ASTM F1904-23

Standard Guide for Testing the Biological Responses to Medical Device Particulate Debris and Degradation Products <emph type="ital">in vivo</emph>

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...

General Information

• Status: Published
• Publication Date: 31-Mar-2023
• ICS: 11.040.40 - Implants for surgery, prosthetics and orthotics
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.16 - Biocompatibility Test Methods

Relations

• Refers: ASTM F561-19 - Standard Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
• Effective Date: 01-Jan-2019
• Refers: ASTM F1903-18 - Standard Practice for Testing for Cellular Responses to Particles <emph type="bdit" >in vitro</emph>
• Effective Date: 01-Oct-2018
• Refers: ASTM F1877-16 - Standard Practice for Characterization of Particles
• Effective Date: 01-Oct-2016
• Refers: ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Apr-2016
• Refers: ASTM F561-13 - Standard Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
• Effective Date: 01-Sep-2013
• Refers: ASTM F561-05a(2010) - Standard Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
• Effective Date: 01-Sep-2010
• Refers: ASTM F1903-10 - Standard Practice for Testing For Biological Responses to Particles <i>In Vitro</i>
• Effective Date: 01-Jun-2010
• Refers: ASTM F748-06(2010) - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Jun-2010
• Refers: ASTM F1877-05(2010) - Standard Practice for Characterization of Particles
• Effective Date: 01-Jun-2010
• Refers: ASTM F619-03(2008) - Standard Practice for Extraction of Medical Plastics
• Effective Date: 01-Aug-2008
• Refers: ASTM F748-06 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Dec-2006
• Refers: ASTM F1877-05 - Standard Practice for Characterization of Particles
• Effective Date: 01-Nov-2005
• Refers: ASTM F1877-05e1 - Standard Practice for Characterization of Particles
• Effective Date: 01-Nov-2005
• Refers: ASTM F561-05a - Standard Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
• Effective Date: 01-Oct-2005
• Refers: ASTM F561-05 - Standard Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
• Effective Date: 01-Jun-2005

Overview

ASTM F1904-23: Standard Guide for Testing the Biological Responses to Medical Device Particulate Debris and Degradation Products in vivo is a significant resource developed by ASTM International. This standard provides guidance for assessing the biocompatibility of materials used in medical devices by focusing on in vivo evaluation of particulate debris and degradation products. The guide addresses the biological impact of particles generated by wear, corrosion, or material breakdown-essential considerations in the lifecycle of implants and other medical devices that come into contact with body tissues.

The standard is designed to help manufacturers, researchers, and regulatory professionals understand and evaluate the local and systemic biological responses, such as foreign body response (FBR) and immune/inflammatory reactions, triggered by medical device debris. By offering principles and recommendations, ASTM F1904-23 informs better study design, outcome predictability, and risk management.

Key Topics

• Biocompatibility Assessment: Guidance for evaluating whether materials and their fragments released from medical devices provoke adverse tissue or immune responses.
• Test Method Selection: Outlines criteria for the appropriate selection of in vivo models and endpoints, such as immunological assays, histopathology, and tissue remodeling.
• Types of Immune Response: Differentiates between innate (e.g., macrophage-driven FBR) and adaptive (delayed-type hypersensitivity) immune reactions, and highlights the need for specific testing of both.
• Debris Characterization: Emphasizes the importance of fully characterizing particles, including source, size, shape, chemistry, and concentration.
• Animal Models: Explores a range of in vivo experimental approaches-air pouch models, subcutaneous cages, and direct injection models-to simulate clinical scenarios and assess localized and systemic responses.
• Data Reporting: Recommends standardized reporting of methodologies, material attributes, routes of administration, and analytical techniques to improve data interpretation and comparison.
• Continuous Improvement: Encourages ongoing refinement of testing protocols in response to new scientific data and clinical experience, acknowledging that predictive accuracy of current methods continues to evolve.

Applications

ASTM F1904-23 is most relevant for industries and professionals involved in:

• Medical Device Development: Particularly for implantable devices (orthopedic, cardiovascular, and other body-contacting devices) where wear debris and degradation products are anticipated.
• Regulatory Submissions: Assisting manufacturers in preparing comprehensive biocompatibility data packages for pre-market approval or clearance.
• Risk Management: Identifying and minimizing potential risks posed by particulate debris and degradation products through careful material selection, device design, and testing.
• Preclinical Research: Designing meaningful animal studies to investigate biological effects of device-derived particles, including immune and inflammatory responses.
• Post-Market Surveillance: Interpreting biological responses to particles retrieved from explanted devices or related clinical cases.

By applying this guide, manufacturers can better predict and mitigate adverse patient outcomes related to material debris, leading to safer and more effective medical devices.

Related Standards

For comprehensive evaluation and to ensure harmonized methodologies, ASTM F1904-23 should be used in conjunction with several related standards:

• ASTM F561: Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
• ASTM F619: Practice for Extraction of Materials Used in Medical Devices
• ASTM F748: Practice for Selecting Generic Biological Test Methods for Materials and Devices
• ASTM F1877: Practice for Characterization of Particles
• ASTM F1903: Practice for Testing for Cellular Responses to Particles in vitro

Additionally, referenced ISO standards provide context for wear simulation, isolation and characterization of device particles, and assessment of orthopedic and cardiovascular implants:

• ISO 14242 & ISO 14243: Wear testing for total hip/knee prostheses
• ISO 17853: Isolation and characterization of polymer and metal wear particles
• ISO 22622: Wear of total ankle-joint prostheses

Keywords: ASTM F1904, biocompatibility, medical device debris, in vivo testing, foreign body response, biological response, particle characterization, implants, immune response, degradation products.

Standards Content (Sample)

