The following page describes some of the specific procedures recommended for biocompatibility testing. This listing does not imply that all procedures are necessary for any given device, nor does it indicate that these are the only available tests.

1. Cytotoxicity (Tissue Culture)
2. Sensitization Assays
3. Irritation Tests
4. Acute Systemic Toxicity
5. Subchronic Toxicity
6. Genotoxicity
7. Implantation Tests
8. Hemocompatibility
9. Carcinogenesis Bioassay
10. Reproductive And Developmental Toxicity
11. Pharmacokinetics
12. Preclinical Safety Testing
13. Histopathology Services

Cytotoxicity (Tissue Culture)

Cell culture assays are used to assess the biocompatibility of a material or extract through the use of isolated cells in vitro. These techniques are useful in evaluating the toxicity or irritancy potential of materials and chemicals. They provide an excellent way to screen materials prior to in vivo tests.

Qualitative Cytotoxicity Tests

There are three qualitative cytotoxicity tests commonly used for medical devices. The Direct Contact procedure is recommended for low density materials, such as contact lens polymers. In this method, a piece of test material is placed directly onto cells growing on culture medium. The cells are then incubated. During incubation, leachable chemicals in the test material can diffuse into the culture medium and contact the cell layer. Reactivity of the test sample is indicated by malformation, degeneration and lysis of cells around the test material.

The Agar Diffusion assay is appropriate for high density materials, such as elastomeric closures. In this method, a thin layer of nutrient-supplemented agar is placed over the cultured cells. The test material (or an extract of the test material dried on filter paper) is placed on top of the agar layer, and the cells are incubated. A zone of malformed, degenerative or lysed cells under and around the test material indicates cytotoxicity.

The MEM Elution assay uses different extracting media and extraction conditions to test devices according to actual use conditions or to exaggerate those conditions.  Extracts can be titrated to yield a semi-quantitative measurement of cytotoxicity. After preparation, the extracts are transferred onto a layer of cells and incubated.  Following incubation, the cells are examined microscopically for malformation, degeneration and lysis of the cells. (See All About Extracts section for more information on the selection of extracting media and conditions.) At least one type of cytotoxicity test should be performed on each component of any device.

Quantitative Cytotoxicity – MTT Assay

Recent regulatory additions (ANSI/AAMI/ISO 10993-5:2009) on biocompatibility for devices state that the qualitative cytotoxicity tests (direct contact, mem elution, agar diffusion) are appropriate for screening purposes, but that quantitative evaluation is preferable.

Annex C of ISO 10993-5:2009 refers to the MTT cytotoxicity assay, which can accurately quantify as few as 950 cells. The MTT is a colorimetric method that measures the reduction of yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide by mitochondial succinate dehydrogenase. Because the cellular reduction is only catalyzed by living cells, it is possible to quantify the percentage of living cells in a solution.

The MTT can be used to evaluate the cytotoxicity of:

• Extractable materials of medical devices
• Toxic compounds
• Toxins and environmental pollutants
• Potential anti-cancer drugs
• Antibodies to examine growth inhibiting potential

The major advantages of the MTT are its quantitative ability, that it can be done on either extracts or by direct contact, and that the results are not subject to analyst interpretation. Additionally, the MTT can be performed on 96-well microplates in a standard reader (such as a Bio-Tek ELx808) allowing for fast screening of multiple samples.

However, it should be noted that while the MTT is recommended, the MTT assay does not discriminate a specific cellular death mechanism – such as apopotosis vs. induced cell death. Additionally, it may underestimate cellular damage and only detect death at the last stages of the cellular dying process.

Sensitization Assays

Sensitization studies help to determine whether a material contains chemicals that cause adverse local or systemic effects after repeated or prolonged exposure. These allergic or hypersensitivity reactions involve immunologic mechanisms. Studies to determine sensitization potential may be performed using either specific chemicals from the test material, the test material itself, or most often, extracts of the test material. The Materials Biocompatibility Matrix recommends sensitization testing for all classes of medical devices.

The Guinea Pig Maximization Test (Magnusson-Kligman Method) is recommended for devices that will have externally communicating or internal contact with the body or body fluids. In this study the test material is mixed with complete Freund’s adjuvant (CFA) to enhance the skin sensitization response.

The Closed Patch Test involves multiple topical doses and is recommended for devices that will contact unbroken skin only.

The Murine Local Lymph Node Assay (LLNA) determinates the quantitative increase in lymphocytes in response to a sensitizer. If a molecule acts as a skin sensitizer, it will induce the epidermal Langherhans cells to transport the allergen to the draining lymph nodes, which in turn causes T-lymphocytes to proliferate and differentiate. From an animal welfare perspective, this test is preferable to the Guinea Pig Maximization Test or the Closed Patch Test.

