F04.16 - Biocompatibility Test Methods

SIGNIFICANCE AND USE
4.1 This practice is a guideline for short-term and long-term assessment of skeletal muscle and bone tissue responses to long-term implant materials. For testing of final finished medical devices, the test article for implantation shall be as for intended use, including packaging and sterilization. The tissue responses to the test article are compared to the skeletal muscle and/or bone tissue response(s) elicited by control materials. The controls consistently demonstrate known cellular reaction and wound healing.
SCOPE
1.1 This practice provides guidelines for biological assessment of tissue responses to nonabsorbable for medical device implants. It assesses the effects of the material that is implanted intramuscularly or intraosseously. The experimental protocol is not designed to provide a comprehensive assessment of the systemic toxicity, immune response, carcinogenicity, or mutagenicity of the material since other standards address these issues. It applies only to materials with projected applications in humans where the materials will reside in bone or skeletal muscle tissue in excess of 30 days. Applications in other organ systems or tissues may be inappropriate and are therefore excluded. Control materials are well recognized with a well-characterized long-term response and can include metals and any one of the metal alloys in Specification F67, F75, F90, F136, F138, or F562, high purity dense aluminum oxide as described in Specification F603, ultra high molecular weight polyethylene as stated in Specification F648, or USP polyethylene negative control.  
1.2 The values stated in SI units, including units officially accepted for use with SI, are to be regarded as standard. No other systems of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This test method is intended to assess a combination of inherent chemical irritation and mechanical irritation for products and materials expected to come into contact with the skin. It is a comparative approach whereby the potential irritation of a test material is compared to that of a reference material similar in form and composition. The reference material should have a known safety and irritation profile.
SCOPE
1.1 The behind-the-knee (BTK) method, using the popliteal fossa of human volunteers as a test site, simultaneously evaluates the inherent chemical irritation and the potential for mechanical irritation of substrates and products that are designed to come into repeated or extended close contact with the skin (see validation references (1-7)).2 This is a bilateral test comparing a test material to a reference material with a known safety profile.  
1.2 This test method shall be used by qualified health care professionals experienced in good clinical practice (GCP) procedures.  
1.3 This test method can be performed using human subjects on either intact or compromised skin. Testing should be performed on intact skin for test substrates or products expected to have contact with normal, intact skin, or for direct comparison to products with a known skin irritation profile. Testing can be performed on compromised skin for test substrates or products that may commonly come into contact with damaged skin (for example, skin with diaper rash, or chapped skin) or skin that is expected to be hydrated.  
1.4 Visual scoring of erythema and dryness is performed by a trained skin grader on a predefined scale.  
1.5 Prior to use in this test, materials shall undergo overall favorable biocompatibility testing consistent with the approach outlined in protocol Practice F748 or ISO 10993-1:2009. As a part of this series of testing, irritation per Practice F719 or ISO 10993-10 shall be conducted.  
1.6 The values stated in inch-pound units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This standard guide is to be used to help assess the biocompatibility of materials used in medical devices (for example, externally communicating, implants, and other body contact medical devices). It is designed to test the effect of particles and other wear debris and/or degradation products on the generation of FBR and other (local and systemic) host responses of immune/inflammatory origin.  
5.2 The appropriateness of the selected testing methods should be carefully considered by the user since not all materials or applications need to be tested by this guide. Existing biocompatibility screening methods may not be fully predictive of the human response, and testing approaches such as those described here are needed for continuous improvement of the predictability of biocompatibility testing. The effectiveness of animal testing in terms of its predictability of human outcomes is dependent on the study design. If possible, study endpoints should be chosen to minimize interspecies variability and to investigate clinically relevant biological responses. While testing approaches should remain at the user’s discretion, the following should be taken into consideration when selecting most appropriate tests and study endpoints.  
5.2.1 Device-induced responses usually involve both innate and adaptive immunities, which raises possible need for specific testing for each of these immune response types.
5.2.1.1 Device-related adaptive immune responses are mostly due to lymphocyte-mediated delayed-type hypersensitivity. In vivo allergenicity to a test material (which can be introduced via different routes) should be assessed by monitoring for any signs of allergic and acute toxicity reactions, for example, scratch, tremor, and dyspnea. In addition, ex vivo analysis on immunophenotyping of the isolated splenocytes/lymphocytes from the same studies should be considered.
5.2.1.2 Device-related innate immune responses are mostly mediated by macrophages and can be assess...
SCOPE
1.1 The purpose of this standard guide is to describe the principles and approaches to testing of medical device debris and degradation products from device materials (for example, particles from wear) for their potential to activate a cascade of biological responses at local and systemic levels in the body. In order to ascertain the role of device debris and degradation products in stimulating such responses, the nature of the responses and the consequences of the responses should be evaluated. This is an emerging area. The continuously updated information gained from the testing results and related published literature is necessary to improve the study designs, as well as predictive value and interpretation of the test results regarding debris/degradation product related responses. Some of the procedures listed here may, on further testing, not prove to be predictive of clinical responses to device-related debris and degradation products. However, only the continuing use of standard protocols will establish the most useful testing approaches with reliable study endpoints and measurement techniques. Since there are many possible and established ways of determining the debris/degradation product related responses in vivo, a single standard protocol is not stated. However, this recommended guide indicates which testing approaches are most applicable per expected biological responses and which necessary information should be supplied with the test results. To address the general role of chronic inflammation in exaggerating device-related foreign body response (FBR), the recommendations in this standard include the assessment of device-related pro-inflammatory responses and subsequent tissue remodeling potential.  
1.2 This document is to provide the users with updated scientific knowledge that may help better characterize medical device debris related responses. It is to help the users to optimize their plans for particle...

SIGNIFICANCE AND USE
5.1 This practice is a guideline for a screening test of candidate materials or assessment of local tissue response to absorbable medical devices which are expected to undergo complete absorption within three years.  
5.2 This practice is similar to those for studies on candidate materials or medical devices that are not absorbable, such as those specified in Practices F763, F981, and F1408; however, analysis of the host response must take into account the effect of degradation and degradation products on the inflammatory response at the local tissue site and on subsequent healing of the implantation site, as well as the potential for adverse distal tissue effects.  
5.3 For testing of absorbable medical devices, the test article for implantation should be in the final finished form as for intended use, including packaging and sterilization (if applicable). Configurations specific to the animal study may be needed. The test article’s surface-area-to-body mass or mass-to-body mass ratios within the animal model should be established by calculating based on surface-area-to-body mass or mass-to-body mass ratios in humans during the device’s intended clinical use. Worst-case clinical dose should be considered in the study design. For implantation studies incorporating evaluation of both local tissue responses and systemic toxicity, exaggerated material surface area or mass-to-body mass ratios (for example, a 2X to 10X safety factor to assess implant safety for regulatory submissions) compared to clinical use (for example, largest device size, maximum number of devices) should be considered, unless otherwise justified. For example, implantation of exaggerated doses may not be feasible in the selected animal model. For some devices, additional animal group(s) for exaggerated conditions should be considered if dose response information is needed. Additionally, for some devices, exaggerated dose at a specific implantation site can also be used to evaluate local tissue res...
SCOPE
1.1 This practice provides experimental protocols for biological assays of tissue reactions to absorbable biomaterials for implant applications. This practice applies only to absorbable materials with projected clinical applications in which the materials will reside in bone or soft tissue longer than 30 days and less than three years. Other standards with designated implantation times are available to address shorter time periods. Careful consideration should be given to the appropriateness of this practice for slowly degrading materials that will remain for longer than three years. It is anticipated that the tissue response to degrading biomaterials will be different from the response to nonabsorbable materials. In many cases, a chronic inflammatory response may be observed during the degradation phase, but the local histology should return to normal after absorption; therefore, the minimal tissue response usually equated with biocompatibility may require long implantations.  
1.2 The time period for implant absorption can depend on variables of chemical composition, implant size, implant location, and animal models. Therefore, the selected time points for assessing tissue effects may be selected based on the rate of absorption.  
1.3 These protocols assess the effects of the material on the animal tissue in which it is implanted. They do not fully assess systemic toxicity, carcinogenicity, reproductive and development toxicity, or mutagenicity of the material. Other standards are available to address these issues.  
1.4 To maximize use of the animals in the study protocol, some aspects of systemic toxicity, including effects of degradation products on different organs and tissues downstream of or surrounding the target site, can be addressed with this practice.  
1.5 Because animal models are not identical to human biology, this practice cannot account for all potential biological hazards, for example the effect o...

SIGNIFICANCE AND USE
4.1 The use of in vivo implantation techniques for characterizing the biocompatibility of implantable materials to be utilized in various medical applications enables the assessment of such materials not achieved by other procedures. Physical characteristics (that is, form, density, hardness, surface finish) can influence the type and severity of the tissue response to the test materials.  
4.2 This practice is intended as a short-term screening procedure for determining the acceptability of candidate materials. It may be utilized prior to using the long-term tests described in Practice F981. It is recommended that for some applications, additional tests, including long-term implantation studies, may be required to assess the final suitability of the candidate materials.  
4.3 This practice may not be appropriate for all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
SCOPE
1.1 This practice provides guidelines for short-term testing or screening of candidate materials, both porous and dense, as to the local effects of the material that is implanted intramuscularly. This method may not be applicable for absorbable materials, depending on the absorption profile of the test material. The tissue reactions will be evaluated in comparison to those evoked by control materials that are accepted as clinical implant materials. This is a short-term (less than 30 days) screening procedure for determining acceptability of candidate materials.  
1.2 This practice, along with other appropriate biological tests (including other appropriate ASTM tests), may be used in the biocompatibility assessment of the candidate materials for use in the fabrication of devices for clinical application.  
1.3 This experimental protocol is not designed to provide a comprehensive assessment of the systemic toxicity, carcinogenicity, or mutagenicity of the material since other standards address these issues.  
