Progress is being made when it comes to moving away from guinea pigs and rabbits, and developing lab-based tests acceptable to FDA.
Thor Rollins, Nelson Laboratories
|In vitro biocompatibility testing (Image courtesy of Nelson Laboratories)|
While there are many obstacles on the path to U.S. regulatory acceptance and validation of in vitro irritation and sensitization models, much progress has been made in developing a medical device specific irritation model.
Every medical device manufacturer must conduct the “Big Three” biocompatibility tests for cytotoxicity, irritation, and sensitization, or provide justification for why the tests were not performed. Cytotoxicity testing is performed in vitro, while irritation and sensitization tests are currently conducted in vivo on guinea pigs and rabbits, something industry is working to change.
Moving to in vitro testing for sensitization is important because the most common test is the guinea pig maximization test. This test is expensive, has a high animal burden, and can take up to nine weeks to complete. In an effort to minimize the time and cost of these tests, and to reduce animal testing, alternatives like in vitro irritation and sensitization testing are being developed. Even with irritation testing, cost and turnaround time could be dramatically reduced with widespread adoption.
The Road to In Vitro Irritation Method Approval
In vitro irritation is already widely accepted in Europe for chemicals and cosmetics. Currently, the medical device industry experts are collaborating to develop an extractable positive control for in vitro irritation testing which may correlate to U.S. regulatory acceptance.
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) has done much of the legwork in creating and validating a plan for in vitro irritation testing of cosmetics. The medical device industry endeavors to now incorporate ECVAM’s validation into a medical device specific validation.
U.S. FDA has indicated that they want a validation to show that a response can be derived via extraction in the form of a positive control before it will approve in vitro sensitization and irritation testing. In other words, FDA would like proof that extraction methods can pull off enough irritants to cause an irritation response. Validating for in vitro can be difficult because, by design, few medical devices contain irritants or sensitizers.
While in vitro skin irritation test methods have historically produced results consistent within vivo data, they have involved the use of chemical solutions, or spiked extracts, as positive controls. To provide a method that more accurately mimics validated in vivo medical device test methods, in which devices are extracted in polar and non-polar solvents, in vitro research has focused on finding an extractable material that will induce a positive skin irritation response in both solutions.
Nelson Laboratories’ recent discovery that heat-pressed polyvinyl chloride (PVC) sheets infused with Genapol X-080 act as an irritant in both polar and non-polar fluid is a major step toward regulatory acceptance. Nelson Laboratories presented its findings and validation data at the 2015 Society of Toxicology (SOT) annual meeting. Regulatory response to the data, which indicates in vitro results are in line with in vivo results, was positive.
How Is In Vitro Testing for Irritation Performed?
In vitro irritation testing is performed by using human-derived epidermal keratinocytes (NHEK), also known as reconstructed human epidermis (RhE). The RhE is cultured in such a way as to form a multilayered, highly differentiated model of the human epidermis. Medical device extracts or chemicals are then applied to the epidermis to predict irritation by conducting an MTT cytotoxicity test. The RhE closely mimics the biochemical and physiological properties of the human epidermis. To acquire the extract to be applied to the epidermis, medical devices are put into polar and non-polar media, allowing elements to be extracted for epidermal testing where irritation can be tested.
Current in vivo irritation testing is performed by extracting leachables from the device in a polar and non-polar medium. That resulting extraction is then injected intracutaneously into rabbit test subjects. Redness and/or swelling would indicate an irritation response, which is ranked according to the level of irritation to determine whether or not the device met the standard. In more than 30 years of testing, what we have observed at Nelson Labs is that most medical device materials are safe and have a low probability of causing an irritation. However, device residuals, like detergents and chemicals from the manufacturing process, may cause an irritation response.
Should the in vitro irritation method gain regulatory approval in the U.S., we believe it would essentially function as an irritation screening process. If irritation is observed in vitro, an animal test would be a secondary verification of that failure. Currently, the cost and turnaround time for in vitro irritation testing is comparable to the cost and time involved with in vivo testing. However, cost and turnaround time could be dramatically reduced with widespread adoption.
What About In Vitro Testing for Sensitization?
The in vitro sensitization test is not as well defined in the industry as the in vitro irritation model. EURL ECVAM is in the process of validating this in vitro method. Nonetheless, the issue is that the in vitro sensitization test requires observation of multiple biomarkers to assess sensitization. Currently, we are working on three different tests for the in vitro sensitization method to gather the endpoints: the Glutathione Reduction Assay, the ARE Gene Expression, and the LDH Cytotoxicity Assay or MTT.
With three tests, the question becomes how do you use three tests to predict sensitization? Answering this question has created controversy in the industry. Some labs have developed algorithms that use the three in vitro sensitization test endpoints to predict sensitization. EURL ECVAM is looking at these endpoints (among others) and working to validate each of them against animal tests to determine which procedure or combination of procedures most accurately mimics the potential clinical application. What method will win out in the end is to be determined.
Current in vivo sensitization testing accepted by FDA commonly involves the guinea pig maximization test. Turnaround time on this test is eight to nine weeks. Because sensitization testing dictates repeat exposure before a biological reaction can be observed, the guinea pig maximization test is the most expensive of the Big Three tests. In addition to limiting animal testing, it is the elimination of this expensive, multi-week test that makes the potential benefits of in vitro sensitization so impressive.
Cytotoxicity Testing Is Already In Vitro in U.S.
The cytotoxicity test is one of the biocompatibility tests currently performed in vitro and accepted by the FDA. Cytotoxicity testing is performed using mouse fibroblast cells, also known as L929 cells. The cells are a nice test platform because L929 cells are adhering cells, meaning they naturally adhere to the walls of the testing flask used to “farm” the cells.
The cytotoxicity test excels in many areas. It is the most sensitive screening test option available. It is also the quickest and the cheapest, primarily because it is an in vitro test and testing is done with cells that develop quickly. This eliminates animal acclimation, which generally takes more time. If you want to analyze a product or material in the R&D phase, cytotoxicity testing is a particularly nice option because it is cheap, fast, and super sensitive. You will know if you have an issue, and it will allow you to make the necessary adjustments for better testing outcomes once the product is ready to go to market.
What Does This All Mean for Device Manufacturers?
All medical device manufacturers must conduct biocompatibility testing. Current biocompatibility test methods generally mean a long, expensive testing process. Once approved by FDA, in vitro testing offers manufacturers a more timely and cost efficient alternative which will also reduce animal testing. Testing that normally takes anywhere from eight to nine weeks can be reduced to two to three weeks thereby significantly lowering costs.
Nelson Laboratories has been conducting in vitro tests for over 20 years, and is actively involved in working with FDA and ISO 10993 representatives to ensure that when in vitro sensitization and irritation method development is complete, the FDA will recognize and accept those submissions.
Skin Irritation/Corrosion, European Chemicals Agency (ECHA), 2013.
Analysis of Interleukin-1α (IL-1α) and Interleukin-8 (IL-8) Expression and Release in In Vitro Reconstructed Human Epidermis for the Prediction of In Vivo Skin Irritation and/or Sensitization, ScienceDirect, 2003.
Extractable Positive Control for In Vitro Skin Irritation Testing of Medical Devices, Nelson Laboratories’ Society of Toxicology Poster Presentation, 2015.
Evaluation of an In Vitro Human Dermal Sensitization Test for Use with Medical Device Extracts, Applied In Vitro Toxicology, 2015.
The Big Three: Tests Manufacturers Need to Know, Medical Design Technology, 2010.
Thor Rollins is a biocompatability specialist at Nelson Laboratories (Salt Lake City).