Irritation Tests

These tests estimate the local irritation potential of devices, materials or extracts, using sites such as skin or mucous membranes, usually in an animal model. The route of exposure (skin, eye, mucosa) and duration of contact should be analogous to the anticipated clinical use of the device, but it is often prudent to exaggerate exposure conditions somewhat to establish a margin of safety for patients.

In the Intracutaneous Test, extracts of the test material and blanks are injected intradermally. The injection sites are scored for erythema and edema (redness and swelling). This procedure is recommended for devices that will have externally communicating or internal contact with the body or body fluids. It reliably detects the potential for local irritation due to chemicals that may be extracted from a biomaterial.

The Primary Skin Irritation test should be considered for topical devices that have external contact with intact or breached skin. In this procedure, the test material or an extract is applied directly to intact and abraded sites on the skin of a rabbit. After a 24-hour exposure, the material is removed and the sites are scored for erythema and edema.

Mucous Membrane Irritation Tests are recommended for devices that will have externally communicating contact with intact natural channels or tissues. These studies often use extracts rather than the material itself. Some common procedures include vaginal, cheek pouch and eye irritation studies. (See All About Extracts section for more information on extracts.)

Acute Systemic Toxicity

By using extracts of the device or device material, the Acute Systemic Toxicity test detects leachables that produce systemic (as opposed to local) toxic effects. The extracts of the test material and negative control blanks are injected into mice (intravenously or intraperitoneally, depending on the extracting media). The mice are observed for toxic signs just after injection and at four other time points. The Materials Biocompatibility Matrix recommends this test for all blood contact devices. It may also be appropriate for any other device that contacts internal tissues.

The Material Mediated Pyrogen test evaluates the potential of a material to cause a pyrogenic response, or fever, when introduced into the blood. Lot release testing for pyrogenicity is done in vitro using the bacterial endotoxin (LAL) test. It must be validated for each device or material. However, for assessing biocompatibility, the rabbit pyrogen test is preferred. The rabbit test, in addition to detecting bacterial endotoxins, is sensitive to material-mediated pyrogens that may be found in test materials or extracts.

Subchronic Toxicity

Tests for subchronic toxicity are used to determine potentially harmful effects from longer-term or multiple exposures to test materials and/or extracts during a period of up to 10% of the total lifespan of the test animal (e.g. up to 90 days in rats). Actual use conditions of a medical device need to be taken into account when selecting an animal model for subchronic toxicity. Appropriate animal models are determined on a case-by-case basis.

Pacific BioLabs offers two standard protocols for subchronic testing that are appropriate for many devices. Both are done in mice. One uses intraperitoneal administration of an extract of the device or device material. The other uses an intravenous route of administration. Implant tests are often performed for different durations appropriate to assess subchronic toxicity of devices and device materials.

Subchronic tests are required for all permanent devices and should be considered for those with prolonged contact with internal tissues.

Genotoxicity

Genotoxicity evaluations use a set of in vitro and in vivo tests to detect mutagens, substances that can directly or indirectly induce genetic damage directly through a variety of mechanisms. This damage can occur in either somatic or germline cells, increasing the risk of cancer or inheritable defects. A strong correlation exists between mutagenicity and carcinogenicity.

Genotoxic effects fall into one of three categories: point mutations along a strand of DNA, damage to the overall structure of the DNA, or damage to the structure of the chromosome (which contains the DNA). A variety of tests have been developed to determine if damage has occurred at any of these levels. These assays complement one another and are performed as a battery.   

The most common test for mutagenicity, the Ames test, detects point mutations by employing several strains of the bacteria Salmonella typhimurium, which have been selected for their sensitivity to mutagens. The Mouse Lymphoma and the HGPRT assays are common procedures using mammalian cells to detect point mutations. The Mouse Lymphoma assay is also able to detect clastogenic lesions in genes (chromosome damage). Assays for DNA damage and repair include both in vitro and in vivo Unscheduled DNA Synthesis (UDS). Cytogenetic assays allow direct observation of chromosome damage. There are both in vitro and in vivo methods, including the Chromosomal Aberration and the Mouse Micronucleus assays. 

ISO 10993-1 specifies an assessment of genotoxic potential for permanent devices and for those with prolonged contact (>24 hours) with internal tissues and blood. Extracorporeal devices with limited contact (<24 hours) may require a genotoxicity evaluation. Generally, devices with long-tem exposure require an Ames test and two in vivomethods, usually the Chromosomal Aberration and Mouse Micronucleus tests. Devices with less critical body contact may be able to be tested using only the Ames test.