1.4 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 provides guidance for the selection of appropriate methods for testing materials for a specific application.  
1.5 The values stated in SI units, including units officially accepted for use with SI, are to be regarded as standard. No other systems of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This practice is a guideline for a short-term screening test for the evaluation of the tissue response to materials that may be selected for implantation in the human body and should be done in accordance with good laboratory practices. This test may be performed prior to long-term testing (for example, Practice F981) to eliminate unsuitable candidate materials early and to avoid unnecessary animal testing.  
4.2 This practice may be used to detect toxic effects of materials in general (see Appendix X1). However, it is particularly suitable for the testing of materials that are intended to have contact with subcutaneous tissues or soft tissues in general. For materials intended to be inserted specifically into muscle tissues, Practice F763 should be considered as a short-term test method.  
4.3 The suggested implant specimens are cylindrical. A special grooved type of cylinder may be used (see Fig. X2.1 of Appendix X2) to allow tissue interlocking that could keep the implant in place and minimize tissue irritation through motion at the interface that otherwise could contribute to increased variance of the results. In case ungrooved cylinders are used (see Fig. X1.2 of Appendix X2), probable motion at the implant/tissue interface must be taken into account. Control cylinders should be shaped like the test cylinders.  
4.4 The type of surface preparation of the specimens can affect the tissue reaction; therefore the preparation procedure should be noted in the report. The test may be used to compare the effect of different surface structures or conditions of the same material or to assess the effect of various treatments of modifications of a material.
Note 1: If this method is used for material research, testing for endotoxin prior to implantation should be considered.
SCOPE
1.1 This practice covers a short-term testing method to screen the subcutaneous tissue reaction to implant candidate materials in small laboratory animals. The material may be dense or porous. This method may not work for absorbable materials, depending on the absorption kinetics. The tissue reactions will be evaluated in comparison to those evoked by control materials that are accepted as clinical implant materials.  
1.2 This practice, along with other appropriate biological tests (including other ASTM test methods), may be used to assess the biocompatibility of candidate materials for use in the fabrication of devices for clinical application. It may also be applied to evaluate the effect of special surface textures and preparations of known materials.  
1.3 This practice does not provide a comprehensive assessment of the systemic toxicity, carcinogenicity, teratogenicity, or mutagenicity of the material. Additional information may be needed on the material in its final finished form, such as implantation assessment at the clinically relevant location.  
1.4 The values stated in SI units, including units officially accepted for use with SI, are to be regarded as standard. No other systems of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 These extraction procedures are the initial part of several test procedures used in the biocompatibility screening of plastics or other materials used in medical devices.  
5.2 The limitations of the results obtained from this practice should be recognized. The choices of the extraction vehicle, duration of immersion, and temperature of the test are necessarily arbitrary. The specification of these conditions provides a basis for standardization and serves as a guide to investigators wishing to compare the relative resistance of various plastics or other materials to extraction vehicles.  
5.3 Correlation of test results with the actual performance or serviceability of materials is necessarily dependent upon the similarity between the testing and end-use conditions (see 12.1.2 and Note 7).  
5.4 Caution should be exercised in the understanding and intent of this practice as follows:  
5.4.1 No allowance or distinction is made for variables such as end-use application and duration of use. Decisions on selection of tests to be done should be made based on Practice F748.  
5.4.2 This practice was originally designed for use with nonporous, solid materials. Its application for other materials, such as those that are porous, absorptive (for example, sponge-like materials that are capable of absorbing liquid), or resorptive, should be considered with caution. Consideration should be given to altering the specified material-to-liquid ratio to allow additional liquid to fully hydrate the material and additional liquid or other methods to fully submerge the test specimen. Additional procedures that fully remove the extract liquid from the test specimen, such as pressure or physically squeezing the material, should also be considered as appropriate. Although no definitions are given in this practice for the following terms, such items as extraction vehicle surface tension at the specified extraction condition and test specimen physical structure should be taken into ...
SCOPE
1.1 This practice covers methods of extraction of medical plastics and may be applicable to other materials. This practice identifies a method for obtaining “extract liquid” for use in determining the biological response in preclinical testing. Further testing of the “extract liquid” is specified in other ASTM standards. The extract may undergo chemical analysis as part of the preclinical evaluation of the biological response, and the material after extraction may also be examined.  
1.2 This practice may be used for, but is not limited to, the following areas: partial evaluation of raw materials, auditing materials within the manufacturing process, and testing final products. This practice may also be used as a reference method for the measurement of extractables in plastics used in medical devices. In general, it is the responsibility of the user of the standard to determine if the methods described in this standard are appropriate for the materials in their device.  
1.3 This practice was initially developed for extraction of medical plastics not intended to undergo degradation or absorption during normal medical device usage. When applied to the extraction of absorbable materials, additional considerations may be necessary in the selection of extraction procedures and fluids.  
1.4 For assessment of compatibility of the Single-use System material with the cell culture medium or the manufacturing processes used for cell-based therapeutics, vaccines, cell-based diagnostics, or other biopharmaceutical products, the user should refer to Guide E3231.  
1.5 The values stated in SI units are to be regarded as standard. The values given in parentheses are mathematical conversions to inch-pound units that are provided for information only and are not considered standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of thi...

SIGNIFICANCE AND USE
4.1 This practice is intended to help assess the biocompatibility of materials used in medical devices. It is an acute toxicological test designed to detect the presence of injurious leachable substances.  
4.2 This practice may not be appropriate for all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.  
4.3 The only limitation applicable is the extract preparation. Refer to Sections 4.3 and 4.4 of Practice F619 for a description of this limitation.
SCOPE
1.1 This practice covers a nonspecific, acute toxicity test used for detecting leachables from materials used in medical devices.  
1.2 The liquids injected into the mouse are those obtained by Practice F619 where the extraction vehicles are saline, vegetable oil, or other liquids simulating human body fluids.  
1.3 Two procedures are outlined: Method A for intravenous injection and Method B for intraperitoneal injection.  
1.4 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 Materials that are to be in contact with the skin should not cause irritation to the skin. Since it is probably the substances leached from a material that cause the irritation, this practice provides for direct material-skin contact testing or for skin exposure to the liquid extract of the test material. The rationale for this rabbit test is that it is a comparatively quick and sensitive method which, through use over the years, has become a generally accepted method. Additionally, the albino rabbit allows for easy visualization of erythema and edema, which are the cardinal signs of skin irritation.
SCOPE
1.1 This practice covers a procedure by which the irritancy of a material may be assessed through contact with abraded and intact skin of rabbits.  
1.2 The results of this practice depend upon the effectiveness with which contact between the skin and the test material is established and maintained. Because of the operator technique included in performing this test, it is important that the test be performed by personnel with appropriate training.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard may involve hazardous materials, operations, and equipment. This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This practice is useful for assessing cytotoxic potential both when evaluating new materials or formulations for possible use in medical applications, and as part of a quality control program for established medical materials and medical devices.  
4.2 This practice assumes that assessment of cytotoxicity potential provides one method for predicting the potential for cytotoxic or necrotic reactions to medical materials and devices during clinical applications to humans. In general, cell culture testing methods have shown good correlation with animal assays when only chemical toxicities are being considered.
Note 1: The results obtained using this method may not predict in vivo behavior which can be influenced by multiple factors such as those arising from site of application or physical properties that may result from design and fabrication.  
4.3 This cell culture test method is suitable for adoption in specifications and standards for materials for use in the construction of medical devices that are intended to have direct contact with tissue, tissue fluids, or blood. However, care should be taken when testing materials that are absorbable, include an eluting or degradable coating, are liquid or gelatinous in nature, are irregularly shaped solid materials, or have a high density or mass, to make sure that the method is applicable. If leachables from the test sample are capable of diffusing through the agar layer, agarose-based methods such as Test Method F895 may be considered as an alternate method, depending on sample characteristics, or in cases where investigators wish to further evaluate the cytotoxic response of cells underlying the test sample.
SCOPE
1.1 This practice covers a reference method of direct contact cell culture testing which may be used in evaluating the cytotoxic potential of materials for use in the construction of medical materials and devices.  
1.2 This practice may be used either directly to evaluate materials or as a reference against which other cytotoxicity test methods may be compared.  
1.3 This is one of a series of reference test methods for the assessment of cytotoxic potential, employing different techniques.  
1.4 Assessment of cytotoxicity is one of several tests employed in determining the biological response to a material, as recommended in Practice F748.  
1.5 The L-929 cell line was chosen because it has a significant history of use in assays of this type. This is not intended to imply that its use is preferred; only that the L-929 is a well characterized, readily available, established cell line that has demonstrated reproducible results in several laboratories.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This practice is to be used to help assess the biocompatibility of materials used in medical devices. It is an acute toxicological test designed to evaluate any irritation caused by device materials by gross assessment.  
4.2 This practice may not be appropriate for all types of implant applications. The user is cautioned to consider the appropriateness of this practice in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
Note 1: Some materials (e.g., absorbables) may result in an extract pH (e.g., ≤2.0 or ≥11.5) that cannot be used with this practice.  
4.3 The only applicable limitation is the extract preparation. Refer to Section 4.3 of Practice F619 for a description of this limitation.
SCOPE
1.1 This practice is an intracutaneous reactivity test used to assess the potential of the material under test to produce irritation following intradermal injections of extracts of the material.  
1.2 The liquids injected into the rabbits are those obtained by Practice F619 where the extraction vehicles are saline, vegetable oil, or other liquids simulating human body fluids.  
1.3 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.  
1.4 The values stated in SI units, including units officially accepted for use with the SI, are to be regarded as standard. No other systems of measurement are included in this standard.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 The objective of this guide is to recommend a panel of biological tests that can be used in addition to the testing recommended in Practice F748. This guide is designed to detect neurotoxicity caused by medical devices that contact nervous tissue.  
