ISO 10993-6:2026 Biological evaluation of medical devices — Part 6: Tests for local effects after implantation

Foreword

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This document was prepared by Technical Committee ISO/TC 194, Biological and clinical evaluation of medical devices, in collaboration with the European Committee for Standardization (CEN) Technical Committee CEN/TC 206, Biological and clinical evaluation of medical devices, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).

This fourth edition cancels and replaces the third edition (ISO 10993-6:2016), which has been technically revised.

The main changes are as follows:

A list of all parts in the ISO 10993 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A complete listing of these bodies can be found at www.iso.org/members.html.

Introduction

The objective of the implantation test methods is to characterize the local tissue response after implantation of a medical device or material (test sample) including integration, degradation, or absorption in an appropriate animal model.

The test sample is implanted into an anatomical site appropriate for the evaluation of the local effects of the medical device (or portion of) in an animal.

The medical device or material local effects are evaluated by a comparison of the tissue response caused by a test sample to that caused by comparative or reference control samples used in medical devices whose clinical acceptability and biocompatibility characteristics have been established.

Careful study design can include other relevant biological effects to reduce the number of animals used to evaluate safety and efficacy while accomplishing all study objectives. Additionally, a long-term systemic toxicity study that is designed to incorporate the methods, biological effects, additional timepoints and outcomes of implantation testing can satisfy the requirements of this document and ISO 10993-11.

1 Scope

This document specifies requirements for implantation test methods for preclinical assessment of the local effects after implantation of medical devices or materials intended for use in medical devices. This document is applicable to the evaluation of local tissue responses from medical devices that are intended to be used where skin or mucosal tissue is breached, when required.

This document is applicable to medical device or materials that require implantation evaluation and can be solid or non-solid (such as porous materials, liquids, gels, pastes, powders, and particulates), absorbable, degradable, non- absorbable, or can be tissue-engineered medical products (TEMPs).

These implantation tests are not intended to evaluate or determine the performance of the test sample in terms of mechanical loading or functional performance. This document also does not provide guidance on methods and study design to satisfy requirements for systemic toxicity, carcinogenicity, teratogenicity or mutagenicity. However, the study designs can be modified to also assess other biological effects.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

• ISO 10993-1 Biological evaluation of medical devices — Part 1: Requirements and general principles for the evaluation of biological safety within a risk management process
• ISO 10993-2 Biological evaluation of medical devices — Part 2: Animal welfare requirements
• ISO 10993-4 Biological evaluation of medical devices — Part 4: Selection of tests for interactions with blood
• ISO 10993-9 Biological evaluation of medical devices — Part 9: Framework for identification and quantification of potential degradation products
• ISO 10993-12:2021 Biological evaluation of medical devices — Part 12: Sample preparation and reference materials
• ISO 10993-16 Biological evaluation of medical devices — Part 16: Toxicokinetic study design for degradation products and leachables

3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 10993-1, ISO 10993-2, ISO 10993-4, ISO 10993-9, ISO 10993-12, ISO 10993-16 and the following apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

3.1 absorb

3.2 comparative control

3.3 coupon

3.4 degradation product

3.5 degrade

3.6 euthanasia

3.7 local effect

3.8 location marker

3.9 reference control

3.10 steady-state

4.1 General

The ISO 10993 series requires animal testing to be avoided unless the required data cannot be obtained by other means. Prior to choosing to pursue implantation studies, consideration to the type of data required, the appropriate method and approach to implantation studies should be given to ensure that the data collected covers the concerns for biological reactivity. It is important that the study be planned in sufficient detail such that all relevant information can be extracted from the use of each animal and each study plan or protocol according to ISO 10993-2, ISO 10993-11 and ISO 10993-16. The implantation tests shall be planned and documented in the study documentation, including the information on the selected test methods, test samples, test animals, implantation sites, test duration, measurement frequencies, assessment methods and evaluation. When relevant vertical standards exist, refer to those as guidance for biological specific evaluation (e.g. ISO 15798, ISO 11979-5, ISO 7405, ISO 14708 series, ISO 5840 series).

All animal studies shall be performed in a facility approved by a nationally recognized organization and in accordance with all appropriate regulations dealing with laboratory animal welfare to conform with the requirements of ISO 10993-2. These studies shall be performed under Good Laboratory Practices or other recognized, quality assurance systems. While pilot implant studies can or cannot be conducted under Good Laboratory Practices, the test facility should have an adequate quality system.

The provisions of this clause shall apply to the test methods specified in Annex A, Annex B, Annex C and Annex D.

