Skin Sensitization: Moving Towards a Validated In Vitro Test

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In the Spotlight

Skin Sensitization: Moving Towards a Validated In Vitro Test

Published: September 7, 2010
Determination of skin sensitization potential is a critical toxicological endpoint in the development and evaluation of ingredients used in fragrance, cosmetic and personal care products. With the deadline to replace animal testing for skin sensitization in the EU quickly approaching, alternative tests are urgently needed to replace animal tests to evaluate the skin sensitization potential of chemicals. The development and validation of in vitro methods to evaluate skin sensitization are moving forward steadily with contributions from the fragrance, cosmetic, and personal care industry, regulatory agencies, contract labs, and animal welfare groups. The European Centre for the Validation of Alternative Methods (ECVAM) has initiated formal phase 3 validation trials of the human cell line activation test (h-CLAT), the myeloid U937 skin sensitization test (MUSST), and the direct peptide reactivity assay (DPRA). Additional technologies, such as the KeratinoSens assay, are undergoing ring trials in preparation for ECVAM review. In response to the burgeoning development of in vitro skin sensitization methods, the Institute for In Vitro Sciences (IIVS), a global leader in the advancement of in vitro methods, is hosting the 2010 In Vitro Alternatives Forum (October 18-19, Old Town Alexandria, VA) with a special emphasis on integrated testing strategies to identify skin sensitizers, featuring lectures from top scientists in the field.

One such expert and co-organizer of the session is Dr. Frank Gerberick of the Procter & Gamble (P&G) Company. We recently posed several questions to Dr. Gerberick regarding this fast-evolving field and the aims of the session at the IIVS workshop.

For those members of the AltTox community who are not overly familiar with this field, could you explain what skin sensitization is and how it relates to allergic contact dermatitis?

Skin sensitization is induced when a susceptible individual is exposed topically to a chemical allergen. This chemical allergen provokes a skin immune response which, if of the required magnitude and quality, will result in the development of contact sensitization. A number of key steps in this process can be identified. Importantly, the proliferation of allergen-responsive T lymphocytes results in clonal expansion of cells that are able to recognize and respond subsequently to the same inducing allergen. It is this selective clonal expansion of T lymphocytes that provides immunological memory and that represents the cellular basis for skin sensitization. If the now-sensitized subject is exposed again to the inducing chemical allergen, at the same or a different site, then this expanded population of specific T lymphocytes will recognize and respond to the allergen in the skin at the site of contact. The activation of T lymphocytes is associated with the release of cytokines and chemokines that collectively stimulate the influx of other leukocytes and initiate the cutaneous inflammatory reaction that is recognized clinically as allergic contact dermatitis.

How close are we to completely replacing the current in vivo skin sensitization test methods with one or more in vitro procedures?

In my opinion, significant progress has been made in recent years in the development of alternative methods for assessing a chemical’s skin sensitization potential. If the evaluation of newer methods continues to progress well, then I would think the use of alternative assays in combination would be available in the coming years for many different chemical classes.

What is the aim of the skin sensitization session at the October Alternatives Forum?

The overall aim of the session is to inform stakeholders about the scientific and validation activities related to the development of in vitro approaches to identify skin sensitizers. Robert Landsiedel (BASF) and I worked with the organizing committee to have global representation in our session and to make sure that the most recent activities in the field of alternatives for skin sensitization are covered. Thus, we have talks focused on current research activities and validation efforts as well as a clinical perspective from a dermatologist’s point of view.

Can you briefly describe a couple of the more promising in vitro tests in this field?

With obvious bias, I feel the direct peptide reactivity assay we have developed at P&G shows promise for utility in skin sensitization testing. In addition, the THP-1 (h-CLAT) and U937 (MUSST) cell-based assays have shown potential for use as well. Each of these methods, which have been supported by COLIPA (the European Cosmetic Industry), is currently under evaluation by ECVAM. It is important to point out, however, that a number of other reactivity assays (Keratinosens) and cell-based methods that are in different stages of development are showing potential as well so it will be very important for all involved to make sure we adequately evaluate these methods and do so in a timely manner.

Can you briefly explain your own work in this field at Procter & Gamble?

Well, I have been working in the field of skin sensitization for 20+ years. In those years I have gone from guinea pigs to mice (LLNA) and now our entire focus is on the development of replacement alternatives. It is important to note that the LLNA offered significant animal welfare benefits including both refinement and reduction. My experience over the years has been that the community of scientists working in this field, including those in industry, academia and government, are extremely talented and are very open to collaboration. Much of the research that I have been privileged to work on has involved scientists from these organizations and who are represented around the world. I have no doubt that we would not be as far along in reaching our goals if not for this amazing collaborative effort.

In other areas of testing, non-animal methods have been developed, validated, and accepted, but they don’t necessarily rely on a deep understanding of the underlying biology. Much of the excitement around the promise of in vitro skin sensitization testing is that we know so much about the biology. Do the most promising replacement alternatives in this field capitalize on this knowledge?

Absolutely, all of the test methods under development that I’m aware of are based on a deep mechanistic understanding. For many years there has been a research focus on trying to understand the underlying chemical and biological mechanisms of skin sensitization. There is no doubt that the results of this research have supported the development of peptide reactivity assays and cell-based dendritic cell and T-cell assays.

What kind of animal and/or human data is being used to assess the predictive capability of the in vitro methods?

Of course the LLNA has been invaluable in the evaluation of alternative methods for skin sensitization testing. The assay is quantitative and allows one to evaluate not only skin sensitization potential but also the degree of sensitization as defined by a chemical’s skin sensitization potency (e.g., strong, moderate or weak). This is required information for toxicologists involved in performing quantitative risk assessments for skin sensitization. Historical human data on skin sensitization is available but is limited. Fortunately, a number of studies that have investigated human data in comparison to LLNA data have reported a good correlation between the two methods. Thus, it is believed that LLNA data will serve us well in our development of alternative methods as long as we don’t forget the purpose of our new methods is to predict human skin sensitization.

Have you been working with the validation centers to structure validation studies and data analysis? What kind of feedback do they provide?

Yes, we have been working with ECVAM on their evaluation of the direct peptide reactivity assay (DPRA). To date, the interaction has been very positive and we are very close to starting a coded study involving two newly trained laboratories. The technical review of our protocol by ECVAM scientists and the interactions with the new laboratories conducting the assay have led to the development of a more robust SOP which will no doubt help us to succeed with our upcoming study.

Are we likely to wind up with one-to-one replacements or are you anticipating an integrated testing approach for at least the short-term?

I believe most scientists working in this field of skin sensitization alternatives believe that an integrated testing strategy (ITS) will be needed to replace the multistep process of skin sensitization. ITS approaches will allow us to use a combination of individual alternative assays (e.g., in vitro, in chemico, in silico) in a smart way to help replace the complex in vivo methods we are currently using for skin sensitization. Exciting work is already underway to develop ITS approaches for utilizing data from the different assays being developed for skin sensitization testing. It is possible that in the future a sophisticated one-to-one replacement will be developed but I believe that in the short-term we will be relying on ITS approaches.

How does the ongoing work in skin sensitization relate to the pathway approach outlined in the National Research Council’s 2007 report on “Toxicity Testing in the 21st Century”?

The research that has been conducted over the past 5 years or more and that will be focused on during the 2010 In Vitro Alternatives Forum has definitely helped to pave the way for “Toxicity Testing in the 21st Century.” I must say these are exciting times to be working in toxicology and I’m sure that taking a new systems biology focus for skin sensitization testing will lead to new and more predictive modeling approaches in the future.