further reviewed on a regular basis to allow for evaluation of experiences with the new in vitro methods, and to evaluate their effect on reduction of animal tests. It will be important to maintain close cooperation among all stakeholders including model producers and assay developers, so that needed improvements or adjustments can be made and any deficiencies corrected.

Summary

Implementation of in vitro methods for skin corrosion and skin irritation testing is continuing to rapidly advance. Four OECD TGs for in vitro skin corrosion and irritation tests are now in place for regulatory use, thus enabling reduction and replacement of animal experiments. Despite a lack of applicability for certain specific regulatory requirements, industry acceptance and use of the existing TGs appear to be strong. Major areas that still require protocol development, validation or improvement include complex classification schemes for corrosion packing group labeling and skin irritation potential of pesticides. Efforts to promote education, training and further implementation of the in vitro tests, as well as mechanisms for monitoring in vitro method use by industry and regulatory agencies, will maximize the effectiveness of the validated in vitro test methods for reducing animal use.

The significant accomplishments of the past and continued spirit of commitment and cooperation by numerous stakeholders provide an encouraging foundation for future efforts to confront the remaining challenges for in vitro skin corrosion/irritation testing.

Acknowledgment: The authors would like to express their thanks to Dr. Manfred Liebsch (ZEBET at the Federal Institute for Risk Assessment, Germany) for review and valuable comments on the essay.
©2010 Helena Kandárová, Yulia Kaluzhny, Mitchell Klausner & Patrick Hayden

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