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.
Designation: F1904 − 23
Standard Guide for
Testing the Biological Responses to Medical Device
Particulate Debris and Degradation Products in vivo
This standard is issued under the fixed designation F1904; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope patibility assessment by considering the testing principles and
methods available in published literature that are appropriate to
1.1 The purpose of this standard guide is to describe the
their products.
principles and approaches to testing of medical device debris
1.3 This standard is not sufficient to address device-related
and degradation products from device materials (for example,
degradation products that result in gas formation or that are
particles from wear) for their potential to activate a cascade of
exclusively represented by nanoparticles, or soluble species
biological responses at local and systemic levels in the body. In
such as dissolved metal ions.
order to ascertain the role of device debris and degradation
products in stimulating such responses, the nature of the 1.4 While devices should be designed and manufactured in
responses and the consequences of the responses should be such a way as to reduce as far as possible the risks posed by
evaluated. This is an emerging area. The continuously updated substances or particles (including wear debris, degradation
information gained from the testing results and related pub- products, and processing residues) that may be released from
the device, this standard guide may help users to identify the
lished literature is necessary to improve the study designs, as
well as predictive value and interpretation of the test results presence of wear debris and degradation products and subse-
quent adverse reactions that may occur.
regarding debris/degradation product related responses. Some
of the procedures listed here may, on further testing, not prove
1.5 Although this guide is based on the available device
to be predictive of clinical responses to device-related debris
debris-related knowledge that is largely based on orthopedic
and degradation products. However, only the continuing use of
devices, most of the recommendations are also applicable to
standard protocols will establish the most useful testing ap-
other (non-orthopedic) device areas.
proaches with reliable study endpoints and measurement tech-
1.6 This standard does not purport to address all of the
niques. Since there are many possible and established ways of
safety concerns, if any, associated with its use. It is the
determining the debris/degradation product related responses
responsibility of the user of this standard to establish appro-
in vivo, a single standard protocol is not stated. However, this
priate safety, health, and environmental practices and deter-
recommended guide indicates which testing approaches are
mine the applicability of regulatory limitations prior to use.
most applicable per expected biological responses and which
1.7 This international standard was developed in accor-
necessary information should be supplied with the test results.
dance with internationally recognized principles on standard-
To address the general role of chronic inflammation in exag-
ization established in the Decision on Principles for the
gerating device-related foreign body response (FBR), the
Development of International Standards, Guides and Recom-
recommendations in this standard include the assessment of
mendations issued by the World Trade Organization Technical
device-related pro-inflammatory responses and subsequent tis-
Barriers to Trade (TBT) Committee.
sue remodeling potential.
2. Referenced Documents
1.2 This document is to provide the users with updated
scientific knowledge that may help better characterize medical 2.1 ASTM Standards:
F561 Practice for Retrieval and Analysis of Medical
device debris related responses. It is to help the users to
optimize their plans for particle characterization and biocom- Devices, and Associated Tissues and Fluids
F619 Practice for Extraction of Materials Used in Medical
Devices
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved April 1, 2023. Published April 2023. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 1998. Last previous edition approved in 2014 as F1904 – 14. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/F1904-23. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F1904 − 23
F748 Practice for Selecting Generic Biological Test Methods of small particles may enhance the amount of extracted
for Materials and Devices substances but, since the response to particles may be related to
F1877 Practice for Characterization of Particles the physical size, shape, composition, and dose, the use of only
F1903 Practice for Testing for Cellular Responses to Par- extracts will not completely address the question of the impact
ticles in vitro
of particle formation on the tissue response, and actual implan-
2.2 ISO Standards: tation or other testing of particles should be included as a part
ISO 14242-1 Implants for surgery—Wear of total hip-joint
of the characterization of tissue response when particle gen-
prostheses—Part 1: Loading and displacement parameters eration is likely during actual usage. These materials or extracts
for wear-testing machines and corresponding environmen-
may be used for the in vivo tests described here or ex vivo / in
tal conditions for test—Amendment 1
vitro approaches described in Practice F1903. Particles and
ISO 14242-3 Implants for surgery—Wear of total hip-joint
other device-related debris/degradation products generated by
prostheses—Part 3: Loading and displacement parameters
alternative methods (for example, from animal studies, clinical
for orbital bearing type wear testing machines and corre-
use, or in vitro studies) may also be used, if appropriately
sponding environmental conditions for test
justified. The method of generation must be described.
ISO 14243-1 Implants for surgery—Wear of total knee-joint
prostheses—Part 1: Loading and displacement parameters
5. Significance and Use
for wear-testing machines with load control and corre-
5.1 This standard guide is to be used to help assess the
sponding environmental conditions for test
biocompatibility of materials used in medical devices (for
ISO 14243-3 Implants for surgery—Wear of total knee-joint
example, externally communicating, implants, and other body
prostheses—Part 3: Loading and displacement parameters
contact medical devices). It is designed to test the effect of
for wear-testing machines with displacement control and
particles and other wear debris and/or degradation products on
corresponding environmental conditions for test
the generation of FBR and other (local and systemic) host
ISO 17853 Wear of implant materials—Polymer and metal
responses of immune/inflammatory origin.
wear particles—Isolation and characterization
ISO 22622 Implants for surgery—Wear of total ankle-joint
5.2 The appropriateness of the selected testing methods
prostheses—Loading and displacement parameters for
should be carefully considered by the user since not all
wear-testing machines with load or displacement control
materials or applications need to be tested by this guide.
and corresponding environmental conditions for test
Existing biocompatibility screening methods may not be fully
predictive of the human response, and testing approaches such
3. Terminology
as those described here are needed for continuous improvement
3.1 Definitions of Terms Specific to This Standard:
of the predictability of biocompatibility testing. The effective-
3.1.1 mechanistic, adj—of or relating to the theory of
ness of animal testing in terms of its predictability of human
mechanism which, in the science of biology, is defined as a
outcomes is dependent on the study design. If possible, study
system of causally interacting parts and processes that produce
endpoints should be chosen to minimize interspecies variability
one or more effects.
and to investigate clinically relevant biological responses.
3.1.2 phagocytosable, adj—capable of being phagocytosed.
While testing approaches should remain at the user’s
discretion, the following should be taken into consideration
4. Summary of Guide
when selecting most appropriate tests and study endpoints.
4.1 Evaluation of biological responses to medical device
5.2.1 Device-induced responses usually involve both innate
debris and degradation products may be performed using
and adaptive immunities, which raises possible need for
specimens from animals being tested in accordance with
specific testing for each of these immune response types.
Practice F748 which provides recommendations for biocom-
5.2.1.1 Device-related adaptive immune responses are
patibility assessment including local and systemic toxicity.
mostly due to lymphocyte-mediated delayed-type hypersensi-
When biocompatibility testing is performed (for example,
tivity. In vivo allergenicity to a test material (which can be
implantation or injection of the test material), evaluation of the
