The Way Forward for In Vitro Skin Irritation Testing

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Skin Irritation-Skin Corrosion

The Way Forward for In Vitro Skin Irritation Testing

Patrick Hayden, MatTek Corporation

Published: December 6, 2007

About the Author(s)
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 has been employed by MatTek Corporation, 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

A major milestone for in vitro skin irritation testing was achieved with the recent release of the Statement on the Validity of In-Vitro Tests for Skin Irritation by the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC). The statement endorsed the use of in vitro skin models and the MTT endpoint as a replacement for the in vivo Draize (rabbit) Skin Irritation Test (OECD TG 404 & Method B.4 of Annex V to Directive 67/548/EEC) for the purpose of distinguishing between R38 (skin irritating) and no-label (non-irritating) chemicals. Thanks and congratulations are in order to all of the many dedicated individuals involved in the long and difficult efforts to make this day a reality, particularly those who served on the validation study Management Team, independent experts of the Peer Review Panel, the sponsoring organization ECVAM/Joint Research Centre, as well as the testing laboratories.

The success of the validation effort notwithstanding, several important challenges and issues for in vitro skin irritation testing remain. Three such challenges/issues will be discussed in this brief commentary: 1) Validation of additional in vitro skin models in order to establish the solid foundation of in vitro skin model supply that will be required to support their use in the regulatory arena; 2) The impact of the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) on the regulatory acceptance and use of the ESAC endorsed in vitro skin irritation test; and 3) The predictive ability of the in vivo Draize rabbit test with respect to human skin irritation hazard, and the use of Draize rabbit data as the gold standard for skin irritation validation studies.

Before beginning, it should be disclosed that the commentator is employed by MatTek Corporation, producer of the EpiDerm™ in vitro skin model.

At present, the ESAC statement fully endorses only one in vitro model (EPISKIN®) as a stand alone replacement for the in vivo Draize test. The EpiDerm™ model is currently endorsed as a validated component in a tiered testing strategy (as described, for example, by OECD TG 404). As history has shown (e.g. setbacks caused by the demise of the SKIN2 model from the marketplace) it will be important to ensure that multiple in vitro models are available to fill the need for in vitro skin irritation testing in the event of availability problems from any particular producer. Furthermore, it is critically important that validated in vitro models are able to deliver long-term reproducibility that will provide consistent, good quality data over time, not just during the validation process (Rispin et al., 2004).

Additional in vitro skin models currently exist, and new models will undoubtedly be introduced into the marketplace. These will need to be validated in future “catch-up” studies. Likewise, additional endpoints (e.g. IL-α) may be considered in future validation studies. Ideally, a “common protocol” that can be applied to any model would be desirable for ease of regulatory acceptance. Therefore, this approach was taken in the ECVAM skin irritation validation study. However, in hindsight, one lesson that was derived from the recent validation study is that a certain amount of flexibility should be possible to accommodate the multiple model systems that will be required in the marketplace to provide a firm foundation to support regulatory use of in vitro models.

As a case in point, the protocol utilized in the recent ECVAM skin irritation validation study, originally developed for use with the EPISKIN® model, and later optimized and applied to the EpiDerm™ model, ultimately did not function adequately with the EpiDerm™ model. That this situation occurred despite the best efforts and intentions of everyone involved in the validation effort underscores the subtle pitfalls and difficult nature of the validation process. It is now recognized that, due to differences in barrier properties between the EPISKIN® and EpiDerm™ models, a modified protocol providing a longer treatment time with test chemicals is required with the EpiDerm™ model in order to achieve similar levels of sensitivity (Kandarova et al., 2007). As a remedy, a follow-up validation of EpiDerm™ is now being conducted with the modified protocol, sponsored by the Centre for Documentation and Evaluation of Alternatives to Animal Experiments (ZEBET) of the (German) Federal Institute for Risk Assessment.

As the preceding example demonstrates, a flexible approach that can accommodate differences in model systems will facilitate successful development and validation of protocols for new models and/or endpoints. Availability of multiple validated in vitro skin model systems will be necessary to avoid past setbacks caused by sudden disruptions or unreliable in vitro skin model supply. Furthermore, demonstration of long-term reproducibility will be necessary to support regulatory use of in vitro skin models.

Regarding the impact of the Globally Harmonized System of Classification and Labeling of Chemicals (GHS), while the ESAC statement on in vitro skin irritation tests is indeed a major achievement, we are not out of the woods yet with regard to universal acceptance of in vitro tests for skin irritation.

The ESAC endorsed test will apply to chemicals marketed in the European Union. However, the United States and many other countries throughout the world will continue to follow the GHS, which at present continues to rely on the in vivo Draize skin irritation test. EU companies marketing chemicals in the US and other countries that adopt the GHS system will need to meet GHS requirements.

The ESAC endorsed in vitro test specifies a chemical as a skin irritant by criteria corresponding to an in vivo Draize score of =2 (OJEC, 1967). In contrast, the GHS specifies a chemical as a skin irritant by criteria corresponding to an in vivo Draize score of =2.3. Additionally under the GHS system, “for those authorities, such as pesticide regulators, wanting more than one designation for skin irritation, an additional mild irritant category (in vivo Draize score =1.5)

©2007 Patrick Hayden

References
Basketter, D.A., York, M., McFadden, J.P. & Robinson, M.K. (2004). Determination of skin irritation potential in the human 4-h patch test. Contact Dermatitis. 51, 1–4.

Jirova, D., Basketter, D., Bendova, H., Marriott, M., Kejlova, K., Kandarova, H., Spiller, E. & Liebsch, M. (2007). Human skin irritation study supports results of 3D human skin model in vitro. OEESC 2007. Golden, Colorado. Downloadable here.

Kandarova, H., Hayden, P., Spiller, E., Klausner, M., Kubilus, J. & Sheasgreen, J. (2007). In vitro skin irritation test: Increasing the sensitivity of the EpiDermTM skin irritation protocol evaluated in the ECVAM skin irritation validation study. Toxicol. Lett. 172(S1), S81.

Occupational Safety & Health Administration (OSHA). (2006). A Guide toThe Globally Harmonized System of Classification and Labeling of Chemicals (GHS). Available here.

Official Journal of the European Communities (OJEC). (1967). Council Directive 67/548/EEC of 27 June 1967 on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. OJEC 196:1-98.

Rispin, A., Harbell, J.W., Klausner, M., Jordan, F.T., Coecke, S., Gupta, K. & Stitzel, K. (2004). Quality assurance for in vitro alternative test methods: Quality control issues in test kit production. Altern. Lab Anim. 32(S1B), 725-729.

Robinson, M.K., Cohen, C., de Fraissinette A.B. Ponec, M., Whittle, E. & Fentem, J.H. (2002). Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products. Food Chem. Toxicol. 40(5), 573-92.

Welss, T., Basketter, D.A. & Schroder, K.R. (2004). In vitro skin irritation: facts and future. State of the art review of mechanisms and models. Toxicol. In Vitro 18(3), 231-243.