4.2 The testing recommendations should be considered for new materials, established materials with different manufacturing methods that could affect nervous tissue response, or materials used in new nervous tissue applications.  
4.3 Chemical characterization can be used to evaluate similarity for materials with a history of clinical use in a similar nervous tissue application.
SCOPE
1.1 Medical devices may cause adverse effects on the structure and/or function of the nervous system. In this guide, these adverse effects are defined as neurotoxicity. This guide provides background information and recommendations on methods for neurotoxicity testing. This guide should be used with Practice F748, and may be helpful where neurotoxicity testing is needed to evaluate medical devices that contact central and/or peripheral nervous system tissue or cerebral spinal fluid (CSF).
Note 1: The results of these in vitro and in vivo tests may not correspond to actual human response.  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 The purpose of this practice is to determine if thrombus formation has occurred by comparing platelet and leukocyte counts in the blood exposed to the test material relative to the blood cell counts in the control blood that has not been exposed to the test material. A large number of platelets and leukocytes becoming entrapped/incorporated in thrombi adhering to the material will be reflected by a decrease in their counts in blood. Thrombogenic materials should not be used for cardiovascular medical devices, unless the purpose of the device is to promote thrombosis.
SCOPE
1.1 This practice assists in the evaluation of cardiovascular device materials for their ability to induce thrombus formation. Thrombus formation is assessed by means of a reduction in human platelets and leukocytes when consumed by thrombus after activation on the material surface. This assay may be part of the hemocompatibility evaluation for devices and materials contacting human blood, as in accordance with ANSI/AAMI/ISO 10993–4. See also Test Method F2382.  
1.2 All safety policies and practices shall be observed during the performance of this practice. All human blood and any materials that had contact with human blood shall be bagged in a biohazard bag, properly labeled with the contents, and disposed of by appropriate means.  
1.3 The human blood should be handled at Biosafety Level 2 (BSL-2) as recommended in the Centers for Disease Control/National Institutes of Health publication, Biosafety in Microbiological and Biomedical Laboratories (BMBL). The human blood donor must have tested negative for Hepatitis B (HBV) and Human Immunodeficiency (HIV) viruses. The blood should be treated like any patient blood and handled/manipulated using standard precautions.
Note 1: The results of this in-vitro test may not correspond to actual human response.  
1.4 The values stated in SI (International System of Units) units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Some specific hazards statements are given in Section 8 on Hazards.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 The propensity of a material to stimulate delayed contact hypersensitivity must be assessed before clinical application of devices containing this material. Delayed hypersensitivity may occur anywhere in the body. Systemic delayed hypersensitivity may have a complex set of reactions and consequences depending on the actual tissue/organ site of reaction. Although the reactions are seldom life-threatening, severe tissue and organ damage my result over time. Skin is the usual test site to determine the propensity of a material to cause delayed hypersensitivity.  
5.2 The standard historical test methods have involved the use of guinea pigs with a cutaneous application and observation of the reaction site. The use of the murine local lymph node assay results in a numerical quantitation of stimulation, rather than subjective evaluation and could be used to determine dose responses.  
5.3 This practice may not be predictive of events occurring during all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
SCOPE
1.1 This practice provides a methodology to use a combination of in vivio and in situ procedures for the evaluation of delayed contact hypersensitivity reactions.  
1.2 This practice is intended to provide an alternative to the use of guinea pigs for evaluation of the ability of a device material to stimulate delayed contact hypersensitivity reactions. This alternative is particularly applicable for materials used in devices that contact only intact skin. However, the guinea pig maximization test is still the recommended method when assessing the delayed hypersensitivity response to metals or when testing substances that do not penetrate the skin but are used in devices that contact deep tissues or breached surfaces. This practice may be used for testing metals, with the exception of nickel-containing metals, unless the unique physicochemical properties of the materials may interfere with the ability of LLNA to detect sensitizing substances.  
1.3 This practice consists of a protocol for assessing an increase in lymphocyte proliferation in the lymph nodes draining the site of test article administration on the ears of mice.  
1.4 The LLNA has been validated only for low-molecular-weight chemicals that can penetrate the skin. The absorbed chemical or metabolite must bind to macromolecules, such as proteins, to form immunogenic conjugates.  
1.5 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.  
1.6 Identification of a supplier of materials or reagents is for the convenience of the user and does not imply a single source. Appropriate materials and reagents may be obtained from many commercial supply houses.  
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This practice is to be used to help assess the biocompatibility of materials used in medical devices. It is designed to test the effect of particles released from medical devices and biomaterials on macrophages or other cells.  
4.2 The appropriateness of the methods should be carefully considered by the user since not all materials or applications need to be tested by this practice.  
4.3 Abbreviations:  
4.3.1 FCS (FBS)—Fetal Calf Serum (Fetal Bovine Serum)  
4.3.2 FGFs—Fibroblast Growth Factors  
4.3.3 HBSS—Hank’s Balanced Salt Solution  
4.3.4 HEPES—A buffering salt (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)  
4.3.5 IL17—Interleukin 17  
4.3.6 IL18—Interleukin 18  
4.3.7 IL1β—Interleukin 1 beta  
4.3.8 IL6—Interleukin 6  
4.3.9 IL8—Interleukin 8  
4.3.10 LAL—Limulus Amebocyte Lysate  
4.3.11 LPS—lipopolysaccharide (endotoxin)  
4.3.12 MCP1—Monocyte Chemotactic Protein-1  
4.3.13 MMPs—Matrix Metalloproteinases  
4.3.14 NO—Nitric Oxide  
4.3.15 PBS—Phosphate Buffered Saline  
4.3.16 PGE2—Prostaglandin E2  
4.3.17 RPMI 1640—Specific Growth Medium (Roswell Park Memorial Institute)  
4.3.18 TGFβ—Transforming growth factor beta  
4.3.19 TNFα–—Tumor Necrosis Factor alpha  
4.3.20 VEGF—Vascular Endothelial Growth Factor
SCOPE
1.1 This practice covers the assessment of cellular responses to wear particles and degradation products from implanted materials that may lead to a cascade of biological responses resulting in damage to adjacent and remote tissues. In order to ascertain the role of particles in stimulating such responses, the nature of the responses, and the consequences of the responses, established protocols are needed. This is an emerging, rapidly developing area, and the information gained from standard protocols is necessary to interpret cellular responses to particles and to determine if these correlate with in vivo responses. Since there are many possible and established ways of determining responses, a single standard protocol is not stated. However, well described protocols are needed to compare results from different investigators using the same materials and to compare biological responses for evaluating (ranking) different materials. For laboratories without established protocols, recommendations are given and indicated with an asterisk (*).  
1.2 Since the purpose of the following test procedures is to predict the response in human tissues, the use of human (preferably macrophage lineage) cells is recommended. However, the use of non-macrophage cell lineage or the use of cells from non-human and non-primate sources may be acceptable. The source of the cells or the cell line used should be justified based on the cellular responses under test and/or tissue of interest. Non-human cells should not be used if there is evidence of possible cross-species difference for specific test results as the results of this in vitro test may not correspond to actual human response.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 The purpose of this test method is to determine the time citrated plasma exposed to medical materials takes to form a clot when exposed to a suspension of phospholipid particles and calcium chloride. In this test method, the test article is the activator. The PTT assay is a general screening test for a medical material’s ability to activate the intrinsic coagulation pathway. Material samples that show a shortened PTT are activators of the intrinsic coagulation pathway.  
4.2 The test article, reference materials, and controls are exposed to human plasma. The plasma is tested on a coagulation device. Each sample tube is assayed in duplicate. The results are reported as a percentage of the negative control.
SCOPE
1.1 This test method covers the screening of circulating blood-contacting device materials for their ability to induce blood coagulation via the intrinsic coagulation pathway. This assay should be part of the hemocompatibility evaluation for devices and materials contacting human blood, as per ANSI/AAMI/ISO 10993-4.  
1.2 All safety policies and practices shall be observed during the performance of this test method.  
1.3 All plasma and any materials that had contact with plasma will be bagged in a biohazard bag, properly labelled with the contents, and disposed of by appropriate means. The plasma should be handled at the Biosafety Level 2 as recommended in the Centers for Disease Control/National Institutes of Health Manual Biosafety in Microbiological Laboratories.  
1.4 The normal pooled human plasma must have tested negative for Hepatitis B (HBV) or Human Immunodeficiency (HIV) viruses. The plasmas should be treated like any patient plasma using standard precautions. The plasma should be handled at the Biosafety Level 2 as recommended in the Centers for Disease Control/National Institutes of Health Manual Biosafety in Microbiological Laboratories.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
2.1 The CHO/HGPRT assay detects forward mutations of the X-linked hypoxanthine-guanine phosphoribosyl transferase (hgprt) locus (coding for the enzyme, HGPRT) in Chinese hamster ovary (CHO) cells. Cells originally derived from Chinese hamster ovary tissue are exposed to a test article and, following an appropriate cell culture regimen, descendants of the original treated population are monitored for the loss of functional HGPRT, presumably due to mutations. Resistance to a purine analogue, 6-thioguanine (6TG) (or less desirably, 8-azaguanine (8AG)), is employed as the genetic marker. HGPRT catalyzes the conversion of the nontoxic 6TG to its toxic ribophosphorylated derivative. Loss of the enzyme or its activity therefore leads to cells resistant to 6TG.  
2.2 Because HGPRT is an enzyme of the purine nucleotide salvage pathway, loss of the enzyme is not a lethal event. Different types of mutational events (base substitutions, frameshifts, deletions, some chromosomal type lesions, and so forth) should theoretically be detectable at the hgprt locus. The CHO/HGPRT assay has been used to study a wide range of mutagens, including radiations (2-4), and a wide variety of chemicals (1), and complex chemical mixtures (5).