introduced via different routes) should be assessed by moni-
tissues surrounding the application site represent the best
toring for any signs of allergic and acute toxicity reactions, for
opportunity for assessing FBR and other local tissue responses.
example, scratch, tremor, and dyspnea. In addition, ex vivo
Bodily fluids such as blood and urine, as well as different organ
analysis on immunophenotyping of the isolated splenocytes/
tissues from the tested animals should be used for the assess-
lymphocytes from the same studies should be considered.
ment of systemic responses. Procedures according to Practice
5.2.1.2 Device-related innate immune responses are mostly
F561 may be used to assess the cellular and tissue responses in
mediated by macrophages and can be assessed by histopatho-
vivo.
logical assessment of the extent of FBR including macrophage
4.2 Biological responses to device-related wear debris and
accumulation around the test material. Supplementary ex vivo
degradation products may be tested using materials or extracts
/ in vitro assessment can be used for additional macrophage-
in accordance with Practice F619. The increased surface area
based testing such as macrophage immunophenotyping (proin-
flammatory M1 and anti-inflammatory/wound healing M2) as
well as debris uptake by phagocytes (phagocytozability) in-
Available from American National Standards Institute (ANSI), 25 W. 43rd St.,
4th Floor, New York, NY 10036, http://www.ansi.org. volving the entire range of test material characteristics.
F1904 − 23
5.2.2 Due to the role of inflammation in extending device- cardiovascular material should take into consideration potential
related FBR and promoting the resultant tissue remodeling, hemolytic, thrombolytic/thrombogenic, and pro-angiogenic ef-
fects.
histopathological assessment should include identification of
immune/inflammatory cell infiltration (with separate counts for
5.2.4.2 Some endpoints currently used in effectiveness as-
the individual cell types representing both innate and adaptive
sessments can be applied to the safety assessment of adverse
responses) as well as corresponding tissue changes (for tissue remodeling (examples of osteogenesis-related study
example, fibrosis, necrosis, ossification or osteolysis, angio-
endpoints can be found in X1.12).
genesis). Identification of immune/inflammatory cells may
5.2.4.3 While not all possible clinical complications can be
involve different approaches including IHC phenotyping as
accurately replicated in animal testing models, the properly
needed. Supplementary ex vivo / in vitro assessment should be
selected study endpoints for in vivo and supplementary in vitro
considered for assessing the balance in release of pro-
testing can enhance the overall predictability of biocompatibil-
inflammatory versus anti-inflammatory cytokines as well as ity testing (more details on the choice of measurable study
generation of hyper-proliferative versus hypo-proliferative tis- endpoints are provided in X1.5).
sue responses.
5.2.5 Rodents and other small animals (for example, rabbit,
guinea pig) are traditionally used for in vivo biocompatibility
5.2.2.1 Since the signs of inflammation and post-
testing models. Use of larger animal models is usually limited
inflammatory tissue changes may not be always apparent,
due to ethical and other concerns and may be reserved for
special attention should be given to the assessment of debris-
models in higher need for imitating similarities with humans
related inflammogenic and tissue remodeling potentials using
(weight, bone and joint structure, etc.).
ex vivo specimens and supplementary in vitro assessment when
needed. Pro-inflammatory cell death (necrosis) should be
5.3 Abbreviations Used:
distinguished from programmed cell death (apoptosis usually
5.3.1 ALVAL—Aseptic lymphocyte-dominated vasculitis-
associated with anti-inflammatory responses) by using cell
associated lesion.
viability and cytotoxicity testing involving cellular staining
5.3.2 CD—Cluster differentiation.
and flow cytometry. Given the importance of phagocytes in
5.3.3 DAMP—Damage-associated molecular pattern.
proper clearance of dying cells, normal non-phlogistic phago-
5.3.4 EDS/EDAX—Energy dispersive X-ray spectroscopy.
cytosis of cells undergoing apoptosis should be distinguished
5.3.5 ELISA—Enzyme-linked immunosorbent assay.
from “frustrated” phlogistic phagocytosis which may result in
5.3.6 FBGC—Foreign body giant cell.
further cell/tissue damage due to the release of damage-
associated molecular patterns (DAMP). See X1.10 for more
5.3.7 FBR—Foreign body response.
details.
5.3.8 FTIR—Fourier-transform infrared (spectroscopy).
5.2.3 Due to the role of the device-tissue interface in
5.3.9 H&E—Hematoxylin and eosin.
shaping biological responses, in vivo models as well as
5.3.10 HMGB1—High-mobility group box 1.
supplementary testing should be aimed to simulate (as much as
5.3.11 HSP—Heat shock protein.
possible) device-specific use environments. In vivo animal
5.3.12 ICAM1—Intercellular adhesion molecule-1.
models with intra-articular applications of a test material may
5.3.13 ICP-MS—Inductively coupled plasma–mass spec-
be beneficial for testing of orthopedic materials, while
trometry.
intracardiac/intravenous applications may be more beneficial
5.3.14 Ig—Immunoglobulin.
for testing of cardio/endovascular materials.
5.3.15 IL—Interleukin.
5.2.3.1 Since many implantable materials come in contact
5.3.16 LAL—Limulus amebocyte lysate.
with blood during their clinical use, the need for hemocompat-
5.3.17 LPS—Lipopolysaccharide (endotoxin).
ibility testing should be considered, especially when develop-
5.3.18 MMP—Matrix metalloproteinase.
ing new materials. Development of new materials for cardio-
vascular applications may benefit from a more detailed 5.3.19 NO—Nitric oxide.
hemocompatibility assessment, which could include
5.3.20 NOS/iNOS—Nitric oxide synthase / Inducible nitic
microcirculation, cell adhesion, and leukocyte-endothelial in-
oxide synthase.
teractions.
5.3.21 PCR—Polymerase chain reaction.
5.2.4 The predictability of testing for a certain material,
5.3.22 ROS—Reactive oxygen species.
including its debris, may benefit from the choice of study
5.3.23 SAA—Serum amyloid A.
endpoints and testing approaches that incorporates clinical
5.3.24 SEM—Scanning electron microscopy.
experience from known therapeutic applications and safety
5.3.25 α-SMA—Alpha-smooth muscle actin.
issues of similar materials.
5.3.26 TBARS—Thiobarbituric acid reactive substances.
5.2.4.1 In general, the study endpoints should be selected
5.3.27 TGF-β—Transforming growth factor-beta.
per their ability to measure immunomodulatory, pro/anti-
5.3.28 TLR—Toll-like receptor.
inflammogenic, and tissue remodeling effects. As the examples
5.3.29 TNF-α—Tumor necrosis factor-alpha.
of more specific choices, testing for an orthopedic material
5.3.30 TRAP—Tartrate-resistant acid phosphatase.
should take into consideration potential tissue changes such as
periprosthetic osteolysis and pseudotumors, while testing for a 5.3.31 VEGF—Vascular endothelial growth factor.
F1904 − 23
6. Characterization Using in Vivo Systems 6.1.2.1 Chemistry (for example, bulk material chemical
composition, additives, impurities, chemical structure such as
6.1 Test and Control Material—Characterize the nature and
crystallinity, surface properties such as protein corona).
the range of the particles and other possible debris or degra-
6.1.2.2 Size (mean and other population characteristics).
dation products (for example, ions) used, including but not
6.1.2.3 Shape (per Practice F1877).
limited to the following.
6.1.2.4 Surface charge (if applicable).
6.1.1 Possible sources of test and control material:
6.1.2.5 Method of sterilization. In the event or when it is
6.1.1.1 The method used to produce the test and control
material for subsequent in vivo biological evaluation shall anticipated that the method of sterilization will confound
medical device debris and/or degradation product generation
generate the range of particles (for example, particle size and
distribution, shape, and amount or dose) and other debris (for and/or release during test, it shall be accounted and considered.
example, generated by instruments used to surgically implant If the presence of bacterial lipopolysaccharide (LPS) was
the product) or degradation products (for example, corrosion, quantified on test or reference materials after sterilization,
breakdown products) reasonably expected from clinical use.
specify the sensitivity of the LPS detection method.
The method of generation shall be described and justified.
6.1.2.6 Concentration of test material as weight, or number
Particles or degradation products can be generated in bench
(of particles), or surface area/device or volumetric dose.
testing (for example, using joint simulator machines or me-
6.2 In Vivo Testing—Biological responses from the animals
chanical fatigue/corrosion testing fixtures) but may also be
exposed to the medical device debris/degradation products
