Originally published December 1996
Standards for medical device biocompatibility testing have become much more clear and useful in recent years. There are now documented procedures for several common biological tests, as well as matrices to help determine which kinds of tests to use. Despite this tremendous progress, however, the standards contradict one another on some important aspects of biocompatibility testing. Some aspects are not covered at all by the existing standards. With such confusing information, it can be difficult to be sure that a medical device meets all necessary requirements for biological safety.
The requirements for biocompatibility testing of biomaterials were adapted from classical toxicology standards. The potential adverse biological effects of biomaterials are, in most cases, the same as those caused by chemicals or drugs: irritation, sensitization, organ and tissue toxicity, mutagenicity, tumors, and reproductive problems.
The standards that have guided biocompatibility testing are the Tripartite Guidance; the International Organization for Standardization (ISO) 10993 standards, which are known as the Biological Evaluation of Medical Devices and remain under development internationally; and the FDA blue book memorandum, #G95-1, which is currently used only in the United States.
The Tripartite Guidance provides useful background information, a brief discussion of the principles that need attention in testing for biocompatibility, and a matrix relating intended use of a device to the categories of biological changes that may be caused by chemicals extractable from biomaterials. It does not describe testing methods. The ISO 10993 standards are being developed in multiple sections that contain all the Tripartite-like information, but also give as much detail as available on suitable test methods for ensuring the absence of undesirable biological effects that may be caused by the chemicals extractable from biomaterials. The relatively new FDA blue book memorandum, a variation on the ISO 10993-1 standard, addresses only the selection of tests.
These three bodies of standards identify 12 principal categories of possible biological effects. The FDA blue book memorandum and ISO 10993-1, "Guidance on Selection of Tests," also offer a matrix that makes it relatively easy to select the categories of biological effects that are of concern for the materials in a particular medical device.
But in several areas of biocompatibility testing, there is a lack of direction in the existing standards. For example, there is in some cases not enough, and in other cases conflicting, information on test methods. The existing standards also do not offer clear guidance on how to prepare the test samples properly or even to characterize the materials for testing.
CHARACTERIZING TEST MATERIALS
Biocompatibility testing is nearly without value unless the test and control articles under study have first been characterized in appropriate ways to permit repeating the studies and to ensure that the clinical devices of concern will be made from materials representative of the tested ones. In a section on general principles, ISO 10993-1 says that in the selection of materials, "the first consideration should be fitness for purpose having regard to the characteristics and properties of the material, which include chemical, toxicological, physical, electrical, morphological, and mechanical properties." According to this standard, the device characteristics that are relevant to biocompatibility testing are "the materials of manufacture, intended additives, process contaminants and residues, leachable substances, degradation products, other components, and their interactions in the final product, and the properties and characteristics of the final product."
Materials characterization as it relates to biocompatibility is likely to receive considerable attention in the years ahead, largely because, for such an important issue, it has received little attention in the past.
It is not enough to subject an uncharacterized material (e.g., one identified only as a "blue PVC tubing") to a biocompatibility test, because such a test would not be repeatable. Lack of characterization would also mean that there would be little or no basis for analytical evaluation of "problem" lots of materials when they are encountered.
Because characterization is so essential, the next revision of ISO 10993-1 circulated for approval this year will contain a flowchart, similar to the one contained in FDA's blue book memorandum, in which the first box will instruct the user to characterize the material before proceeding to biological testing.
Characterization can be accomplished with many methods, depending on the criticality of the materials. It may mean determining physical properties or confirming major components with infrared analysis or other fingerprinting techniques. It may include physicochemical tests to determine the weight of nonvolatile residues present in an aqueous or alcoholic extract prepared under controlled conditions or tests to identify specific extractables and their relative quantities.
The phrase materials characterization is actually used in two related but distinctly different contexts. In the first context, it refers to knowing the qualities and components of a material, so that biological test results can be associated with a particular formulation for future reference.
In the second context, the phrase refers to a step in the risk-assessment process. This type of characterization is described in a 48-page draft released in December 1995 by FDA's Center for Devices and Radiological Health entitled "Guidance for the Biological Evaluation of Materials." According to the "new paradigm" described in this document, materials are first characterized by identifying and quantifying their principal available chemicals. The industry literature is then searched to determine the relative toxicity of those components. Judgments are made about the safety of medical devices based on the toxicity of the chemicals they contain and the amounts of those chemicals that are available to patients. If only minimal quantities of extractables are present, no further testing is required.
In principle, this is a very logical approach. It avoids unnecessary, often duplicative testing by using the results of existing toxicity studies. It replaces, whenever possible, empirical in vitro or in vivo testing with systematic, scientific calculations.
