Biocompatibility Testing

Cytotoxicity in vitro screening assays are a sensitive method to rapidly assess the biocompatibility of the test material (extract) when in contact with a specific cell culture. The qualitative version with L929 cells complies with both ISO10993-5 and USP (paragraph 87).

The neutral red uptake (NRU), (ISO 10993-5:2009 (E)), is an in vitro assay designed to evaluate the cytotoxic potential of medical devices using mouse fibroblast BALB/c 3T3 cells. Toxic substances can modify the cell surface or the lysosomal membrane, causing lysosomal fragility and other adverse changes that gradually become irreversible. Such adverse changes cause inhibition of cell growth and/or cell death, which will decrease the amount of NR retained by the culture.

Figure 1: Example of a 96-well plate of a positive control (sodium dodecyl sulfate) used in the Neutral Red Uptake assay (NRU). NT= Non-treated (Blue). Since viable cells retain the NR, the intensity of the colored wells (pink) is directly associated to high cell viability.

Sensitization biocompatibility testing for medical devices is used to determine the allergic or sensitizing capacity to the repeated or prolonged exposure of a test material. Sensitization is characterized by delayed reactions that are not localized, independent of dose.

• Local lymph node assay (LLNA)
• Guinea pig maximization test (GPMT)

Irritation biocompatibility testing consists of in vivo screens that evaluate the potential of test materials – or their extracts – to cause irritation on the exposed part of the body. Standard studies are single-exposure evaluations. Based on clinical product use, studies may be customized for evaluation of repeat exposures.

• Topical irritation (skin, buccal cavity, vagina, urethra, rectum)
• Intracutaneous reactivity: Single or cumulative exposure in accordance with the intended clinical use

Bacterial endotoxin testing or rabbit material-mediated pyrogen testing detects endotoxin on the biomaterial surface and any related pyrogen reaction via endotoxin contamination with a known specified toxin limit.

Genetic toxicology biocompatibility testing for medical devices evaluates the potential of the test article to induce gene mutations or chromosome damage using a battery of bacterial, mammalian cells in vitro, and in vivo test systems. This critical information constitutes an essential part of preclinical studies because genetic damage can cause an increase in the incidence of heritable diseases and cancer in human populations. Physical or chemical agents that induce such effects by interacting with genetic material and altering their structure are considered genotoxic.

Charles River uses the polar and non-polar extracts methodology. The obtained test samples treat the test systems in the following assays, as per OECD guidelines modified for medical devices, and performed under GLP conditions.

Genotoxicity biocompatibility testing for medical devices:

• Bacterial Reverse Mutation Test (Ames)
• In Vitro Mouse Lymphoma Assay TK
• In Vitro Micronucleus Test
• In Vitro Chromosome Aberration Test
• In Vivo Rat Micronucleus Test
• In Vivo Mouse Micronucleus Test

In vivo systemic tests evaluate the impairment or activation of a system – rather than the impairment of individual cells or organs. Acute systemic toxicity tests evaluate the test article material for systemic toxic effects as a result of a single, acute exposure.

• Acute and repeated exposure, available with a wide range of dose routes in appropriate species

For devices in direct contact with circulating blood (regardless of contact duration), hemolysis, complement activation, and thrombogenicity biocompatibility testing may be performed. Hemocompatibility tests are more commonly used to evaluate the effects of blood-contacting medical devices. Hemolysis determines the degree of erythrocyte (red blood cell) lysis and the release of hemoglobin caused by medical devices in vitro. Both direct (implantation) and indirect (extract) methods for material/surface-mediated hemolysis may be conducted. Charles River offers two in vitro hemocompatibility tests:

• In vitro hemolysis
• In vitro coagulation

Carcinogenicity and reproductive toxicity testing is rarely required for medical devices. However, when necessary, Charles River’s capabilities and experience with carcinogenicity testing of pharmaceuticals enables us to offer this service to medical device developers when necessary.

Biocompatibility testing for medical devices is a regulatory safety requirement that ensures that a device is compatible with the biologic system where it will be implanted or used.

The US FDA, TGA, and Regulatory Bodies in Europe follow several regulations. Europe and Asia follow the ISO 10993 as the main source for guidance of the testing protocols. The US FDA has adopted some of the ISO 10993 guidelines, but for some testing it follows the USP, ASTM and US FDA “Blue Book.”

The gold standard for biocompatibility testing is to perform a device chemical characterization followed by a TRA (toxicological risk assessment). For more insight, please review our Biocompatibility Evaluation of Orthopedic Biomaterials and Medical Devices: A Review of Safety and Efficacy Models.

To support global submissions, all their protocols must be compliant with OECD GLPs in addition to US FDA GLPs. In addition, laboratories that offer medical device biocompatibility testing are widely expected to have ISO/IEC 17025 accreditation.