When selecting a battery of genotoxicity tests, you should consider the requirements of the specific regulatory agency where your submission will be made. Because of the high cost of genotoxicity testing, Pacific BioLabs strongly recommends that you consult your FDA reviewer before you authorize testing.

Implantation Tests

Implant studies are used to determine the biocompatibility of medical devices or biomaterials that directly contact living tissue other than skin (e.g. sutures, surgical ligating clips, implantable devices, etc.). These tests can evaluate devices, which, in clinical use, are intended to be implanted for either short-term or long-term periods.  Implantation techniques may be used to evaluate both absorbable and non-absorbable materials. To provide a reasonable assessment of safety, the implant study should closely approximate the intended clinical use.

The dynamics of biochemical exchange and cellular and immunologic responses may be assessed in implantation studies, especially through the use of histopathology. Histopathological analysis of implant sites greatly increases the amount of information obtained from these studies. 

Hemocompatibility

Materials used in blood contacting devices (e.g. intravenous catheters, hemodialysis sets, blood transfusion sets, vascular prostheses) must be assessed for blood compatibility to establish their safety. In practice, all materials are to some degree incompatible with blood because they can either disrupt the blood cells (hemolysis) or activate the coagulation pathways (thrombogenicity) and/or the complement system.

The hemolysis assay is recommended for all devices or device materials except those which contact only intact skin or mucous membranes.  This test measures the damage to red blood cells when they are exposed to materials or their extracts, and compares it to positive and negative controls. 

Coagulation assays measure the effect of the test article on human blood coagulation time. They are recommended for all devices with blood contact. The Prothrombin Time Assay (PT) is a general screening test for the detection of coagulation abnormalities in the extrinsic pathway.
The Partial Thromboplastin Time Assay (PTT) detects coagulation abnormalities in the intrinsic pathway.

The most common test for thrombogenicity is the in vivo method. For devices unsuited to this test method, ISO 10993-4 requires tests in each of four categories: coagulation, platelets, hematology, and complement system.

Complement activation testing is recommended for implant devices that contact circulatory blood. This in vitro assay measures complement activation in human plasma as a result of exposure of the plasma to the test article or an extract. The measure of complement actuation indicates whether a test article is capable of inducing a complement-induced inflammatory immune response in humans.

Other blood compatibility tests and specific in vivo studies may be required to complete the assessment of material-blood interactions, especially to meet ISO requirements. 

DEVICES OR COMPONENTS WHICH CONTACT CIRCULATING BLOOD AND THE CATEGORIES OF APPROPRIATE TESTING

External Communicating Devices

a- Hemolysis testing only

Implant Devices

a – Hemolysis testing only

Carcinogenesis Bioassay

These assays are used to determine the tumorigenic potential of test materials and/or extracts from either a single or multiple exposures, over a period consisting of the total lifespan of the test system (e.g. two years for rat, 18 months for mouse, or seven years for dog).

Carcinogenicity testing of devices is expensive, highly problematic, and controversial. Manufacturers can almost always negotiate an alternative to full scale carcinogenicity testing of their devices.

Reproductive And Developmental Toxicity

These studies evaluate the potential effects of test materials and/or extracts on fertility, reproductive function, and prenatal and early postnatal development. They are often required for devices with permanent contact with internal tissues.

Pharmacokinetics

Pharmacokinetic or ADME (Absorption/Distribution/Metabolism/Excretion) studies are used to investigate the metabolic processes of absorption, distribution, biotransformation, and elimination of toxic leachables and potential degradation products from test materials and/or extracts. They are especially appropriate for bioabsorbable materials or for drug/device combinations. Our toxicology team is happy to work with you in setting up the appropriate PK or ADME study for your product. 

Preclinical Safety Testing

The objectives of preclinical safety studies are to define pharmacological and toxicological effects not only prior to initiation of human studies but throughout clinical development. Both in vitro and in vivo studies can contribute to this characterization. Pacific BioLabs has extensive experience in designing and running successful preclinical safety studies. 

Histopathology Services

Implant studies are often the most direct evaluation of device biocompatibility. The test material is placed in direct contact with living tissue. After an appropriate period, the implant site is recovered and examined microscopically for tissue reaction. The histopathologist can detect and describe many types of tissue and immune system reactions.

Similarly, in subchronic and chronic studies, various organs and tissues are harvested at necropsy and evaluated microscopically for toxic effects. Many of these studies also call for clinical chemistry analysis of specimens or serum samples form the test animals.