However, this approach is years away from being useful, except in isolated situations, because there are insufficient toxicity data for the range of chemical species recoverable from material formulations used in medical devices. Also, although there is much information on systemic responses, not enough data are available to predict local tissue responses.
To achieve true standardization of test methods, it is essential to control and standardize the preparation of the test and control articles. This is particularly true in the case of biomaterials, which must often be subjected to extraction with specific fluids under specified conditions to prepare suitable dosage forms. ISO 10993-12, "Sample Preparation and Reference Materials," deals with sample preparation and eventually should be the only standard in the series that does so. For now, many of the other ISO 10993 standards also address sample preparation, sometimes in confusing and potentially contradictory ways. It is likely that this will be corrected in the ongoing revision process.
Once the materials are characterized and the samples prepared for testing, the next step, selecting specific test methods, can be difficult, depending on the categories of biological effects for which a device is to be tested. Some test methods have been in use for many years, are generally accepted in the industry, and are described in great detail in the various ISO 10993 standards. Thus, if cytotoxicity testing is required (as it is for the materials in every device category), ISO 10993-5, "Tests for Cytotoxicity: In Vitro Methods," provides protocol-like directions for the use of several alternative laboratory methods (elution and overlay are the ones most commonly used). The standard describes sample preparation; appropriate cell lines; culture media; and methods of handling, incubating, and scoring cells. It requires that triplicate flasks be used for each sample (more replicates than laboratories have typically used) and lists the information that must be included in the test report.
For other categories of biological effects, the amount of detail included in test methods or protocols described in the respective ISO standards varies according to the availability of accepted methods that either have been validated or have simply become widely used and accepted by the medical device industry. In general, the more familiar the tests are by virtue of their years of use, the more detail is presented in the standards. Thus, ISO 10993-10, "Tests for Irritation and Sensitization," describes the intracutaneous injection test, the primary skin irritation test, and the maximization sensitization test in protocol-like detail. All three of these tests are based on evaluations that have been widely used in the pharmaceutical, cosmetic, and medical device industries for more than 20 years. This standard also includes a listing of additional irritation tests (e.g., oral, vaginal, penile, and rectal models) that have been used somewhat less extensively.
Like the requirement for triplicate flasks in the cytotoxicity standard, there are unique aspects even of the traditional tests as interpreted by the authors of ISO 10993-10. For example, the ISO intracutaneous test method is not the same as the one described in most pharmacopoeial texts. For example, the ISO version requires that three rather than two animals be used for each test article and describes a scoring scheme that is a hybrid of the one used for the intracutaneous test and the one used for the primary skin irritation test. In the case of the Magnusson-Kligman sensitization method, the ISO version contains numerous small variations from the test originally described by its developers in 1969.
The standard also varies from common practice in some small details. For example, ISO requires that the fur on guinea pigs be clipped the day before they are treated with the test or control article. In practice, however, clipping the fur on the day of treatment has become standard in many laboratories and is considered preferred, especially by technicians who conduct the tests.
For systemic toxicity, ISO standard 10993-11, "Tests for Systemic Toxicity," is of little value to the industry because it contains no details applicable to biomaterials testing, instead referring to methods that were written specifically for chemicals and drugs. The standard references U.S. and European pharmacopoeias as well as the standards and methods of FDA, the Environmental Protection Agency, the American Society for Testing and Materials (ASTM), and the Organization for Economic Cooperation and Development (OECD) guidelines for testing chemicals. These various reference sources are simply listed in the ISO standard without any experimental details in sections on subjects such as acute systemic toxicity, acute oral application, subchronic dermal application, chronic toxicity, and carcinogenicity. Though the principles behind chemical and drug tests are generally sound, they can be applied to the study of medical devices only after modification. Some subjects covered in the standard, such as acute application by inhalation, are virtually never used in testing medical devices.
ISO 10993-3, "Tests for Genotoxicity, Carcinogenicity, and Reproductive Toxicity," also contains no experimental detail and refers to the OECD guidelines. However, the standard does provide some general advice about the extent of testing required for devices that may have problematic biological effects. For example, in the case of genotoxicity, the standard says that "a series of in vitro tests shall be used. This series shall include at least three assays. At least two of these should preferably use mammalian cells as the target. The tests should preferably cover the three levels of genotoxic effects: DNA effects, gene mutations, and chromosomal aberrations."