4.2.1

Test and control samples shall be in a final finished state as described in ISO 10993-12 (unless used in a pilot study). The implant size and shape shall be documented and justified. For certain devices, clinically relevant dosing can be considered. A justification should be provided to demonstrate mimicking a clinically relevant dose in the implantation study.

Test samples for various implant sites are described in Annex A, Annex B, Annex C and Annex D. Physical properties (such as form, density, hardness, surface porosity and texture), geometrical characteristics (form, shape and size) and composition can influence the character of the tissue response to the test material and shall be recorded and taken into account when the response is characterized. Comparative control samples should be matched as closely as reasonably possible for physical properties and geometrical (form and shape) characteristics. Otherwise, a reference control (well characterized material with a well characterized response) can be used, such as high density polyethylene (HDPE).

Additional control samples may be included in the study to help interpret tissue reactions [e.g. in case of combination drugs, an additional control such as a placebo device (without drug) can be considered].

4.2.2

Each implant shall be manufactured, processed, cleaned of contaminants and sterilized by the method intended for the final product and this shall be confirmed in the study documentation. After final preparation and sterilization, the implant samples shall be handled aseptically and in such a way as to ensure that they are not damaged or contaminated in any way prior to or during implantation.

The use of smaller test samples or test coupons that are consistent with the requirements as described in ISO 10993-12 can be relevant if the medical device to be tested cannot be tested as is, due to size or complex geometry. The test samples or test coupons shall contain all tissue-contacting materials and surface finishes as in the final, finished medical device. For medical devices comprising two or more different materials, implanted test samples should be of similar composition, surface finish and include each individual material to the final product. With multiple materials, it can be necessary to implant several coupons together, each containing a subset of device materials. The potential for synergies and interactions of different materials in the final product should be considered in the choice of test sample.

NOTE

Refer to the respective annexes for guidance on the number of test and control samples for each material and implantation period.

4.2.3

For materials used as scaffolds for tissue-engineered medical products, it can be appropriate not to use the final preparation pre-populated with cells or proteins, as the immune reaction of the animal to human cellular or protein components of such products and the reaction of the cells to the animal can interfere with the resulting local tissue response, making it difficult to interpret.

NOTE 1

Implantation in an appropriate immune-deficient animal can be an option to avoid the xenograft responses, if justified.

NOTE 2

Products with living cells are not considered medical devices in all jurisdictions.

4.2.4

For multicomponent material device designed to be cured prior to placement, the components shall be mixed before use and allowed to set before implantation. However, materials that are designed to polymerize in situ (e.g. bone cements, many dental materials, tissue sealants and glues) shall be introduced in a manner such that in situ polymerization occurs. The procedure used shall be justified and documented.

4.3 Selection of control samples

The process of implanting any material in tissue induces some degree of cellular response. In the evaluation of material local effects after implantation, the choice of control samples is important in determining the acceptability of the observed tissue reaction to the test sample. Evaluation should be performed by comparing the tissue reaction to that of a similar material (comparative control) whose clinical acceptability and biocompatibility characteristics have been established. The geometrical and physical characteristics such as shape, size, curvature and especially the surface condition (including porosity and texture) of the control article(s), should be as similar to that of the implant test samples as is practical. Any deviations shall be explained, justified and recorded. If no appropriate comparative control is available, a reference control can be used. For further guidance, see ISO 10993-12. For example, reference materials as specified in ISO 10993-12:2021 can provide a good control for solid, smooth, non-absorbable materials. Conversely, the reaction can likely be different if it is used to compare against a non-absorbable or degradable hernia mesh due to the differing physical form characteristics. Ideally, a commercially available material should be used as a comparative control so the tissue reaction is similar to what has been clinically proven. For non-absorbable medical devices or materials, a comparative control shall be used made of a stable non-absorbable comparative material (see Annex A, Annex B, Annex C and Annex D).

Since absorbable materials encounter changing tissue responses as degradation proceeds at rates that differ based on composition, processing and sterilization techniques, similar absorbable comparative medical device or material controls should be considered.

The absorption rate of the control material or medical device should be similar to the test material or medical device (for more information, see ISO/TS 37137-1). Alternatively, a non-absorbable comparative control can be used, if justified (e.g. there are no clinically relevant absorbable controls). If a comparative control is used, typically reference materials as specified in ISO 10993-12:2021 are also implanted into the animal for comparison and serving as a procedural control yielding a total of three separate articles included in the study. For novel materials or medical devices, if a commercially available comparative material or medical device does not exist, the choice of a control that is as close as possible is preferred. The nature of any adverse inflammatory response or high reactivity rating or similar histopathological evaluation conclusion should be discussed in the context of the control article chosen. The choice of control shall be documented and justified. If no control or a sham control is used, this also shall be documented and justified.

