When extracts of a medical device are required for a biocompatibility test protocol, a current practice is to follow the guidance in ISO 10993 Biological evaluation of medical devices–Part 12: Sample preparation and reference materials. This international standard assumes that the amount of extractable substance(s) is/are related to the period of extraction, the temperature, the ratio of surface area or mass of material to the volume of extractant, the nature of the solvent, and ultimately, to the interaction of solute and solvent. Because extraction is such a complex process, this guidance document concludes that the material extraction conditions used “shall be appropriate to the nature and use of the final product.” Specifics of the current guidance, and concepts and strategies for process maximization are discussed in this article. More specifically, discussion is provided for exaggerated surface area testing and concentrated extract testing, and the potential benefits of each method.

Routine Extraction Time and Temperature

The period of extraction should be sufficient to maximize the amount of material extracted. In practice, standard time and temperature conditions to provide a measure of the hazard potential of a device or material are recommended in lieu of specific chemical analyses. However, an alternative practice cited in ISO 10993-12 (Annex C), particularly when increased test sensitivity is desired, is repeated extraction followed by concentration to obtain sufficient extractable substance(s) for analysis. It should be noted, however, that concentration of the extracts may result in loss of volatile materials such as ethylene oxide. Consequently, the effects of higher temperatures or other conditions on extraction kinetics and the identity of the extractant(s) should be considered carefully, and potentially monitored, if accelerated or exaggerated extraction is used. Standard extraction temperatures and times (with permissible ranges) are as follows:

The extraction temperature should also be selected to maximize the amount of extractable substances as well as to simulate any extremely high temperatures the device may incur during clinical use. However, this simulation should not initiate significant degradation of the material.

The extraction temperature is dependent upon the physiochemical characteristics of the device material(s). For example, for polymers, the extraction temperature chosen should be below the glass transition temperature (T_g). If the T_g is below the use temperature, the extraction temperature should be below the melting temperature. It is generally accepted that materials that are used only at body temperature should be extracted at temperatures that provide the maximum leachables without material degradation (e.g., collagen can be extracted at 37ºC whereas ceramic implants can be extracted at 121ºC).

Surface Area to Volume Ratio

Standard surface area (e.g., projected area) excluding indeterminate surface irregularity and extractant volumes are generally recommended as noted in Table I.

Irregular shapes and/or indeterminate surface areas may be tested at a mass-to-volume ratio of 0.2 g/mL (polymers) and 0.1 g/mL (elastomers). Mass-to-volume extractions may also be appropriate for any device material or component, for which a surface area can also be calculated, when it increases or exaggerates the exposure above surface area to volume extractions to create a worst-case exposure condition.

Mass-to-volume and surface-area-to-volume extraction ratios other than those listed in Table I may be used, provided they simulate the conditions during clinical use or result in a relevant measure of the hazard potential (e.g., see later discussion of exaggerated surface-area-to-volume testing). Coated or surface-treated materials, elastomers, composites, laminates, etc., should be tested intact whenever possible. Other materials should be cut into small pieces before extraction to assure submersion in the extract liquid.

An acceptable method for testing absorbents and hydrocolloids involves first determining the “absorption capacity” of the material (i.e., the amount of extractant absorbed by 0.1 gram or 1.0 cm² of the material). Then, in performing the material extraction, add this additional volume to each 0.1 gram or 1.0 cm² in an extraction mixture.

Extraction Medium

Medium employed for the extraction of medical device materials has varied as a direct function of the test protocol employed. For example, an assessment of the hemolytic potential for device materials will routinely employ blood as one extraction medium for direct contact materials and physiologic saline for indirect contact materials. Some investigators have used a 50:50 mixture of alcohol:saline to simulate the extraction properties of blood, and then tested these extracts in the protocol for indirect contact materials.  Most guidance documents list the following mediums as appropriate extraction solutions:

Extractions may be performed under static or agitation conditions. When agitation is considered to be appropriate, the method should be specified and justified. Liquid extracts shall, when possible, be used immediately after preparation to prevent sorption onto the extraction container or other changes in composition. If an extract is stored longer than 24-hours, then the stability of the extract under the conditions of storage should be verified. The extract should not routinely be processed by filtration, centrifugation, or other methods to remove suspended particulates. However, if such processing is necessary, the rationale shall be justified.

Phase Equilibrium

The phase equilibrium of a material at a temperature controls the relative amounts of amorphous and crystalline phases present. For the amorphous phase, the glass transition temperature (T_g) dictates the polymer chain mobility and the diffusion rate in the phase. Usually, the diffusion rate is considerably higher above the T_g compared with that below. The diffusion rate is lowest in the crystalline phase. If the protocoled extraction conditions alter the phase equilibrium of the material, it should be understood that it may affect the amount and type of extractables. For this reason, phase equilibrium should always be considered when extraction protocols are designed. Thermal transitions in polymers can be readily measured by differential scanning calorimetry (DSC).