produced by other validated techniques, taking into account the
under test should be evaluated in comparison to those derived
proposed intended use. General recommendations for isolation
from controls. The following controls should be considered to
and characterization of wear particles from bench testing are
assist with data analysis of medical device debris/degradation
included in Practice F561 and ISO 17853. Additionally, stan-
products under test: negative and positive controls (for
dards are available that provide recommended loading and
example, reference products) as well as a sham procedure
displacement parameters for wear testing for specific anatomi-
control with no exposure to any test or reference material.
cal locations to generate corresponding wear particles (for
example, ISO 14242-1 and 14242-3 for total hip replacement
6.2.1 In Vivo Models—One or more of these models with
devices; ISO 14243-1 and ISO 14243-3 for total knee replace- different routes of exposure to the wear debris/degradation
ment devices; ISO 22622 for total ankle replacement devices).
products can be used if appropriately qualified (see 6.4):
Device-related particles and other debris or degradation prod-
6.2.1.1 Air Pouch Model—This intradermal model has an
ucts may also be generated in vivo from a test material in
established utility for simulating synovium and identifying
clinical use or animal studies which replicate the conditions of
particle-related immune responses. This model may be adapted
a relevant end-use application. Once particles are isolated and
for testing of devices/materials that come into contact with
characterized using techniques such as in Practice F561 and
tissues other than synovium, but its relevance to other in vivo
ISO 17853, they should be processed to remove or reduce (that
systems should be validated. The volume of air and the time
is, to a biologically insignificant level) any contaminants (for
allowed before introduction of the particles into the created
example, endotoxin, chemicals used in the isolation or charac-
pouches, the time points for sample collection, and the ob-
terization process) and then sterilized as needed (see 6.1.2.5) to
served effects should be specified. This model is more appro-
allow them to be used in subsequent biological testing.
priate for the assessment of acute immune responses; its
6.1.1.2 Test articles can be prepared according to Practice
appropriateness for the assessment of prolonged immune and
F619.
inflammatory responses needs to be validated for the length of
6.1.1.3 Purchased or generated controls (including reference time of implantation. The assessment of immune responses in
materials) should consist of particles with characteristics (for this model is based on evaluation of the exudates collected
example, chemical composition, charge, size, shape, and dose) from the pouches. In addition to conventional testing for cell
that correspond to the test particles and thus could aid in
death/viability, this model allows a detailed assessment of the
achieving their comprehensive evaluation. In addition to selec- induced leukocytosis, for example, percentages of
tion per physical/chemical characteristics with regard to the lymphocytes, monocytes, neutrophils, and macrophages, cor-
test samples, controls should be selected, when possible, per
roborated with the use of flow cytometry and cell-specific
expected biological effects of a test material; the choice and markers. Evaluation of the debris/degradation product induced
appropriateness of selected control(s) should be explained. The
leukocyte infiltration may be enhanced by measuring addi-
selected controls should correspond to the range of test
tional immune responses such as the induction of cytokines
material related debris/degradation products. Specifically, con-
(for example, tumor necrosis factor alpha (TNFα)) or matrix
trols should include both phagocytosable and non-
metalloproteinases (for example, MMP9). The enhanced air
phagocytosable types (for example, per size: <10 μm and
pouch model may constitute a surrogate test for histopathology
≥10 μm, respectively) that were found within the range of
analysis and (semi)quantitative assessment of immune reac-
generated test particles, to enable comparative analysis of the
tions and tissue inflammation. In addition to its use for particle
test particle effects in terms of their phagocytozability.
debris, this model may be used to test the effects of test
materials that are not particles, including gaseous device/
6.1.2 The following attributes and corresponding methods
should be reported for test and control materials: material related products. As an additional criterion of its
F1904 − 23
suitability as a standard model, the air pouch model can be evaluated in comparison with those from control animals. See
executed with limited costs and basic personnel and infrastruc- also X1.5 in this standard for additional considerations regard-
ture. ing the characterization of device-related debris/degradation
6.2.1.2 Cages—Cages made of porous materials such as products.
stainless steel mesh or porous polytetrafluoroethylene (PTFE)
6.3 Control Animals—In the conduct of testing with any of
can be implanted subcutaneously or intraperitoneally with a
the above described models, appropriate control animals who
test material inside the cage. The cage material and the implant
receive any vehicles, carriers, or other treatments received by
location chosen should be specified and explained. The fluid
the experimental models, to control for the effects of factors
accumulating in the cage should be sampled at various time
other than the presence of the particles, should be included as
intervals which should also be specified and explained. The
well.
cage and contained material removed at the termination of the
6.4 Method Qualification:
experiment should be evaluated for cell adhesion, cell types,
6.4.1 For any of the methods described above, the following
and other study endpoints including soluble products (the time
should be developed to support qualification for use with
chosen for termination and the choice of subsequent measure-
medical device debris/degradation products:
ments should be explained per specifics of a test material).
6.4.1.1 Provide detailed protocols and discuss any optimi-
Fluid containing a large number of red blood cells should be
zations needed as compared to published methods. Describe
discarded as representing blood and not cage fluid. As further
the applicability of the method for the device-specific end use
limitations of this model, encapsulation of the cage may impact
(for example, treatment period optimization, size and amount/
the fluid collection and corresponding cell and protein re-
dose of particles, debris materials that may be incompatible
sponses; more importantly, some of the observed effects may
with the test system).
be due to the cage itself and not a test or control material.
6.4.1.2 Specify and justify with supporting data, including
6.2.1.3 Bone Implant Chamber—This is a modification of
the criteria to be used to interpret test results as compared to
the cage system which allows determination of the effect of
controls.
particles and the resulting biological response on bone remod-
eling and therefore is more appropriate for testing of devices/
7. Testing Approaches, Data Analysis, and Reporting—
materials with orthopedic applications.
Biological Responses
6.2.1.4 Direct Implantation/Injection—Direct applications
of a test material via implantation (or injection if relevant to
7.1 For all test and control samples, the following should be
device use) are practically devoid of the delivery system
considered for analysis and reporting:
related effects that may complicate interpretation of the test
7.2 Cellular/Tissue Response—Cell accumulation at the site
results from cage/chamber-based models. Intraperitoneal,
of the particles should be evaluated for relative numbers and
intravenous, intramuscular, and subcutaneous are the favored
types of cells. Standard paraffin or plastic embedded sections
routes in injection models; intra-articular injection is usually
are usually sufficient to identify neutrophils, lymphocytes,
reserved for materials with orthopedic end applications. In
macrophages, foreign body giant cells (FBGC), osteoclasts,
general, the end use application should govern the route of
osteoblasts, osteocytes, eosinophils, etc. Traditional Hema-
injection/implantation as well as the choice and scope of
toxylin and Eosin (H&E) staining can be used in most
organs and tissues utilized for testing.
biocompatibility-related histology and cytology applications.
NOTE 1—Careful dose selection and monitoring is essential to ensure
In some cases, special histological procedures, or immunohis-
animal welfare and minimization of adverse outcomes.
tochemical stains such as those described in Practice F561, or
6.2.1.5 At the termination of the study, all sites used in these
flow cytometry may be needed, for example, to confirm the