Thus, although in the past FDA has considered the Ames bacterial reverse mutation test sufficient to ensure the absence of genotoxic effects, two additional in vitro tests with mammalian cells must be included to satisfy international requirements. The ISO standard also describes materials that require carcinogenicity testing: materials that produce a positive in vitro genetic toxicity effect on mammalian cells, resorbable materials, and materials that will remain in the body for 30 days or longer. Carcinogenicity test methods are based on modified OECD guidelines designed for implantation of the maximum implantable dose and some fraction of that dose in separate groups of animals. Intrauterine devices, energy-depositing devices, and resorbable materials are identified as candidates for reproductive toxicity tests, also based on information from the OECD guidelines. In practice, genotoxicity tests are commonly performed to qualify materials, but carcinogenicity and reproductive effects studies are seldom considered necessary based on the history of the materials and their chemical composition. Very few of the many biomaterials in use today have been subjected to rigorous carcinogenicity or reproductive effects studies.
ISO 10993-6, "Tests for Local Effects after Implantation," recognizes the potential for implantation of materials in a wide variety of alternative animal species including mice, rats, guinea pigs, rabbits, dogs, sheep, goats, and pigs. The two-rabbit muscle implant method has its origins in the United States Pharmacopeia implant test for plastic pharmaceutical containers and is still widely used. However, ISO variations on the USP test may now include the use of more animals, different species, longer implant intervals, additional implant sites, and microscopic examination of tissue reactions. The choice of species is based on implant size and test duration in relation to the expected life span of the animals. Multiple test periods are generally specified "to ascertain that a steady state has been reached with respect to biological response." Thus, for short-term implantation in rabbits, 1-, 4-, and 12-week test periods are prescribed; for long-term implantation, 12, 26, 52, and 78 weeks.
Some materials produce adverse effects only in the immediate postimplantation period, presumably because of the effects of surface chemicals that are rapidly dissipated in the body. Other materials produce few or no acute effects, but as they undergo biodegradation, their breakdown products eventually cause irritation. The authors of ISO 10993-6 intended for local effects to be evaluated by comparing the tissue response caused by a test article to that caused by materials used in medical devices whose clinical acceptability has been established.
Because there is a lack of standardized testing models for studying the effects of biomaterials on blood, ISO 10993-4, "Selection of Tests for Interaction with Blood," deals largely in generalities. The standard provides information on the classification of products that require biological testing and the categories of hematologic effects that may be caused by biomaterials, and offers lists of commercially available assays for various hematological effects, informative annexes with general information on devices and laboratory tests, and an extensive bibliography. In the sense that it does not give procedures or how-to information, the standard is not yet a standard at all; it is more of a review article.
However, the document does contain a useful list of five test categories that need to be considered for various kinds of blood-contact devices, and it appears that as validated methods become available, they could logically be added to the standard under one of these categories, such as the effects on thrombosis, coagulation, platelets, hematology, and immunology.
In the past, FDA has generally accepted hemolysis testing and sometimes thromboresistance testing as sufficient evidence for hemocompatibility. But, as the lack of detail in the ISO 10993-4 standard indicates, there is not yet enough information in this area. The ASTM hemolysis test (which was recently modified and is undergoing reapproval) is an excellent test for hemolytic activity. Several methods now exist for studying effects on complement activation, which is an immunologic effect. Methods are under development for studying effects on the coagulation proteins in decalcified plasma and on the formed elements of blood in vitro and in vivo. Numerous models exist for studying the thrombogenic properties of materials, but most are relatively unsatisfactory because they are difficult to perform, are subject to experimental variables that are difficult to control, and require samples in special forms that may be unavailable. These are perplexing difficulties, particularly because most materials used in medical devices are thrombogenic and most blood-contact materials are used clinically only for patients who have received anticoagulant drugs.
OUTLOOK FOR THE FUTURE
Methods for biocompatibility assessment of medical devices are evolving. The ISO 10993 standards, the FDA blue book memorandum, and the "Japanese Guidelines for Basic Biological Tests of Medical Materials and Devices," issued in 1995, are all steps toward providing specific methods that can be used by device manufacturers and recognized by regulatory systems worldwide. But, because there is disagreement among those in charge of regulations, there is not yet clear direction on all the issues. For example, there is confusion on characterizing materials for testing, preparing the samples, and testing methods. There is also need for detailed standardization on the selection of tests according to the end use of a device, methods of sample preparation, number and kinds of tests to be performed once test categories have been selected, and definition of a positive and a negative test result. In addition, those who assess safety data for regulatory review have yet to agree on when specific testing must be performed, especially costly, time-consuming testing, to what extent existing data and literature can be used in support of device safety, and by what degree specific testing protocols may vary from standards and still be considered acceptable. So the search continues for the most practical, affordable, and valid ways to ensure the biological safety of medical devices.
Richard F. Wallin, DVM, PhD, is the president of NAmSA (Northwood, OH).