SCOPE
1.1 This guide highlights some of the more relevant biological concepts as they are currently understood, and summarizes the critical technical aspects for acceptable bioassay performances as they currently are perceived and practiced. The Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay (1) 2 has been widely applied to the toxicological evaluation of industrial and environmental chemicals.  
1.2 This guide concentrates on the practical aspects of cell culture, mutagenesis procedures, data analysis, quality control, and testing strategy. The suggested approach represents a consensus of the panel members for the performance of the assay. It is to be understood, however, that these are merely general guidelines and are not to be followed without the use of sound scientific judgement. Users of the assay should evaluate their approach based on the properties of the substances to be tested and the questions to be answered.  
1.3 Deviation from the guidelines based on sound scientific judgement should by no means invalidate the results obtained.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This guide is not intended to specify the exact method of conducting a test for any particular material but only to present some of the criteria that should be considered in method design and possible problems that could lead to misleading results. In the development of the actual test protocol, it is recommended that recognized tumorigenesis bioassay procedures be consulted.  
4.2 The recommendations given in this guide may not be appropriate for all applications or types of implant materials. These recommendations should be utilized by experienced testing personnel in conjunction with other pertinent information and the requirements of the specific material application.
SCOPE
1.1 This guide is intended to assist the biomaterials testing laboratory in the conduct and evaluation of tumorigenicity tests to evaluate the potential for new materials to evoke a neoplastic response. The procedure is generally reserved only for those materials which have not previously been used for human implantation for a significant period of time.  
1.2 Assessment of tumorigenicity is one of several procedures employed in determining the biological response to a material as recommended in Practice F748. It is assumed that the investigator has already determined that this type of testing is necessary for a particular material before consulting this guide. The recommendations of Practice F748 should be considered before a study is commenced.  
1.3 Whenever possible, it is recommended that a battery of genotoxicity procedures be initiated and proposed as an alternative to an in-vivo tumorigenicity bioassay. Genotoxicity assays may also be considered as initial screening procedures due to the sensitivity of the assays, the significant reduction in time to gain valuable data, and the desire to reduce the use of animals for testing. Genotoxicity assays that may be considered are outlined in Guides E1262, E1263, E1280, and E2186, and Practices E1397 and E1398. Additionally, other genotoxicity testing which might be considered (but which do not yet have ASTM test methods) include Salmonella/Mammalian-Microsomal Plate Incorporation Mutagenicity Assay, In Vivo Cytogenetics Bone Marrow Chromosomal Damage Assay, BALB/3T3 Morphological Transformation of Mouse Embryo Cells, and the Mouse Micronucleus Assay. The investigator is advised to consider carefully the appropriateness of a particular method for his application after a review of the published literature.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Inappropriate activation of complement by blood-contacting medical devices may have serious acute or chronic effects on the host. This practice is useful as a simple, inexpensive screening method for determining functional whole complement activation by solid materials in vitro.  
5.2 This practice is composed of two parts. In Part A (Section 11), human serum is exposed to a solid material. Complement may be depleted by the classical or alternative pathways. In principle, nonspecific binding of certain complement components also may occur. The alternative pathway can deplete later acting components common to both pathways, that is components other than C1, C4, and C3 (1) .4 In Part B (Section 12), complement activity remaining in the serum after exposure to the test material is assayed by classical pathway-mediated lysis of sensitized RBC.  
5.3 Assessment of in vitro whole complement activation, as described here, provides one method for predicting potential complement activation by medical materials intended for clinical application in humans when the material contacts the blood. Other test methods for complement activation are available, including assays for specific complement components and their split products (see X1.3 and X1.4).  
5.4 This in vitro test method is suitable for adoption in specifications and standards for screening solid materials for use in the construction of medical devices intended to be implanted in the human body or placed in contact with human blood.
SCOPE
1.1 This practice provides a protocol for rapid,  in vitro screening for whole complement activating properties of solid materials used in the fabrication of medical devices that will contact blood.  
1.2 This practice is intended to evaluate the acute  in vitro whole complement activating properties of solid materials intended for use in contact with blood. For this practice, the words “serum” and “complement” are used interchangeably (most biological supply houses use these words synonymously in reference to serum used as a source of complement).  
1.3 This practice consists of two procedural parts. Procedure A describes exposure of solid materials to a standard lot of human serum, using a 0.1-mL serum/13 x 100-mm disposable test tube. Cellulose acetate powders and fibers are used as examples of test materials. Procedure B describes assaying the exposed serum for significant functional whole complement depletion as compared to control samples.  
1.4 This practice does not address function, elaboration, or depletion of individual complement components, nor does it address the use of plasma as a source of complement.  
1.5 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for other aspects of biocompatibility.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 In selecting a new material for human contact in medical applications, it is important to ensure that the material will not stimulate the immune system to produce an allergic reaction. The reaction would be due to substances which could leach out of a material. Therefore, this practice provides for using material extracts. The rationale for this practice is based on the fact that the guinea pig has been shown to be the best animal model for human allergic contact dermatitis. The use of Freund’s complete adjuvant and sodium lauryl sulfate tends to enhance the potential of a material to cause an allergy. Therefore, this test, while not guaranteeing that a material is nonallergenic, is the most severe animal test in common use today.
SCOPE
1.1 This practice is intended to determine the potential for a substance, or material extract, to elicit contact dermal allergenicity.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 The presence of hemolytic material in contact with the blood may cause loss of, or damage to, red blood cells and may produce increased levels of free plasma hemoglobin capable of inducing toxic effects or other effects which may stress the kidneys or other organs.  
5.2 This practice may not be predictive of events occurring during all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
SCOPE
1.1 This practice provides a protocol for the assessment of hemolytic properties of materials used in the fabrication of medical devices that will contact blood.  
1.2 This practice is intended to evaluate the acute in vitro hemolytic properties of materials intended for use in contact with blood.  
1.3 This practice consists of a protocol for a hemolysis test under static conditions with either an extract of the material or direct contact of the material with blood. It is recommended that both tests (extract and direct contact) be performed unless the material application or contact time justifies the exclusion of one of the tests.  
1.4 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application. Test Method E2524 provides a protocol using reduced test volumes to assess the hemolytic properties of blood-contacting nanoparticulate materials; this may include nanoparticles that become unbound from material surfaces.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 The biological response to materials in the form of small particles, as from wear debris, often is significantly different from that to the same materials as larger implant components. The size and shape (morphology) of the particles may have a major effect on the biological response; therefore, this practice provides a standardized nomenclature for describing particles. Such a unified nomenclature will be of value in interpretation of biological tests of responses to particles, in that it will facilitate separation of biological responses associated with shape from those associated with the chemical composition of debris.  
5.2 The quantity, size, and morphology of particles released as wear debris from implants in vivo may produce an adverse biological response which will affect the long term survival of the device. Characterization of such debris will provide valuable information regarding the effectiveness of device designs or methods of processing components and the mechanisms of wear.  
5.3 The morphology of particles produced in laboratory tests of wear and abrasion often is affected by the test conditions, such as the magnitude and rate of load application, device configuration, and test environment. Comparison of the morphology and size of particles produced in vitro with those produced in vivo will provide valuable information regarding the degree to which the method simulates the in vivo condition being modeled.
SCOPE
1.1 This practice covers a series of procedures for characterization of the morphology, number, size, and size distribution of particles. The methods utilized include sieves, optical, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and electrooptical.  
1.2 These methods are appropriate for particles produced by a number of different methods. These include wear test machines (Test Method F732), total joint simulation systems (Guides F1714 and F1715), abrasion testing, methods for producing particulates, such as shatter boxes or pulverizers, commercially available particles, and particles harvested from tissues in animal or clinical studies.  
1.3 The debris may include metallic, polymeric, ceramic, or any combination of these.  
1.4 The digestion procedures to be used and issues of sterilization of retrieved particles are not the subject of this practice.  
1.5 A classification scheme for description of particle morphology is included in Appendix X3.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 As a precautionary measure, removed debris from implant tissues should be sterilized or minimally disinfected by an appropriate means that does not adversely affect the particulate material.  
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This test method is useful for assessing the cytotoxic potential of new materials and formulations and as part of a quality control program for established medical devices and components.  
4.2 This test method assumes that assessment of cytotoxicity provides useful information to aid in predicting the potential clinical applications in humans. Cell culture methods have shown good correlation with animal assays and are frequently more sensitive to cytotoxic agents.  
4.3 This cell culture test method is suitable for incorporation into specifications and standards for materials to be used in the construction of medical devices that are to be implanted into the human body or placed in contact with tissue fluids or blood on a long-term basis.  
4.4 Some biomaterials with a history of safe clinical use in medical devices are cytotoxic. This test method does not imply that all biomaterials must pass this assay to be considered safe for clinical use (Practice F748).
SCOPE
1.1 This test method is appropriate for materials in a variety of shapes and for materials that are not necessarily sterile. This test method would be appropriate in situations in which the amount of material is limited. For example, small devices or powders could be placed on the agar and the presence of a zone of inhibition of cell growth could be examined.  
1.1.1 This test method is not appropriate for leachables that do not diffuse through agar or agarose.  
1.1.2 While the agar layer can act as a cushion to protect the cells from the specimen, there may be materials that are sufficiently heavy to compress the agar and prevent diffusion or to cause mechanical damage to the cells. This test method would not be appropriate for these materials.  
1.2 The L-929 cell line was chosen because it has a significant history of use in assays of this type. This is not intended to imply that its use is preferred, only that the L-929 is an established cell line, well characterized and readily available, that has demonstrated reproducible results in several laboratories.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 The objective of this practice is to recommend appropriate biological endpoint assessments (which may or may not require testing) to establish a reasonable level of confidence concerning the biological response to a material or device, while at the same time avoiding unnecessary testing.  