in vivo studies should be carefully evaluated for infection, identity of lymphocytes and macrophages as the main cell
since the presence of infection may have a major impact on the
types involved in adaptive and innate immune responses,
testing outcome by simulating and mimicking many test respectively. Overall cellular response should be characterized
material-related inflammatory responses (more details are pro-
as focal or diffuse; its extent can be further evaluated, for
vided in 7.2.1). In many cases, evaluation for possible infection example, using a scale of 0 to 5 with 0 being no cell
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F1904 − 14 F1904 − 23
Standard PracticeGuide for
Testing the Biological Responses to Particles Medical
Device Particulate Debris and Degradation Products in vivo
This standard is issued under the fixed designation F1904; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. 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.
2. Referenced Documents
2.1 ASTM Standards:
F561 Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
F619 Practice for Extraction of Materials Used in Medical Devices
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F1877 Practice for Characterization of Particles
3. Summary of Practice
3.1 Biological responses to particles testing may be done using specimens from animals being tested in accordance with the
Practice F748 matrix for irritation and sensitivity, or for implantation. If particles were implanted during the testing procedures or
generated during the experimental time period, the response to those particles may form a part of the overall investigation of
response to particles. Blood, organs, or tissues from the animals may be used.
This practiceguide is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods.
Current edition approved March 1, 2014April 1, 2023. Published May 2014April 2023. Originally approved in 1998. Last previous edition approved in 20082014 as
F1904 – 98 (2008).F1904 – 14. DOI: 10.1520/F1904-14.10.1520/F1904-23.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F1904 − 23
3.2 Biological responses to particles may be tested using the actual particulate materials or extracts in accordance with Practice
F619. The increased surface area of small particles may enhance the amount of extracted substances but, since the response to
particles may be related to the physical size, shape and composition, the use of only extracts will not completely address the
question of the impact of particle formation on the tissue response and actual implantation or other testing of particles should be
included as a part of the characterization of tissue response when particle generation is likely during actual usage. These materials
or extracts may be used in in vivo tests or for the in vitro tests. Particles generated by other methods may also be used. The method
of generation shall be described.
4. 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.
5. Responses from In Vivo Systems
5.1 Particles—Define the nature of the particles used:
5.1.1 Source,
5.1.2 Chemistry,
5.1.3 Size (mean and range),
5.1.4 Shape,
5.1.5 Surface charge (if known),
5.1.6 Method of sterilization,
5.1.7 If the presence of bacterial lipopolysaccharide (LPS) was determined, specify how this was done and the sensitivity of the
method. (LAL testing with a sensitivity of at least 0.06 EU is recommended),
5.1.8 Concentration of particles used as weight, or number, or surface area/implant, and
5.1.9 Polystyrene particles, spherical, 1 to 5 μm in size may be used as reference particles.
F1904 − 23
5.1.10 Practice F1877 may be useful in defining the nature of the particles.
5.2 Biological System—One or more of these sites should be used:
5.2.1 Air Pouch Model— This is a model to simulate synovial tissue. The volume of air and the time allowed before introduction
of the particles should be specified. This model needs to be validated for length of time of implantation and relevance to other in
vivo systems.
5.2.2 Cages—Cages made of porous materials such as stainless steel mesh or porous teflon can be implanted with a test material
inside the cage. These may be implanted subcutaneously or intraperitoneally. The material and the implant location chosen should
be specified. The fluid accumulating in the cage can be sampled at various time intervals. The time intervals shall be specified. The
cage and contained material is removed at the termination of the experiment (specify the time chosen) and evaluated for cell
adhesion, cell type, and products. Fluid containing a large number of red blood cells should be discarded since it represents blood,
not cage fluid.
5.2.3 Bone Implant Chamber—This is a modification of the cage system and allows determination of the effect of particles and
the resulting biological response on bone remodeling
5.2.4 Direct Injection— Intraperitoneal, intravenous, intramuscular, and subcutaneous are the favored routes. The end use
application should govern the route of injection and the organ or tissue utilized in this test. Inhalation may be suitable for some
end use applications.
5.2.5 Other Methods—The use of other biological systems, animal models,or methods of implantation may be appropriate,
depending upon the intended use of the material.
5.2.6 Examination of tissue at implant retrieval from animal models or clinical conditions is dealt with in Practice F561, and
Practice F1877 may be used to describe the morphology of the particles that may be present in or extracted from those tissues.
Some of the procedures defined here are also applicable to these tissues.
5.2.7 All sites used in these studies should be carefully evaluated for infection and inflammation at the termination of the study.
The presence of infection or inflammation will have a major impact on the outcome since it stimulates many responses.
5.2.8 Control Animals—In the conduct of testing with any of the above described models, appropriate control animals who receive
any vehicles, carriers, other treatments received by the experimental models, to control for the effects of factors other than the
presence of the particles, should be included as well.
5.3 Biological Response—One or more of the following should be performed:
5.3.1 Cell accumulation at the site of the particles should be evaluated for the relative number and type of cells. Standard paraffin
or plastic embedded sections are usually sufficient to identify acute inflammatory cells, lymphocytes, macrophages, foreign body
giant cells, osteoclasts, osteoblasts, osteocytes, eosinophils, etc. But in some cases special histological procedures, or
immunohistochemical stains such as those described in Practice F561, or flow cytometry may be needed to confirm the identity
of lymphocytes and macrophages. An evaluation scale of 0 to 5 with 0 being no cell response, 1 being accumulation of a few cells,
2 being a mild response with some cell accumulation, 3 being a moderate response, 4 being a large response, and 5 being a severe
response is recommended. It should also be noted whether the response is focal or diffuse.
5.3.1.1 Transport of particles to relevant draining organs and histologic responses in these organs should be determined, especially
when direct injection is used. The relevant organs would be spleen, liver, and kidney. In some cases, the lung may also be an
appropriate draining organ when it is reasonable to suspect that particles could enter the venous return portion of the vascular
system. The draining nodes should be harvested if identifiable. Some types of particles are distinctive (for example, carbon fibers),
but lymph nodes and lung commonly contain particles and bits of birefringent material that may be confused with particles used
in the experiment. Light microscopy with and without polarized light can be suggestive of particle migration, but other methods
(for example, EDS) may be necessary to confirm the composition of the migrating particles. Organs from control animals should
also be evaluated.
5.3.2 Soluble Cell Products Elaborated—This is a rapidly emerging area of technology. Histochemical and immunohistochemical
techniques can be used to great advantage in these studies. Reliable reagents, kits, or hybridization protocols are available to detect
F1904 − 23
cellular products such as cytokines, prostaglandins, immunoglobulins, as well as the lymphocyte CD markers and some HLA
markers. It is not necessary to measure all possible cellular products and the selection should be based on whether there is emphasis
on the response of macrophages or other cells involved in the non-specific immune response, or on the specific immune response.
NOTE 1—The identification and study of reactive cellular products is a rapidly expanding field and any listing of specific products from which to choose
would necessarily become obsolete quickly. An immunologist should be consulted to assist in the selection of substances for which testing should be
performed.