Strategies for Extraction Process Maximization

An overwhelming majority of medical devices submitted for registration pass all biocompatibility testing for several reasons:

Ironically, these points suggest that a large number of research animals may be used unnecessarily to meet the perceived “requirement” of an established “guidance” document. Also ironically, this action is in direct violation of ISO 10993-2 (Animal Welfare Requirements), which exists to reduce, refine, and replace animal experimentation with alternative methods. Consequently, two strategies to maximize the testing are discussed below. These include exaggerated-surface-area-to-volume extraction ratios and concentrated extract testing. Either of these strategies could address the deficiency identified as “insufficient sample concentration”.

What is Exaggerated Surface-Area-to-Volume Medical Device Testing?

Exaggerated surface-area-to-volume medical device testing is a principle that defines the test article as the combined unique “material extraction ratios” of all components of a device in a single extraction vessel. The relative concentration or potency of the final exaggerated surface-area-to-volume solution becomes the additive surface areas (or mass) of the unique materials per mL of extraction vessel volume. Typically, all unique material surface areas (or mass) follow the material extraction ratios of ISO 10993 Part 12 (Sample Preparation), regardless of their proportion-contribution to the device. 

Example. A device has four materials (ABCD) with potential for interaction either by direct contact (e.g., juxtapositional) or fluid-path (solution-flow) contact. Materials A (10% proportion of the device) and B (50% proportion of the device) have a thickness of > 5 mm while materials C (10% proportion of the device) and D (30% proportion of the device) have a thickness < 5 mm. The exaggerated-surface-area-to-volume extraction therefore, irrespective of material proportion-contribution, includes 3 cm²/mL of material A + 3 cm²/mL of material B + 6 cm²/mL of material C + 6 cm²/mL of material D = 18 cm²/mL of material A+B+C+D. In every case, the greater of the material extraction ratio or the clinical exposure ratio unique to each device material should be employed to address a worst case exposure. In other words, if the clinical exposure to material D was known to be 9 cm²/mL, then that would be the amount added to the extraction vessel instead of 6 cm²/mL.

Precedence exists for medical device exaggerated surface-area-to-volume testing since all current medical device regulatory standards emphasize the importance of evaluating the final-finished device (i.e., a composite of all materials/components) to test for processing effects and potential material interactions. As defined, exaggerated surface-area-to-volume testing is an extension of these standards, applying an additional safety factor (increased material concentration) to the testing protocol.

Process maximization for extraction is evident if data are archived for use in other future device applications. For example, if material A was qualified for use at an exposure ratio of 6 cm²/mL in device X, and it is now desired to use the same material A at a lower exposure ratio (e.g., 4 cm²/mL) in a different device Y, then a reduced biocompatibility testing panel may be possible because material A had been previously qualified at the higher exposure ratio. On the other hand, if material A had originally been tested at its device proportion-contribution ratio (e.g., 10% of 3 cm²/mL = 0.3 cm²/mL) then full testing could be necessary to approve the higher potential exposure in device Y. In summary, the surface-area or mass of a material added to any extraction vessel dictates its subsequent “level of approval status” for other device applications, and the higher the extraction ratio the greater the future applications with reduced or no additional testing requirements.

Would the higher “material extraction ratios” employed in the testing protocol significantly increase the potential for an adverse test result that might not be seen with lower “clinically relevant” exposure extraction testing?

Increased concentration of an extraction solution could be expected to increase the potential for adverse effect. However, because most medical device materials are relatively inert anyway, and the chemical additives are usually nontoxic at their prescribed formulation concentrations (or, more importantly, their leaching/bioavailability concentrations), the risk of adverse effect from the described exaggerated surface-area-to-volume may be judged small enough to be deemed acceptable. If adverse effects are produced, it would be necessary, and feasible, to reduce the extraction ratios to the lower “clinically relevant” exposure conditions. When any doubt exists, it would be prudent to use a staged testing approach, applying in vitro tests before in vivo. In vitro test failures might require the investigator to reconsider the extraction protocol parameters and potentially resort to conventional methods, e.g., material device-proportionate extraction.

Would the potential extracted-chemical interactions resulting from the higher “material extraction ratios” employed in the exaggerated surface-area-to-volume testing protocol be different (and biologically significant) from those seen with lower “clinically relevant” exposure extraction testing?

By design, mixture (composite) testing or final-finished device testing accounts for potential chemical interactions. The potential for different interactions always exists and is multifactorial (e.g., a function of specific materials, and extraction time, temperature, volume, and surface area or concentration). However, by changing only the concentration the unique “material extraction ratio” composite sample may be compared by “chemical characterization” (analytical chemistry) profile to other extraction ratio profiles, including the clinically relevant ratio profile. Theoretically, when no additional factors interplay, the profile of a “material extraction” ratio (exaggerated-surface-area-to-volume sample) should be the same as the clinically relevant ratio, multiplied by the increase in surface area.