4.2 This practice is intended to provide guidance to the materials investigator in selecting the proper procedures to be carried out for the screening of new or modified materials. Because each material and each implant situation involves its own unique circumstances, these recommendations should be modified as necessary and do not constitute the only assessment that will be required for a material. Nor should these guidelines be interpreted as minimum requirements for any particular situation. While an attempt has been made to provide recommendation for different implant circumstances, some of the recommended assessment may not be necessary or reasonable for a specific material or application.
SCOPE
1.1 This practice recommends generic biological test methods for materials and devices according to end-use applications. While chemical testing for extractable additives and residual monomers or residues from processing aids is necessary for most implant materials, such testing is not included as part of this practice. The reader is cautioned that the area of materials biocompatibility testing is a rapidly evolving field, and improved methods are evolving rapidly, so this practice is by necessity only a guideline. A thorough knowledge of current techniques and research is critical to a complete evaluation of new materials.  
1.2 These test protocols are intended to apply to materials and medical devices for human application. Biological evaluation of materials and devices, and related subjects such as pyrogen testing, batch testing of production lots, and so on, are also discussed. Tests include those performed on materials, end products, and extracts. Rationale and comments on current state of the art are included for all test procedures described.  
1.3 The biocompatibility of materials used in single or multicomponent medical devices for human use depends to a large degree on the particular nature of the end-use application. Biological reactions that are detrimental to the success of a material in one device application may have little or no bearing on the successful use of the material for a different application. It is, therefore, not possible to specify a set of biocompatibility test methods which will be necessary and sufficient to establish biocompatibility for all materials and applications.  
1.4 The evaluation of tissue engineered medical products (TEMPs) may, in some cases, involve different or additional testing beyond those suggested for non-tissue-based materials and devices. Where appropriate, these differences are discussed in this practice and additional tests described.  
1.5 The ethical use of research animals places the obligation on the individual investigator to determine the most efficient methods for performing the necessary testing without undue use of animals. Where adequate prior data exists to substantiate certain types of safety information, these guidelines should not be interpreted to mean that testing should be unnecessarily repeated.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

ABSTRACT
This practice is intended to determine the potential for a substance, or material extract, to elicit contact dermal allergenicity. It is intended as an alternative to the Guinea Pig Maximization Test (GPMT), given the limitations on dosage form and tendency for false positives associated with the latter test. The split adjuvant method is used when topical application is considered relevant, and the dosage form is a solid, liquid, extract, paste, or gel. The method includes four induction doses applied over a period of time to the same shaved or depilated site on guinea pigs, followed by occlusive patching. Freund's Complete Adjuvant (FCA) is injected near the dose site on a specific time, (second induction dose). Following a rest period, animals are challenged at a previously unexposed site, and the reaction evaluated at regular intervals. The closed patch method is used when topical application is relevant, but the preferred dosage form does not permit injection under the skin or intradermally, and the discomfort involved with extended occlusive patching and adjuvant use is to be avoided. It involves repeated induction doses over a period of time at the same shaved/depilated site, followed each time by occlusive wrapping. After a rest period, animals are challenged at previously untreated sites, and their reactions evaluated after a period of time.
SIGNIFICANCE AND USE
5.1 In selecting a material for human contact in medical applications, it is important to ensure that the material will not stimulate the immune system to produce an allergic reaction under relevant exposure conditions. Extractable chemicals produced by skin contact or during physiological exposures may cause allergic reactions. Therefore, this practice provides for evaluations of solid or semisolid dosage forms using material extracts or direct evaluation of the test article. The rationale for this animal model is based on the fact that the guinea pig has been shown to be an appropriate animal model for predicting human contact dermatitis. Its tractable nature, its availability from reputable suppliers, the historical database of information already acquired using this species, and the correlation of such results to data on known human allergens, all contribute to its widespread use for allergenicity studies (1-5).4  
5.2 The need for sensitization procedures other than the maximization test (Practice F720) is based on: (1) the need for a route of exposure more similar to use conditions; (2) concern over the use of adjuvant because of its recruitment of cell types to the test site which are not typically involved in immunologic reactions, and because of the discomfort this causes in the animals; (3) absence of a proper FCA-irritant control group in the traditional maximization design; and (4) the frequency of false positives often encountered with the GPMT. Both of these tests are internationally accepted (1).
SCOPE
1.1 This practice is intended to determine the potential for a substance, or material extract, to elicit contact dermal allergenicity.  
1.2 This practice is intended as an alternative to the Guinea Pig Maximization Test (GPMT), given the limitations on dosage form and tendency for false positives associated with the latter test. See Rationale and References.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This practice is a guideline for a short-term screening test for the evaluation of the tissue response to materials that may be selected for implantation in the human body and should be done in accordance with good laboratory practices. This test may be performed prior to long-term testing (for example, Practice F981) to eliminate unsuitable candidate materials early and to avoid unnecessary animal testing.  
4.2 This practice may be used to detect toxic effects of materials in general (see Appendix X1). However, it is particularly suitable for the testing of materials that are intended to have contact with subcutaneous tissues or soft tissues in general. For materials intended to be inserted specifically into muscle tissues, Practice F763 should be considered as a short-term test method.  
4.3 The suggested implant specimens are cylindrical. A special grooved type of cylinder may be used (see Fig. X2.1 of Appendix X2) to allow tissue interlocking that could keep the implant in place and minimize tissue irritation through motion at the interface that otherwise could contribute to increased variance of the results. In case ungrooved cylinders are used (see Fig. X1.2 of Appendix X2), probable motion at the implant/tissue interface must be taken into account. Control cylinders should be shaped like the test cylinders.  
4.4 The type of surface preparation of the specimens can affect the tissue reaction; therefore the preparation procedure should be noted in the report. The test may be used to compare the effect of different surface structures or conditions of the same material or to assess the effect of various treatments of modifications of a material.
Note 1: If this method is used for material research, testing for endotoxin prior to implantation should be considered.
SCOPE
1.1 This practice covers a short-term testing method to screen the subcutaneous tissue reaction to implant candidate materials in small laboratory animals. The material may be dense or porous. This method may not work for absorbable materials, depending on the absorption kinetics. The tissue reactions will be evaluated in comparison to those evoked by control materials that are accepted as clinical implant materials.  
1.2 This practice, along with other appropriate biological tests (including other ASTM test methods), may be used to assess the biocompatibility of candidate materials for use in the fabrication of devices for clinical application. It may be also applied to evaluate the effect of special surface textures and preparations of known materials.  
1.3 This practice does not provide a comprehensive assessment of the systemic toxicity, carcinogenicity, teratogenicity, or mutagenicity of the material. Additional information may be needed on the material in its final finished form, such as implantation assessment at the clinically relevant location.  
1.4 The values stated in SI units, including units officially accepted for use with SI, are to be regarded as standard. No other systems of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 Materials that are to be in contact with the skin should not cause irritation to the skin. Since it is probably the substances leached from a material that cause the irritation, this practice provides for direct material-skin contact testing or for skin exposure to the liquid extract of the test material. The rationale for this rabbit test is that it is a comparatively quick and sensitive method which, through use over the years, has become a generally accepted method. Additionally, the albino rabbit allows for easy visualization of erythema and edema, which are the cardinal signs of skin irritation.
SCOPE
1.1 This practice covers a procedure by which the irritancy of a material may be assessed through contact with abraded and intact skin of rabbits.  
1.2 The results of this practice depend upon the effectiveness with which contact between the skin and the test material is established and maintained. Because of the operator technique included in performing this test, it is important that the test be performed by personnel with appropriate training.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard may involve hazardous materials, operations, and equipment. This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This test method is intended to assess a combination of inherent chemical irritation and mechanical irritation for products and materials expected to come into contact with the skin. It is a comparative approach whereby the potential irritation of a test material is compared to that of a reference material similar in form and composition. The reference material should have a known safety and irritation profile.
SCOPE
1.1 The Behind-the-Knee (BTK) method, using the popliteal fossa of human volunteers as a test site, simultaneously evaluates the inherent chemical irritation, and the potential for mechanical irritation of substrates and products that are designed to come into repeated or extended close contact with the skin (see validation references 1-7).2 This is a bilateral test comparing a test material to a reference material with a known safety profile.  
1.2 This test method shall be used by qualified health care professionals experienced in good clinical practice (GCP) procedures.  
1.3 This test method can be performed using human subjects on either intact or compromised skin. Testing should be performed on intact skin for test substrates or products expected to have contact with normal, intact skin, or for direct comparison to products with a known skin irritation profile. Testing can be performed on compromised skin for test substrates or products that may commonly come into contact with damaged skin (for example, skin with diaper rash, or chapped skin) or skin that is expected to be hydrated.  
1.4 Visual scoring of erythema and dryness is performed by a trained skin grader on a pre-defined scale.  
1.5 Prior to use in this test, materials shall undergo overall favorable biocompatibility testing consistent with the approach outlined in protocol Practice F748 or ISO 10993-1:2009. As a part of this series of testing, irritation per Practice F719 or ISO 10993-10 shall be conducted.  
1.6 The values stated in inch-pound units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.  
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 The purpose of this test method is to determine the time citrated plasma exposed to medical materials takes to form a clot when exposed to a suspension of phospholipid particles and calcium chloride. In this test method, the test article is the activator. The PTT assay is a general screening test for a medical material’s ability to activate the intrinsic coagulation pathway. Material samples that show a shortened PTT are activators of the intrinsic coagulation pathway.  
4.2 The test article, reference materials, and controls are exposed to human plasma. The plasma is tested on a coagulation device. Each sample tube is assayed in duplicate. The results are reported as a percentage of the negative control.
SCOPE
1.1 This test method covers the screening of circulating blood-contacting device materials for their ability to induce blood coagulation via the intrinsic coagulation pathway. This assay should be part of the hemocompatibility evaluation for devices and materials contacting human blood, as per ANSI/AAMI/ISO 10993-4.  