5.3.3 When other products from the cellular response are being detected, they should be specified and the method used specified.
5.4 Effects of the particles on other systems such as bone remodeling, chondrocyte function, cartilage repair, and synovial tissue
function and repair are also important studies. The methods used should be fully described.
6. Report Section and Data Analysis
6.1 The histologic response should be compared to that of normal tissues with no particles and to that of tissues receiving the
polystyrene reference particles, if used as reference particles. This may be done by counting, by digitization, by cell analyzer, or
by estimation in the field of view. In some circumstances, the presence or absence of marker or response will suffice. In some
++
circumstances, the quantitation of the response may be obtained with data on responses such as Ca released, enzyme levels, DNA
or RNA levels, etc.
6.2 The report should include a description of the methods used, route of administration and source of the particles, and other
details of the experimental protocol sufficient to allow the results to be interpreted in the context of the testing methods used.
7. Keywords
7.1 biocompatibility; biological response; in vivo; interleukins; particles
APPENDIX
(Nonmandatory Information)
X1. RATIONALE
X1.1 The primary purpose of this practice is to describe methodologies to determine the biological response to particles using in
vivo responses.
X1.2 It is well recognized that the biological responses to particles could be different from those to solid materials. The interaction
of the particles with cells in the tissues, notably macrophages and other phagocytic cells, is a key to the final biological response.
X1.3 The interaction of particles with host tissues has been an active research area for many years. Many investigators have
developed procedures for doing these studies. This practice is intended to delineate the information necessary for interpretation of
the results from these various studies and to describe methodology appropriate for investigators developing such studies.
X1.4 The interaction of the biological system with particles will lead to the accumulation of various cells that may produce soluble
mediators that influence the progression of the immune response. Studies such as the ones described here are needed to determine
the importance of this response in the biocompatibility and biocompatibility testing of materials.
F1904 − 23
X1.5 This practice was revised in 2014 to incorporate new information and update some of the information originally included
in the 1998 version.
ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentioned
in this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the risk
of infringement of such rights, are entirely their own responsibility.
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if not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standards
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1. 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 characterization and biocompatibility assessment
by considering the testing principles and methods available in published literature that are appropriate to their products.
1.3 This standard is not sufficient to address device-related degradation products that result in gas formation or that are exclusively
represented by nanoparticles, or soluble species such as dissolved metal ions.
1.4 While devices should be designed and manufactured in such a way as to reduce as far as possible the risks posed by substances
or particles (including wear debris, degradation products, and processing residues) that may be released from the device, this
standard guide may help users to identify the presence of wear debris and degradation products and subsequent adverse reactions
that may occur.
1.5 Although this guide is based on the available device debris-related knowledge that is largely based on orthopedic devices, most
of the recommendations are also applicable to other (non-orthopedic) device areas.
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.
F1904 − 23
2. Referenced Documents
2.1 ASTM Standards:
F561 Practice for Retrieval and Analysis of Medical Devices, and Associated Tissues and Fluids
F619 Practice for Extraction of Materials Used in Medical Devices
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F1877 Practice for Characterization of Particles
F1903 Practice for Testing for Cellular Responses to Particles in vitro
2.2 ISO Standards:
ISO 14242-1 Implants for surgery—Wear of total hip-joint prostheses—Part 1: Loading and displacement parameters for
wear-testing machines and corresponding environmental conditions for test—Amendment 1
ISO 14242-3 Implants for surgery—Wear of total hip-joint prostheses—Part 3: Loading and displacement parameters for orbital
bearing type wear testing machines and corresponding environmental conditions for test
ISO 14243-1 Implants for surgery—Wear of total knee-joint prostheses—Part 1: Loading and displacement parameters for
wear-testing machines with load control and corresponding environmental conditions for test
ISO 14243-3 Implants for surgery—Wear of total knee-joint prostheses—Part 3: Loading and displacement parameters for
wear-testing machines with displacement control and corresponding environmental conditions for test
ISO 17853 Wear of implant materials—Polymer and metal wear particles—Isolation and characterization
ISO 22622 Implants for surgery—Wear of total ankle-joint prostheses—Loading and displacement parameters for wear-testing
machines with load or displacement control and corresponding environmental conditions for test
3. Terminology
3.1 Definitions of Terms Specific to This Standard:
3.1.1 mechanistic, adj—of or relating to the theory of mechanism which, in the science of biology, is defined as a system of
causally interacting parts and processes that produce one or more effects.
3.1.2 phagocytosable, adj—capable of being phagocytosed.
4. Summary of Guide
4.1 Evaluation of biological responses to medical device debris and degradation products may be performed using specimens from
animals being tested in accordance with Practice F748 which provides recommendations for biocompatibility assessment including
local and systemic toxicity. When biocompatibility testing is performed (for example, implantation or injection of the test material),
evaluation of the tissues surrounding the application site represent the best opportunity for assessing FBR and other local tissue
responses. Bodily fluids such as blood and urine, as well as different organ tissues from the tested animals should be used for the
assessment of systemic responses. Procedures according to Practice F561 may be used to assess the cellular and tissue responses
in vivo.
4.2 Biological responses to device-related wear debris and degradation products may be tested using materials or extracts in
accordance with Practice F619. The increased surface area of small particles may enhance the amount of extracted substances but,
since the response to particles may be related to the physical size, shape, composition, and dose, the use of only extracts will not
completely address the question of the impact of particle formation on the tissue response, and actual implantation or other testing
of particles should be included as a part of the characterization of tissue response when particle generation is likely during actual
usage. These materials or extracts may be used for the in vivo tests described here or ex vivo / in vitro approaches described in
Practice F1903. Particles and other device-related debris/degradation products generated by alternative methods (for example, from
animal studies, clinical use, or in vitro studies) may also be used, if appropriately justified. The method of generation must be
described.
5. 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.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
F1904 − 23
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 immunopheno-
typing 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 assessed by histopathological
assessment of the extent of FBR including macrophage accumulation around the test material. Supplementary ex vivo / in vitro
assessment can be used for additional macrophage-based testing such as macrophage immunophenotyping (proinflammatory M1
and anti-inflammatory/wound healing M2) as well as debris uptake by phagocytes (phagocytozability) involving the entire range
of test material characteristics.
5.2.2 Due to the role of inflammation in extending device-related FBR and promoting the resultant tissue remodeling,
histopathological assessment should include identification of immune/inflammatory cell infiltration (with separate counts for the
individual cell types representing both innate and adaptive responses) as well as corresponding tissue changes (for example,
fibrosis, necrosis, ossification or osteolysis, angiogenesis). Identification of immune/inflammatory cells may involve different
