For medical device testing, an in vitro approach for assessing skin sensitization potential

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For medical device testing, an in vitro approach for assessing skin sensitization potential

The publication of a study by Coleman and colleagues (2015) that evaluates the SenCeeToxTM as a possible in vitro alternative for assessing skin sensitization in medical devices was recently named “Best Published Paper” by the Society of Toxicology Medical Device Specialty Section.

Skin sensitization tests are among the tests recommended by regulatory agencies, such as the FDA, for medical device evaluations. Traditionally, at least one of three validated in vivo assays is used: the Guinea Pig Maximization Test, the Buehler Test, and/or the Murine Local Lymph Node Assay (LLNA).

Coleman, et al. (2015) note that political and regulatory pressures to reduce animal testing are spurring a search for in vitro alternatives to these traditional methods. In the last two years, EURL ECVAM has recommended three in vitro alternatives as valid for assessing different aspects of skin sensitization: the ARE-Nrf2 Luciferase Test Method (e.g., KeratinoSensTM), the Direct Peptide Reactivity Assay (DPRA), and the Human Cell Line Activation Test (h-CLAT). However, skin sensitization is a complex physiological sequence, and currently, a single in vitro test method cannot capture all of the key events. For that reason, organizations including EURL ECVAM and Cosmetics Europe have been developing Integrated Test Strategies (ITS) that combine in vitro, in chemico, and in silico methods to generate a more complete picture of sensitizer potential.

This is a welcome advancement, but as the authors of this article point out, all of the approved in vitro methods for evaluating skin sensitization were validated using pure test substances. In medical devices, potential sensitizers are often extremely diluted, mixed with other substances. The current study was designed to determine if the SenCeeTox assay could detect sensitizers at such low concentrations, which would make it a viable in vitro alternative for skin sensitization testing on medical device extracts.

The authors evaluated ten known sensitizers. Six were incorporated into medical device silicone (so that the resulting chemical concentration was 10%), and four were added to plain silicone. These ten preparations were then injected into both sesame oil and saline solvents, to create 20 test solutions. EpiDerm 3-D tissues were exposed to these preparations for 24 hours, and then assessed for viability, glutathione reactivity, and gene expression controlled by Nrf2/ARE.

Only three preparations reduced tissue viability (Diethyl maleate in sesame oil, and 2,4-Dinitrochlorobenzene in both saline and sesame oil). GSH binding (as measured by the glutathione reactivity assay) generally followed expected patterns: GSH depletion was greatest for strong sensitizers, and lowest with weak sensitizers. Variation in gene expression mostly followed expected trends: in most cases, strong sensitizers produced the highest amplitudes.

The authors also developed a proprietary algorithm to assign an estimated in vivo EC3 score and potency assessment to each test substance, based on results from the viability, reactivity, and gene expression analyses. As the authors explain, the EC3 value is determined from the LLNA assay, and is “the concentration (in percent) required to induce a positive threshold response. The correlation of the EC3 score and the potency has been established by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC)… .” The SenCeeTox assay predicted sensitization (extreme, strong, and moderate) or no sensitization with 80% accuracy for each test material, and predicted potency with 40% accuracy. (When the authors merged “strong” and “extreme” sensitizers into one category, potency prediction accuracy increased to 50%.)

The authors conclude that “the SenCeeTox assay combined with 3D EpiDerm tissues may be a useful in vitro model for accurately predicting the dermal sensitization potential of medical device extracts,” but recommend additional evaluation to determine whether it can work as a standalone method. Either way, they believe the model could be “a screening tool for dermal sensitization testing of medical devices, and thus reduce the need for animals while saving time and money.”

Article cited: Coleman K. P., McNamara L. R., Grailer T. P., Willoughby J. A. Sr., Keller D. J., Patel P., Thomas S., & Dilworth C. (2015). Evaluation of an in vitro human dermal sensitization test for use with medical device extracts. Applied In Vitro Toxicol 1(2): 118-130.
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