1.2 All safety policies and practices shall be observed during the performance of this test method.  
1.3 All plasma and any materials that had contact with plasma will be bagged in a biohazard bag, properly labelled with the contents, and disposed of by appropriate means. The plasma should be handled at the Biosafety Level 2 as recommended in the Centers for Disease Control/National Institutes of Health Manual Biosafety in Microbiological Laboratories.  
1.4 The normal pooled human plasma must have tested negative for Hepatitis B (HBV) or Human Immunodeficiency (HIV) viruses. The plasmas should be treated like any patient plasma using standard precautions. The plasma should be handled at the Biosafety Level 2 as recommended in the Centers for Disease Control/National Institutes of Health Manual Biosafety in Microbiological Laboratories.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 The purpose of this test method is to determine the time citrated plasma exposed to medical materials takes to form a clot when exposed to a suspension of phospholipid particles and calcium chloride. In this test method, the test article is the activator. The PTT assay is a general screening test for a medical material’s ability to activate the intrinsic coagulation pathway. Material samples that show a shortened PTT are activators of the intrinsic coagulation pathway.  
4.2 The test article, reference materials, and controls are exposed to human plasma. The plasma is tested on a coagulation device. Each sample tube is assayed in duplicate. The results are reported as a percentage of the negative control.
SCOPE
1.1 This test method covers the screening of circulating blood-contacting device materials for their ability to induce blood coagulation via the intrinsic coagulation pathway. This assay should be part of the hemocompatibility evaluation for devices and materials contacting human blood, as per ANSI/AAMI/ISO 10993-4.  
1.2 All safety policies and practices shall be observed during the performance of this test method.  
1.3 All plasma and any materials that had contact with plasma will be bagged in a biohazard bag, properly labelled with the contents, and disposed of by appropriate means. The plasma should be handled at the Biosafety Level 2 as recommended in the Centers for Disease Control/National Institutes of Health Manual Biosafety in Microbiological Laboratories.  
1.4 The normal pooled human plasma must have tested negative for Hepatitis B (HBV) or Human Immunodeficiency (HIV) viruses. The plasmas should be treated like any patient plasma using standard precautions. The plasma should be handled at the Biosafety Level 2 as recommended in the Centers for Disease Control/National Institutes of Health Manual Biosafety in Microbiological Laboratories.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 The use of in vivo implantation techniques for characterizing the biocompatibility of materials to be utilized in various medical applications provides a unique assessment of such materials not achieved by other procedures. Physical characteristics (that is, form, density, hardness, surface finish) can influence the character of the tissue response to the test materials.  
5.2 This practice is intended as a rapid screening procedure for determining the acceptability of candidate materials. It would be invoked prior to using the long-term tests described in Practice F981. It is understood that for some applications additional tests, including long-term implantation studies, may be required to assess the final suitability of the candidate materials.  
5.3 This practice may not be appropriate for all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
SCOPE
1.1 This practice provides guidelines for short-term testing or screening of candidate materials, both porous and dense, as to the effects of the material on animal tissue in which it is implanted. This is a rapid screening procedure for determining acceptability of candidate materials.  
1.2 This practice, along with other appropriate biological tests (including other appropriate ASTM tests) may be used in the biocompatibility assessment of the candidate materials for use in the fabrication of devices for clinical application.  
1.3 This experimental protocol is not designed to provide a comprehensive assessment of the systemic toxicity, carcinogenicity, teratogenicity, or mutagenicity of the material since other standards deal with these issues.  
1.4 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 provides guidance for the selection of appropriate methods for testing materials for a specific application.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This practice covers a test protocol for comparing the local tissue response evoked by biomaterials, from which medical implantable devices might ultimately be fabricated, with the local tissue response elicited by control materials currently accepted for the fabrication of surgical devices. The materials may include metals (and metal alloys), dense aluminum oxide, and polyethylene that are standardized on the basis of acceptable, well recognized, long-term response. The controls consistently produce cellular reaction and wound healing to a degree that has been found to be acceptable to the host.
SCOPE
1.1 This practice provides a series of experimental protocols for biological assays of tissue reaction to nonabsorbable biomaterials for surgical implants. It assesses the effects of the material on animal tissue in which it is implanted. The experimental protocol is not designed to provide a comprehensive assessment of the systemic toxicity, immune response, carcinogenicity, teratogenicity, or mutagenicity of the material since other standards deal with these issues. It applies only to materials with projected applications in humans where the materials will reside in bone or soft tissue in excess of 30 days and will remain unabsorbed. It is recommended that short-term assays, according to Practice F763, first be performed. Applications in other organ systems or tissues may be inappropriate and are therefore excluded. Control materials will consist of any one of the metal alloys in Specifications F67, F75, F90, F136, F138, or F562, high purity dense aluminum oxide as described in Specification F603, ultra high molecular weight polyethylene as stated in Specification F648 or USP polyethylene negative control.  
1.2 This practice is a combination of Practice F361 and Practice F469. The purpose, basic procedure, and method of evaluation of each type of material are similar; therefore, they have been combined.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This practice is a guideline for a screening test for the evaluation of the local tissue response to materials that may be selected for implantation into the human body and which are expected to undergo absorption within three years.  
4.2 This practice is similar to that for studies on candidate materials that are not absorbable, such as those specified in Practices F763, F981, and F1408; however, analysis of the host response must take into account the effect of degradation and degradation products on the inflammatory response at the local tissue site and on subsequent healing of the implantation site.  
4.3 The material to be tested should be in the final finished form as for intended use, including sterilization. Material/body ratios should be relevant to that of intended device use. Material surface area or mass to body mass ratios of 1X, 10X, and 50X if applicable, are recommended.  
4.4 Materials that are designed for use in devices with in situ polymerization shall be introduced in a manner such that in situ polymerization occurs. Testing of individual precursor components is not recommended.
SCOPE
1.1 This practice provides experimental protocols for biological assays of tissue reactions to absorbable biomaterials for implant applications. This practice applies only to absorbable materials with projected clinical applications in which the materials will reside in bone or soft tissue longer than 30 days and less than three years. Other standards with designated implantation times are available to address shorter time periods. Careful consideration should be given to the appropriateness of this practice for slowly degrading materials that will remain for longer than three years. It is anticipated that the tissue response to degrading biomaterials will be different from the response to nonabsorbable materials. In many cases, a chronic inflammatory response may be observed during the degradation phase, but the local histology should return to normal after absorption; therefore, the minimal tissue response usually equated with “biocompatibility” may require long implantations.  
1.2 The time period for implant absorption will vary depending on chemical composition implant size, implant location, and test subject species; therefore, the implantation times for examination of tissue response will be linked to the rate of absorption. No single implantation time is indicated in this practice.  
1.3 These protocols assess the effects of the material on the animal tissue in which it is implanted. The experimental protocols do not fully assess systemic toxicity, carcinogenicity, teratogenicity, or mutagenicity of the material. Other standards are available to address these issues.  
1.4 To maximize use of the animals in the study protocol, all toxicological findings should be recorded. There are some aspects of systemic toxicity, including effects of degradation products on the target organs, that can be addressed with this practice, and these effects should be documented fully.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This practice is to be used to help assess the biocompatibility of materials used in medical devices. It is designed to test the effect of particles from the materials on the host tissues.  
4.2 The appropriateness of the methods should be carefully considered by the user since not all materials or applications need to be tested by this practice. The validity of these studies in predicting the human response is not known at this time and studies such as those described here are needed.  
4.3 Abbreviations Used:  
4.3.1 CD—Cluster differentiation.  
4.3.2 DNA—Deoxyribonucleic acid.  
4.3.3 EDS—Energy dispersive X-ray spectroscopy.  
4.3.4 EU—Endotoxin unit.  
4.3.5 HLA—Human leukocyte antigens.  
4.3.6 LAL—Limulus amebocyte lysate.  
4.3.7 LPS—Lipopolysaccharide (endotoxin).  
4.3.8 RNA—Ribonucleic acid.
SCOPE
1.1 This practice covers the production of wear particles and degradation products from implanted materials that may lead to a cascade of biological responses resulting in damage to adjacent and remote tissues. In order to ascertain the role of particles in stimulating such responses, the nature of the responses, and the consequences of the responses, established protocols are needed. This is an emerging, rapidly developing area and the information gained from standard protocols is necessary to interpret responses. Some of the procedures listed here may, on further testing, not prove to be predictive of clinical responses to particulate debris. However, only the use of standard protocols will establish which are useful techniques. Since there are many possible and established ways of determining responses, a single standard protocol is not stated. However, this recommended practice indicates which necessary information should be supplied with test results. For laboratories without established protocols, recommendations are given and indicated with an asterisk (*).  
1.2 This standard is not designed to provide a comprehensive assessment of the systemic toxicity, carcinogenicity, teratogenicity, or mutagenicity of the material.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
5.1 These extraction procedures are the initial part of several test procedures used in the biocompatibility screening of plastics used in medical devices.  
5.2 The limitations of the results obtained from this practice should be recognized. The choice of extraction vehicle, duration of immersion, and temperature of the test is necessarily arbitrary. The specification of these conditions provides a basis for standardization and serves as a guide to investigators wishing to compare the relative resistance of various plastics to extraction vehicles.  
5.3 Correlation of test results with the actual performance or serviceability of materials is necessarily dependent upon the similarity between the testing and end-use conditions (see 12.1.2 and Note 4).  
5.4 Caution should be exercised in the understanding and intent of this practice as follows:  
5.4.1 No allowance or distinction is made for variables such as end-use application and duration of use. Decisions on selection of tests to be done should be made based on Practice F748.  