Concentrated Extract Testing (CET)

CET refers to the chemical or mechanical process whereby a test sample(s) is/are concentrated to directly improve the sample potential to elicit a measurable response in the test system, as well as to indirectly improve the sensitivity of the test system. The process for CET may employ changes in any of the conventional extraction parameters, including time, temperature, extraction medium, etc. to achieve maximum extractables, with subsequent sample concentration as necessary. CET is designed to achieve multiples of the clinically relevant exposure (e.g., increased safety margin) reduced (concentrated) to a physiological dosage volume.

Is There Precedence for CET?

Yes. For example, the Japanese Ministry of Health and Welfare (MHW) currently recommends a version of CET for the Guinea Pig Maximization Test (GPMT) for skin sensitization. This particular MHW protocol requires repeated extractions (using organic solvents) of device material(s) to achieve a prescribed residue W/W% of the original device. The organic solvent is then removed by rotary evaporation, the residue dissolved in an appropriate delivery vehicle (e.g., vegetable oil, DMSO, acetone, etc.), and the sample processed through the conventional GPMT protocol.  

The current revision of ISO 10993–11 (Systemic Toxicity) also recommends CET in some cases to maximize exposure and material safety assurance (e.g., to increase the exposure safety margin).

CET Vs. Exaggerated Surface-Area-to Volume Testing

CET serves a similar purpose to exaggerated-surface-area-to-volume but potentially provides significantly higher levels of leachables for testing. CET may be defined as one or more material composites subjected to additional worst-case extraction conditions, e.g., usually prolonged extraction times or organic solvents (e.g., methanol and acetone) to maximize leachables, accomplished through concentration or even recycling of extraction solutions. CET may also have application when the total clinical or exaggerated-surface-area-to-volume (or mass) of a single device (multiple components) exceeds the volume capacity of the standard extraction vessel, or when multiple or similar devices are tested together, or when all devices for a specific clinical procedure are tested together.

Example. Four materials of a device, when considered at the exaggerated-surface-area-to-volume ratio (or mass), are found to exceed the volume capacity of the extraction vessel. The investigator decides that, rather than extract in a larger volume to submerge all parts and then concentrate the extract solution-sample, the process will be divided into two stages. First, 3 cm²/mL of material A + 3 cm²/mL of material B are extracted for 24 hours at 70ºC. Following removal of materials A+B from the extraction vessel/solution, 6 cm²/mL of material C + 6 cm²/mL of material D are added to the same vessel/solution and extracted for 24 hours at 70ºC. The resulting test solution = 18 cm²/mL of material A+B+C+D, where all materials have been extracted for 24 hours and leachable chemicals from materials A+B have been subjected to an additional 24 hours of 70ºC temperature. Chemical confirmation of the stability of the extracts from materials A+B at 24 and 48 hours are provided.

CET Validation

Although emphasis on this scientific need is increasing, it must be noted that current guidelines do not presently require chemical/material characterization of test samples, and that potential resulting material interactions are accepted as innate properties (and consequences) of the final device configuration. One approach, however, may be to characterize and compare the individual device composites being considered for CET such that composite A + composite B = composite A+B without the qualitative/quantitative loss of either constituent.

Are current analytical limits of detection capable of detecting biologically significant differences in CET chemical characterization profiles?

Few of the chemicals incorporated in the formulations of the materials employed for construction of medical devices have known toxicities in the parts per million (ppm) range, and this has been demonstrated repeatedly through risk assessment and actual clinical and consumer experience. Moreover, there are very few, if any, chemical entities employed in device construction that are toxic below their theoretical limit of detection.

A medical device may be defined as multiple components which, when combined, constitute a unique sample with a unique spectral “fingerprint”. Assuring that that fingerprint remains clinically relevant will facilitate the application of the maximization processes described here. An increased focus on material and chemical characterization is necessary.

The Clinical Margin of Safety

It is always a useful exercise to calculate the actual clinical margin of safety for any medical device extraction strategy. To do this, one simply needs to calculate the total surface area (or mass, as worst case) of all fluid-contacting materials and the actual volume of fluid exposure. Using a simple 6 gm tubing connector (i.e., new material to be qualified) as an example, and knowing that 1 L of a physiological fluid will pass through the connector fluid-path lumen within a 24 h period, the clinical exposure is calculated as 6 g/L, or 0.006 g/mL. Appreciating that a typical extraction for biocompatibility testing of this new component/material might include extraction at 0.2 g/mL, the clinical margin of safety is 0.2 g/0.006 g = 33. In other words, the typical prescribed extraction ratio of 0.2 g/mL, not acounting for the exaggerated effects of increased extraction temperature and time, and diffent solvent polarities, exceeds the actual clinical exposure by 33 times. While this is an acceptable margin of safety, use of the CET described above can increase this safety margin many fold. Minimally, it should be assured that any device extraction strategy should be designed so as to exceed the actual clinical exposure.