approaches including IHC phenotyping as needed. Supplementary ex vivo / in vitro assessment should be considered for assessing
the balance in release of pro-inflammatory versus anti-inflammatory cytokines as well as generation of hyper-proliferative versus
hypo-proliferative tissue responses.
5.2.2.1 Since the signs of inflammation and post-inflammatory tissue changes may not be always apparent, special attention should
be given to the assessment of debris-related inflammogenic and tissue remodeling potentials using ex vivo specimens and
supplementary in vitro assessment when needed. Pro-inflammatory cell death (necrosis) should be distinguished from programmed
cell death (apoptosis usually associated with anti-inflammatory responses) by using cell viability and cytotoxicity testing involving
cellular staining and flow cytometry. Given the importance of phagocytes in proper clearance of dying cells, normal non-phlogistic
phagocytosis of cells undergoing apoptosis should be distinguished from “frustrated” phlogistic phagocytosis which may result in
further cell/tissue damage due to the release of damage-associated molecular patterns (DAMP). See X1.10 for more details.
5.2.3 Due to the role of the device-tissue interface in shaping biological responses, in vivo models as well as supplementary testing
should be aimed to simulate (as much as possible) device-specific use environments. In vivo animal models with intra-articular
applications of a test material may be beneficial for testing of orthopedic materials, while intracardiac/intravenous applications may
be more beneficial for testing of cardio/endovascular materials.
5.2.3.1 Since many implantable materials come in contact with blood during their clinical use, the need for hemocompatibility
testing should be considered, especially when developing new materials. Development of new materials for cardiovascular
applications may benefit from a more detailed hemocompatibility assessment, which could include microcirculation, cell adhesion,
and leukocyte-endothelial interactions.
5.2.4 The predictability of testing for a certain material, including its debris, may benefit from the choice of study endpoints and
testing approaches that incorporates clinical experience from known therapeutic applications and safety issues of similar materials.
5.2.4.1 In general, the study endpoints should be selected per their ability to measure immunomodulatory, pro/anti-inflammogenic,
and tissue remodeling effects. As the examples of more specific choices, testing for an orthopedic material should take into
consideration potential tissue changes such as periprosthetic osteolysis and pseudotumors, while testing for a cardiovascular
material should take into consideration potential hemolytic, thrombolytic/thrombogenic, and pro-angiogenic effects.
F1904 − 23
5.2.4.2 Some endpoints currently used in effectiveness assessments can be applied to the safety assessment of adverse tissue
remodeling (examples of osteogenesis-related study endpoints can be found in X1.12).
5.2.4.3 While not all possible clinical complications can be accurately replicated in animal testing models, the properly selected
study endpoints for in vivo and supplementary in vitro testing can enhance the overall predictability of biocompatibility testing
(more details on the choice of measurable study endpoints are provided in X1.5).
5.2.5 Rodents and other small animals (for example, rabbit, guinea pig) are traditionally used for in vivo biocompatibility testing
models. Use of larger animal models is usually limited due to ethical and other concerns and may be reserved for models in higher
need for imitating similarities with humans (weight, bone and joint structure, etc.).
5.3 Abbreviations Used:
5.3.1 ALVAL—Aseptic lymphocyte-dominated vasculitis-associated lesion.
5.3.2 CD—Cluster differentiation.
5.3.3 DAMP—Damage-associated molecular pattern.
5.3.4 EDS/EDAX—Energy dispersive X-ray spectroscopy.
5.3.5 ELISA—Enzyme-linked immunosorbent assay.
5.3.6 FBGC—Foreign body giant cell.
5.3.7 FBR—Foreign body response.
5.3.8 FTIR—Fourier-transform infrared (spectroscopy).
5.3.9 H&E—Hematoxylin and eosin.
5.3.10 HMGB1—High-mobility group box 1.
5.3.11 HSP—Heat shock protein.
5.3.12 ICAM1—Intercellular adhesion molecule-1.
5.3.13 ICP-MS—Inductively coupled plasma–mass spectrometry.
5.3.14 Ig—Immunoglobulin.
5.3.15 IL—Interleukin.
5.3.16 LAL—Limulus amebocyte lysate.
5.3.17 LPS—Lipopolysaccharide (endotoxin).
5.3.18 MMP—Matrix metalloproteinase.
5.3.19 NO—Nitric oxide.
5.3.20 NOS/iNOS—Nitric oxide synthase / Inducible nitic oxide synthase.
5.3.21 PCR—Polymerase chain reaction.
5.3.22 ROS—Reactive oxygen species.
F1904 − 23
5.3.23 SAA—Serum amyloid A.
5.3.24 SEM—Scanning electron microscopy.
5.3.25 α-SMA—Alpha-smooth muscle actin.
5.3.26 TBARS—Thiobarbituric acid reactive substances.
5.3.27 TGF-β—Transforming growth factor-beta.
5.3.28 TLR—Toll-like receptor.
5.3.29 TNF-α—Tumor necrosis factor-alpha.
5.3.30 TRAP—Tartrate-resistant acid phosphatase.
5.3.31 VEGF—Vascular endothelial growth factor.
6. Characterization Using in Vivo Systems
6.1 Test and Control Material—Characterize the nature and the range of the particles and other possible debris or degradation
products (for example, ions) used, including but not limited to the following.
6.1.1 Possible sources of test and control material:
6.1.1.1 The method used to produce the test and control material for subsequent in vivo biological evaluation shall generate the
range of particles (for example, particle size and distribution, shape, and amount or dose) and other debris (for example, generated
by instruments used to surgically implant the product) or degradation products (for example, corrosion, breakdown products)
reasonably expected from clinical use. The method of generation shall be described and justified. Particles or degradation products
can be generated in bench testing (for example, using joint simulator machines or mechanical fatigue/corrosion testing fixtures)
but may also be produced by other validated techniques, taking into account the proposed intended use. General recommendations
for isolation and characterization of wear particles from bench testing are included in Practice F561 and ISO 17853. Additionally,
standards are available that provide recommended loading and displacement parameters for wear testing for specific anatomical
locations to generate corresponding wear particles (for example, ISO 14242-1 and 14242-3 for total hip replacement devices; ISO
14243-1 and ISO 14243-3 for total knee replacement devices; ISO 22622 for total ankle replacement devices). Device-related
particles and other debris or degradation products may also be generated in vivo from a test material in clinical use or animal
studies which replicate the conditions of a relevant end-use application. Once particles are isolated and characterized using
techniques such as in Practice F561 and ISO 17853, they should be processed to remove or reduce (that is, to a biologically
insignificant level) any contaminants (for example, endotoxin, chemicals used in the isolation or characterization process) and then
sterilized as needed (see 6.1.2.5) to allow them to be used in subsequent biological testing.
6.1.1.2 Test articles can be prepared according to Practice F619.
6.1.1.3 Purchased or generated controls (including reference materials) should consist of particles with characteristics (for
example, chemical composition, charge, size, shape, and dose) that correspond to the test particles and thus could aid in achieving
their comprehensive evaluation. In addition to selection per physical/chemical characteristics with regard to the test samples,
controls should be selected, when possible, per expected biological effects of a test material; the choice and appropriateness of
selected control(s) should be explained. The selected controls should correspond to the range of test material related
debris/degradation products. Specifically, controls should include both phagocytosable and non-phagocytosable types (for
example, per size: <10 μm and ≥10 μm, respectively) that were found within the range of generated test particles, to enable
comparative analysis of the test particle effects in terms of their phagocytozability.
6.1.2 The following attributes and corresponding methods should be reported for test and control materials:
6.1.2.1 Chemistry (for example, bulk material chemical composition, additives, impurities, chemical structure such as crystallinity,
surface properties such as protein corona).
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6.1.2.2 Size (mean and other population characteristics).
6.1.2.3 Shape (per Practice F1877).
6.1.2.4 Surface charge (if applicable).
6.1.2.5 Method of sterilization. In the event or when it is anticipated that the method of sterilization will confound medical device
debris and/or degradation product generation and/or release during test, it shall be accounted and considered. If the presence of
bacterial lipopolysaccharide (LPS) was quantified on test or reference materials after sterilization, specify the sensitivity of the LPS