5.4.2 This practice was originally designed for use with nonporous, solid materials. Its application for other materials, such as those that are porous, or absorptive, or resorptive, should be considered with caution. Consideration should be given to altering the specified material to liquid ratio to allow additional liquid to fully hydrate the material and additional liquid or other methods to fully submerge the test article. Additional procedures that fully remove the extract liquid from the test article, such as pressure or physically squeezing the material, should also be considered as appropriate. Although no definitions are given in this practice for the following terms, such items as extraction vehicle surface tension at the specified extraction condition and plastic specimen physical structure should be taken into account.  
5.5 Test Methods D543, D570, and D1239 may be useful in providing supplemental information.
SCOPE
1.1 This practice covers methods of extraction of medical plastics and may be applicable to other materials. This practice identifies a method for obtaining “extract liquid” for use in determining the biological response in preclinical testing. Further testing of the “extract liquid” is specified in other ASTM standards. The extract may undergo chemical analysis as part of the preclinical evaluation of the biological response, and the material after extraction may also be examined.  
1.2 This practice may be used for, but is not limited to the following areas: partial evaluation of raw materials, auditing materials within the manufacturing process, and testing final products. This practice may also be used as a referee method for the measurement of extractables in plastics used in medical devices.  
1.3 1.3 This practice was initially developed for extraction of medical plastics not intended to undergo degradation or absorption during normal medical device usage. When applied to the extraction of absorbable materials, additional considerations may be necessary in the selection of extraction procedures and fluids.  
1.4 The values stated in inch-pound units are to be regarded as standard. The values given in parentheses are mathematical conversions to SI units that are provided for information only and are not considered standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
5.1 The presence of hemolytic material in contact with the blood may cause loss of, or damage to, red blood cells and may produce increased levels of free plasma hemoglobin capable of inducing toxic effects or other effects which may stress the kidneys or other organs.  
5.2 This practice may not be predictive of events occurring during all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
SCOPE
1.1 This practice provides a protocol for the assessment of hemolytic properties of materials used in the fabrication of medical devices that will contact blood.  
1.2 This practice is intended to evaluate the acute in vitro hemolytic properties of materials intended for use in contact with blood.  
1.3 This practice consists of a protocol for a hemolysis test under static conditions with either an extract of the material or direct contact of the material with blood. It is recommended that both tests (extract and direct contact) be performed unless the material application or contact time justifies the exclusion of one of the tests.  
1.4 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application. Test Method E2524 provides a protocol using reduced test volumes to assess the hemolytic properties of blood-contacting nanoparticulate materials; this may include nanoparticles that become unbound from material surfaces.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
5.1 Inappropriate activation of complement by blood-contacting medical devices may have serious acute or chronic effects on the host. This practice is useful as a simple, inexpensive screening method for determining functional whole complement activation by solid materials in vitro.  
5.2 This practice is composed of two parts. In Part A (Section 11), human serum is exposed to a solid material. Complement may be depleted by the classical or alternative pathways. In principle, nonspecific binding of certain complement components also may occur. The alternative pathway can deplete later acting components common to both pathways, that is components other than C1, C4, and C3 (1) .4 In Part B (Section 12), complement activity remaining in the serum after exposure to the test material is assayed by classical pathway-mediated lysis of sensitized RBC.  
5.3 Assessment of in vitro whole complement activation, as described here, provides one method for predicting potential complement activation by medical materials intended for clinical application in humans when the material contacts the blood. Other test methods for complement activation are available, including assays for specific complement components and their split products (see X1.3 and X1.4).  
5.4 This in vitro test method is suitable for adoption in specifications and standards for screening solid materials for use in the construction of medical devices intended to be implanted in the human body or placed in contact with human blood.
SCOPE
1.1 This practice provides a protocol for rapid,  in vitro screening for whole complement activating properties of solid materials used in the fabrication of medical devices that will contact blood.  
1.2 This practice is intended to evaluate the acute  in vitro whole complement activating properties of solid materials intended for use in contact with blood. For this practice, the words “serum” and “complement” are used interchangeably (most biological supply houses use these words synonymously in reference to serum used as a source of complement).  
1.3 This practice consists of two procedural parts. Procedure A describes exposure of solid materials to a standard lot of human serum, using a 0.1-mL serum/13 x 100-mm disposable test tube. Cellulose acetate powders and fibers are used as examples of test materials. Procedure B describes assaying the exposed serum for significant functional whole complement depletion as compared to control samples.  
1.4 This practice does not address function, elaboration, or depletion of individual complement components, nor does it address the use of plasma as a source of complement.  
1.5 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for other aspects of biocompatibility.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

SIGNIFICANCE AND USE
4.1 This guide is not intended to specify the exact method of conducting a test for any particular material but only to present some of the criteria that should be considered in method design and possible problems that could lead to misleading results. In the development of the actual test protocol, it is recommended that recognized tumorigenesis bioassay procedures be consulted.  
4.2 The recommendations given in this guide may not be appropriate for all applications or types of implant materials. These recommendations should be utilized by experienced testing personnel in conjunction with other pertinent information and the requirements of the specific material application.
SCOPE
1.1 This guide is intended to assist the biomaterials testing laboratory in the conduct and evaluation of tumorigenicity tests to evaluate the potential for new materials to evoke a neoplastic response. The procedure is generally reserved only for those materials which have not previously been used for human implantation for a significant period of time.  
1.2 Assessment of tumorigenicity is one of several procedures employed in determining the biological response to a material as recommended in Practice F748. It is assumed that the investigator has already determined that this type of testing is necessary for a particular material before consulting this guide. The recommendations of Practice F748 should be considered before a study is commenced.  
1.3 Whenever possible, it is recommended that a battery of genotoxicity procedures be initiated and proposed as an alternative to an in-vivo tumorigenicity bioassay. Genotoxicity assays may also be considered as initial screening procedures due to the sensitivity of the assays, the significant reduction in time to gain valuable data, and the desire to reduce the use of animals for testing. Genotoxicity assays that may be considered are outlined in Guides E1262, E1263, E1280, and E2186, and Practices E1397 and E1398. Additionally, other genotoxicity testing which might be considered (but which do not yet have ASTM test methods) include Salmonella/Mammalian-Microsomal Plate Incorporation Mutagenicity Assay, In Vivo Cytogenetics Bone Marrow Chromosomal Damage Assay, BALB/3T3 Morphological Transformation of Mouse Embryo Cells, and the Mouse Micronucleus Assay. The investigator is advised to consider carefully the appropriateness of a particular method for his application after a review of the published literature.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This practice is a guideline for a short-term screening test for the evaluation of the tissue response to materials that may be selected for implantation in the human body. This test may be performed prior to longterm testing (for example, Practice F981) to eliminate unsuitable candidate materials early and to save further animal testing.  
4.2 This practice may be used to detect toxic effects of materials in general (see Appendix X1). However, it is particularly suitable for the testing of materials that are intended to have contact with subcutaneous tissues or soft tissues in general. For materials intended to be inserted specifically into muscle tissues, Practice F763 should be considered as a short term test method.  
4.3 The suggested implant specimens are cylindrical. A special grooved type of cylinder may be used (see Fig. X2.1 of Appendix X2) to allow tissue interlocking that could keep the implant in place and minimize tissue irritation through motion at the interface that otherwise could contribute to increased variance of the results. In case ungrooved cylinders are used (see Fig. X1.2 of Appendix X2), probable motion at the implant/tissue interface must be taken into account. Control cylinders should be shaped like the test cylinders.  
4.4 The type of surface preparation of the specimens can affect the tissue reaction, therefore the preparation procedure should be noted in the report. The test may be used to compare the effect of different surface structures or conditions of the same material or to assess the effect of various treatments of modifications of a material.
SCOPE
1.1 This practice covers a short-term testing method to screen the subcutaneous tissue reaction to metallic or other implant candidate materials in small laboratory animals. The material may be dense or porous. The tissue reactions will be evaluated in comparison to those evoked by control materials that are accepted as clinical implant materials.  
1.2 This practice, along with other appropriate biological tests (including other ASTM test methods), may be used to assess the biocompatibility of candidate materials for use in the fabrication of devices for clinical application. It may be also applied to evaluate the effect of special surface textures and preparations of known materials.  
1.3 This experimental protocol is not designed to provide a comprehensive assessment of the systemic toxicity, carcinogenicity, teratogenicity, or mutagenicity of the material.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
2.1 The CHO/HGPRT assay detects forward mutations of the X-linked hypoxanthine-guanine phosphoribosyl transferase (hgprt) locus (coding for the enzyme, HGPRT) in Chinese hamster ovary (CHO) cells. Cells originally derived from Chinese hamster ovary tissue are exposed to a test article and, following an appropriate cell culture regimen, descendants of the original treated population are monitored for the loss of functional HGPRT, presumably due to mutations. Resistance to a purine analogue, 6-thioguanine (6TG) (or less desirably, 8-azaguanine (8AG)), is employed as the genetic marker. HGPRT catalyzes the conversion of the nontoxic 6TG to its toxic ribophosphorylated derivative. Loss of the enzyme or its activity therefore leads to cells resistant to 6TG.  
2.2 Because HGPRT is an enzyme of the purine nucleotide salvage pathway, loss of the enzyme is not a lethal event. Different types of mutational events (base substitutions, frameshifts, deletions, some chromosomal type lesions, and so forth) should theoretically be detectable at the hgprt locus. The CHO/HGPRT assay has been used to study a wide range of mutagens, including radiations (2-4), and a wide variety of chemicals (1), and complex chemical mixtures (5).
SCOPE
1.1 This guide highlights some of the more relevant biological concepts as they are currently understood, and summarizes the critical technical aspects for acceptable bioassay performances as they currently are perceived and practiced. The Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay (1) 2 has been widely applied to the toxicological evaluation of industrial and environmental chemicals.  
1.2 This guide concentrates on the practical aspects of cell culture, mutagenesis procedures, data analysis, quality control, and testing strategy. The suggested approach represents a consensus of the panel members for the performance of the assay. It is to be understood, however, that these are merely general guidelines and are not to be followed without the use of sound scientific judgement. Users of the assay should evaluate their approach based on the properties of the substances to be tested and the questions to be answered.  
1.3 Deviation from the guidelines based on sound scientific judgement should by no means invalidate the results obtained.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
5.1 The propensity of a material to stimulate delayed contact hypersensitivity must be assessed before clinical application of devices containing this material. Delayed hypersensitivity may occur anywhere in the body. Systemic delayed hypersensitivity may have a complex set of reactions and consequences depending on the actual tissue/organ site of reaction. Although the reactions are seldom life-threatening, severe tissue and organ damage my result over time. Skin is the usual test site to determine the propensity of a material to cause delayed hypersensitivity.  
5.2 The standard historical test methods have involved the use of guinea pigs with a cutaneous application and observation of the reaction site. The use of the murine local lymph node assay results in a numerical quantitation of stimulation, rather than subjective evaluation and could be used to determine dose responses.  
5.3 This practice may not be predictive of events occurring during all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
SCOPE
1.1 This practice provides a methodology to use an in-situ procedure for the evaluation of delayed contact hypersensitivity reactions.  
1.2 This practice is intended to provide an alternative to the use of guinea pigs for evaluation of the ability of a device material to stimulate delayed contact hypersensitivity reactions. This alternative is particularly applicable for materials used in devices that contact only intact skin. However, the guinea pig maximization test is still the recommended method when assessing the delayed hypersensitivity response to metals or when testing substances that do not penetrate the skin but are used in devices that contact deep tissues or breached surfaces. This practice may be used for testing metals, with the exception of nickel-containing metals, unless the unique physicochemical properties of the materials may interfere with the ability of LLNA to detect sensitizing substances.  
1.3 This practice consists of a protocol for assessing an increase in lymphocyte proliferation within the nodes draining the site of administration on the ears of mice.  
1.4 The LLNA has been validated only for low-molecular-weight chemicals that can penetrate the skin. The absorbed chemical or metabolite must bind to macromolecules, such as proteins, to form immunogenic conjugates.  
1.5 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.  
1.6 Identification of a supplier of materials or reagents is for the convenience of the user and does not imply a single source. Appropriate materials and reagents may be obtained from many commercial supply houses.  
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 In selecting a new material for human contact in medical applications, it is important to ensure that the material will not stimulate the immune system to produce an allergic reaction. The reaction would be due to substances which could leach out of a material. Therefore, this practice provides for using material extracts. The rationale for this practice is based on the fact that the guinea pig has been shown to be the best animal model for human allergic contact dermatitis. The use of Freund’s complete adjuvant and sodium lauryl sulfate tends to enhance the potential of a material to cause an allergy. Therefore, this test, while not guaranteeing that a material is nonallergenic, is the most severe animal test in common use today.
SCOPE
1.1 This practice is intended to determine the potential for a substance, or material extract, to elicit contact dermal allergenicity.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This practice is to be used to help assess the biocompatibility of materials used in medical devices. It is an acute toxicological test designed to detect the presence of injurious leachable substances.  
4.2 This practice may not be appropriate for all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.  
4.3 The only applicable limitation is the extract preparation. Refer to Sections 4.3 and 4.4 of Practice F619 for a description of this limitation.
SCOPE
1.1 This practice is a nonspecific, acute toxicity test used to help determine the biocompatibility of materials used in medical devices.  
1.2 The liquids injected into the rabbits are those obtained by Practice F619 where the extraction vehicles are saline, vegetable oil, or other liquids simulating human body fluids.  
1.3 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

SIGNIFICANCE AND USE
4.1 The purpose of this test method is to determine if medical materials exposed to human whole blood will adversely affect the platelet and leukocyte counts in whole blood. A large number of platelets and leukocytes as part of thrombi adhering to the material will be reflected by a decrease in their counts in blood. Thrombogenic materials should not be used for cardiovascular medical devices, unless the purpose of the device is to promote thrombosis.
SCOPE
1.1 This test method assists in the evaluation of cardiovascular device materials for their ability to induce thrombus formation. Thrombus formation is assessed by means of a reduction in human platelets and leukocytes when consumed by thrombus after activation on the material surface. This assay may be part of the hemocompatibility evaluation for devices and materials contacting human blood, as per ANSI/AAMI/ISO 10993–4. See also Test Method F2382.  
1.2 All safety policies and practices shall be observed during the performance of this test method. All human blood and any materials that had contact with human blood shall be bagged in a biohazard bag, properly labeled as the contents, and disposed of by appropriate means.  
1.3 The human blood should be handled at Biosafety Level 2 as recommended in the Centers for Disease Control/National Institutes of Health Manual Biosafety in Microbiological Laboratories. The human blood donor must have tested negative for Hepatitis B (HBV) and Human Immunodeficiency (HIV) viruses. The blood should be treated like any patient blood in using universal precautions.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Some specific hazards statements are given in Section 7 on Hazards.

SIGNIFICANCE AND USE
4.1 The objective of this guide is to recommend a panel of biological tests that can be used in addition to the testing recommended in Practice F748. This guide is designed to detect neurotoxicity caused by medical devices that contact nervous tissue.  
4.2 The testing recommendations should be considered for new materials, established materials with different manufacturing methods that could affect nervous tissue response, or materials used in new nervous tissue applications.  
4.3 Chemical characterization can be used to evaluate similarity for materials with a history of clinical use in a similar nervous tissue application.
SCOPE
1.1 Medical devices may cause adverse effects on the structure and/or function of the nervous system. In this guide, these adverse effects are defined as neurotoxicity. This guide provides background information and recommendations on methods for neurotoxicity testing. This guide should be used with Practice F748, and may be helpful where neurotoxicity testing is needed to evaluate medical devices that contact nervous system tissue or cerebral spinal fluid (CSF).  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 Materials that are to be in contact with the skin should not cause irritation to the skin. Since it is probably the substances leached from a material that cause the irritation, this practice provides for direct material-skin contact testing or for skin exposure to the liquid extract of the test material. The rationale for this rabbit test is that it is a comparatively quick and inexpensive method which, through use over the years, has become a generally accepted method.
SCOPE
1.1 This practice covers a procedure by which the irritancy of a biomaterial may be assessed through contact with abraded and intact skin of rabbits.  
1.2 The results of this practice depend upon the effectiveness with which contact between the skin and the test material is established and maintained. Because of the operator technique included in performing this test, it is important that the test be performed by personnel with appropriate training.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
5.1 This practice is useful for assessing the cytotoxic potential both when evaluating new materials or formulations for possible use in medical applications, and as part of a quality control program for established medical devices.  
5.2 This practice is used for assessing the cytotoxic potential of materials intended for the fabrication of inserts or implants in the orofacial region.  
5.3 This practice is restricted to normal non-transformed, human orofacial tissues using cells cultured in human serum factors and does not depend upon cells and serum from non-human sources.  
5.4 This practice incorporates procedures to monitor the quality of ingredient materials and the uniformity of the production process for formulating stock compositions.  
5.5 This practice may be useful to determine the effects of age and radiation, and the state of carcinogenicity on the sensitivity of HED tissues to materials and devices used for orofacial prostheses.
SCOPE
1.1 This practice describes a procedure to assess the cytotoxic potential of materials for use in the construction of medical materials and devices using human excised donor (HED) tissues and their derived primary cells taken from the orofacial region.  
1.2 This practice may be used either directly to evaluate materials or as a reference against which other cytotoxicity methods may be compared.  
1.3 This practice is one of a series of reference methods for assessment of cytotoxic potential, employing different techniques.  
1.4 Assessment of cytotoxicity is one of several procedures employed in determining the biological response to a material, as recommended in Practice F748.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This practice is intended to help assess the biocompatibility of materials used in medical devices. It is an acute toxicological test designed to detect the presence of injurious leachable substances.  
4.2 This practice may not be appropriate for all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.  
4.3 The only limitation applicable is the extract preparation. Refer to Sections 4.3 and 4.4 of Practice F619 for a description of this limitation.
SCOPE
1.1 This practice covers a nonspecific, acute toxicity test used for detecting leachables from materials used in medical devices.  
1.2 The liquids injected into the mouse are those obtained by Practice F619 where the extraction vehicles are saline, vegetable oil, or other liquids simulating human body fluids.  
1.3 Two procedures are outlined: Method A for intravenous injection and Method B for intraperitoneal injection.  
1.4 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

SIGNIFICANCE AND USE
4.1 In selecting a new material for human contact in medical applications, it is important to ensure that the material will not stimulate the immune system to produce an allergic reaction. The reaction would be due to substances which could leach out of a material. Therefore, this practice provides for using material extracts. The rationale for this practice is based on the fact that the guinea pig has been shown to be the best animal model for human allergic contact dermatitis. The use of Freund’s complete adjuvant and sodium lauryl sulfate tends to enhance the potential of a material to cause an allergy. Therefore, this test, while not guaranteeing that a material is nonallergenic, is the most severe animal test in common use today.
SCOPE
1.1 This practice is intended to determine the potential for a substance, or material extract, to elicit contact dermal allergenicity.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This practice is to be used to help assess the biocompatibility of materials used in medical devices. It is an acute toxicological test designed to detect the presence of injurious leachable substances.  
4.2 This practice may not be appropriate for all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.  
4.3 The only applicable limitation is the extract preparation. Refer to Sections 4.3 and 4.4 of Practice F619 for a description of this limitation.
SCOPE
1.1 This practice is a nonspecific, acute toxicity test used to help determine the biocompatibility of materials used in medical devices.  
1.2 The liquids injected into the rabbits are those obtained by Practice F619 where the extraction vehicles are saline, vegetable oil, or other liquids simulating human body fluids.  
1.3 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.