detection method.
6.1.2.6 Concentration of test material as weight, or number (of particles), or surface area/device or volumetric dose.
6.2 In Vivo Testing—Biological responses from the animals exposed to the medical device debris/degradation products under test
should be evaluated in comparison to those derived from controls. The following controls should be considered to assist with data
analysis of medical device debris/degradation products under test: negative and positive controls (for example, reference products)
as well as a sham procedure control with no exposure to any test or reference material.
6.2.1 In Vivo Models—One or more of these models with different routes of exposure to the wear debris/degradation products can
be used if appropriately qualified (see 6.4):
6.2.1.1 Air Pouch Model—This intradermal model has an established utility for simulating synovium and identifying
particle-related immune responses. This model may be adapted for testing of devices/materials that come into contact with tissues
other than synovium, but its relevance to other in vivo systems should be validated. The volume of air and the time allowed before
introduction of the particles into the created pouches, the time points for sample collection, and the observed effects should be
specified. This model is more appropriate for the assessment of acute immune responses; its appropriateness for the assessment
of prolonged immune and inflammatory responses needs to be validated for the length of time of implantation. The assessment of
immune responses in this model is based on evaluation of the exudates collected from the pouches. In addition to conventional
testing for cell death/viability, this model allows a detailed assessment of the induced leukocytosis, for example, percentages of
lymphocytes, monocytes, neutrophils, and macrophages, corroborated with the use of flow cytometry and cell-specific markers.
Evaluation of the debris/degradation product induced leukocyte infiltration may be enhanced by measuring additional immune
responses such as the induction of cytokines (for example, tumor necrosis factor alpha (TNFα)) or matrix metalloproteinases (for
example, MMP9). The enhanced air pouch model may constitute a surrogate test for histopathology analysis and (semi)quantitative
assessment of immune reactions and tissue inflammation. In addition to its use for particle debris, this model may be used to test
the effects of test materials that are not particles, including gaseous device/material related products. As an additional criterion of
its suitability as a standard model, the air pouch model can be executed with limited costs and basic personnel and infrastructure.
6.2.1.2 Cages—Cages made of porous materials such as stainless steel mesh or porous polytetrafluoroethylene (PTFE) can be
implanted subcutaneously or intraperitoneally with a test material inside the cage. The cage material and the implant location
chosen should be specified and explained. The fluid accumulating in the cage should be sampled at various time intervals which
should also be specified and explained. The cage and contained material removed at the termination of the experiment should be
evaluated for cell adhesion, cell types, and other study endpoints including soluble products (the time chosen for termination and
the choice of subsequent measurements should be explained per specifics of a test material). Fluid containing a large number of
red blood cells should be discarded as representing blood and not cage fluid. As further limitations of this model, encapsulation
of the cage may impact the fluid collection and corresponding cell and protein responses; more importantly, some of the observed
effects may be due to the cage itself and not a test or control material.
6.2.1.3 Bone Implant Chamber—This is a modification of the cage system which allows determination of the effect of particles
and the resulting biological response on bone remodeling and therefore is more appropriate for testing of devices/materials with
orthopedic applications.
6.2.1.4 Direct Implantation/Injection—Direct applications of a test material via implantation (or injection if relevant to device use)
are practically devoid of the delivery system related effects that may complicate interpretation of the test results from
cage/chamber-based models. Intraperitoneal, intravenous, intramuscular, and subcutaneous are the favored routes in injection
models; intra-articular injection is usually reserved for materials with orthopedic end applications. In general, the end use
application should govern the route of injection/implantation as well as the choice and scope of organs and tissues utilized for
testing.
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NOTE 1—Careful dose selection and monitoring is essential to ensure animal welfare and minimization of adverse outcomes.
6.2.1.5 At the termination of the study, all sites used in these in vivo studies should be carefully evaluated for infection, since the
presence of infection may have a major impact on the testing outcome by simulating and mimicking many test material-related
inflammatory responses (more details are provided in 7.2.1). In many cases, evaluation for possible infection signs could be limited
to macroscopic assessment of the test sites; in some questionable cases, additional assessments (for example, microscopic
evaluation, blood/tissue culture) may be used as needed.
6.2.2 Other Methods—The use of other biological systems, animal models, or methods of implantation may be appropriate
depending upon the intended use of the material.
6.2.3 Characterization of device-related debris/degradation products retrieved from in vivo studies can be performed using Practice
F1877. Sample collection and processing methods applicable to testing of the tissue and biofluid specimens retrieved from both
clinical and animal studies can be found in Practice F561. The specimens from test animals should be evaluated in comparison
with those from control animals. See also X1.5 in this standard for additional considerations regarding the characterization of
device-related debris/degradation products.
6.3 Control Animals—In the conduct of testing with any of the above described models, appropriate control animals who receive
any vehicles, carriers, or other treatments received by the experimental models, to control for the effects of factors other than the
presence of the particles, should be included as well.
6.4 Method Qualification:
6.4.1 For any of the methods described above, the following should be developed to support qualification for use with medical
device debris/degradation products:
6.4.1.1 Provide detailed protocols and discuss any optimizations needed as compared to published methods. Describe the
applicability of the method for the device-specific end use (for example, treatment period optimization, size and amount/dose of
particles, debris materials that may be incompatible with the test system).
6.4.1.2 Specify and justify with supporting data, including the criteria to be used to interpret test results as compared to controls.
7. Testing Approaches, Data Analysis, and Reporting—Biological Responses
7.1 For all test and control samples, the following should be considered for analysis and reporting:
7.2 Cellular/Tissue Response—Cell accumulation at the site of the particles should be evaluated for relative numbers and types
of cells. Standard paraffin or plastic embedded sections are usually sufficient to identify neutrophils, lymphocytes, macrophages,
foreign body giant cells (FBGC), osteoclasts, osteoblasts, osteocytes, eosinophils, etc. Traditional Hematoxylin and Eosin (H&E)
staining can be used in most biocompatibility-related histology and cytology applications. In some cases, special histological
procedures, or immunohistochemical stains such as those described in Practice F561, or flow cytometry may be needed, for
example, to confirm the identity of lymphocytes and macrophages as the main cell types involved in adaptive and innate immune
responses, respectively. Overall cellular response should be characterized as focal or diffuse; its extent can be further evaluated,
for example, using a scale of 0 to 5 with 0 being no cell response, 1 being accumulation of a few cells, 2 being a mild response
with some cell accumulation, 3 being a moderate response, 4 being a large response, and 5 being a severe response with extensive
immune/inflammatory cell accumulation. Further guidance on histopathological evaluation with (semi)quantitative scoring of
cellular responses can also be found in other standards such as ISO 10993-6).
7.2.1 It should also be noted whether the response includes signs of infection that may mask or mimic inflammatory responses
due to a test material. Specifically, the presence of neutrophils should be interpreted with caution. In early stages, neutrophil
accumulatio
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