5.1.1

The test sample shall be implanted into or onto the tissues most relevant to the intended clinical use of the material. The justification for the choice of sample numbers, tissue, implantation sites and test period shall be documented. Test methods for various implantation sites are given in Annex A, Annex B, Annex C and Annex D. If other implantation sites are chosen, the general scientific principles behind the test methods described in Annex A, Annex B, Annex C and Annex D shall still be adhered to and a justification be provided for selecting an anatomical location that does not align with the use of the device.

NOTE

For some devices, there are vertical standards prescribing specific implant studies to evaluate local tissue responses, e.g. for intraocular lens implant (see ISO 11979-5) and dental devices (see ISO 7405). The studies described in the standards can be used to satisfy the requirements in ISO 10993-6.

5.1.2

For absorbable materials, the implantation site shall be marked in a manner suitable for identification of the site at the end of the designated time periods, taking into consideration the growth and aging of the implanted animal(s). The use of a non-invasive permanent skin marker or a template marking the placement of the sample is recommended as a single application for short-term study intervals only. This method of marking can come off as skin exfoliates and requires remarking as often as needed to maintain the marked site. Additionally, in many laboratory animals, the loose highly mobile skin limits the precision of marking sites that are deeper than skin. In most circumstances, a location marker comprising an appropriate non-absorbable inert or biocompatible material (e.g. HDPE 1 mm by 2 mm by 5 mm, polypropylene suture, gold band, clips) may be used to identify the location of the implant site. These location markers should be far enough away from each implant site to not induce changes in the local tissue reaction to the implanted material. If this is not possible, i.e. the marker of the implanted material is adjacent to or on the edge of the implanted material, then the tissue reaction to this marker should not be included in the evaluation of the tissue reaction to the implanted material.

5.1.3

A sham surgical procedure can be used to evaluate the impact of the procedure on the tissue involved; in these cases, the specific justification shall be provided. If a sham surgical procedure is performed, the same implantation procedure that is used for the test or control should be used for the sham procedure.

5.2.1

All aspects of animal care and accommodation shall be performed in accordance with ISO 10993-2.

5.2.2

Animals are used to evaluate local effects following implantation and are described as animal models in the context of this document. In general, small laboratory animals such as mice, rats, guinea-pigs or rabbits are preferred. The use of larger animals such as dogs, sheep, goats or pigs may be justified based upon special scientific considerations of the test sample under study, length of study in relation to animal life expectancy, or if needed to accommodate implant size, with whole device testing or applicable sham defect size.

5.2.3

Select an animal species in line with the principles set out in ISO 10993-2, giving due consideration to the size of the implant test samples, the number of implants per animal, the intended duration of the test in relation to the expected lifespan of the animals, as well as potential species' differences regarding biological responses. The number of animals and implant sites within should be the minimum to account for site to site and animal to animal variability.

5.2.4

For short-term testing, animals such as rodents or rabbits are commonly used. For long-term testing, animals such as rodents, rabbits, dogs, sheep, goats, pigs and other animals with a relatively long life expectancy are suitable.

5.2.5

Before starting an animal study with degradable materials, relevant information from in vitro degradation studies should be considered for estimating relevant retrieval timing. For absorbable materials, a pilot study in rodents can be considered to determine the expected rate of degradation and the implantation duration needed to reach the steady-state before embarking on studies in larger animals.

NOTE

Guidance regarding in vitro degradation characterization can be found in ISO 13781 and ASTM F1635^[23^] for hydrolysable polymeric constructs and in ASTM F3268^[25^] for absorbable metallic constructs. General guidance regarding the linkage of in vitro observations with the determination of relevant in vivo retrieval intervals can be found in ISO/TS 17137. Additional guidance on selection of implantation time points for absorbable materials can be found in ISO/TS 37137-1.

5.2.6

The samples of test and control materials shall be implanted under the same conditions in animals of the same species and of the same age, sex and strain in corresponding anatomical sites. The number and size of implants inserted into an animal depends on the size of the species and the anatomical location. Whenever possible, the control and the test samples should be implanted into the same animal unless systemic toxicity effects are also evaluated in the study. In cases where both implantation and systemic toxicity effects are assessed, control and test samples shall be implanted into separate animals. Guidance on evaluation for systemic toxicity can be found in ISO 10993-11.

5.2.7

When a neuroimplantation study (see Annex D) is conducted, control and test samples shall not be placed in the same animal. Histological assessments should follow best practices for processing and analysis of central nervous system tissues (see Annex D and Reference [70]) or peripheral nervous tissues (see Annex E and Reference [70]).

5.3.1

The test period shall be determined by the likely clinical exposure time or be continued until or beyond when a steady-state with respect to the biological response has been reached. The time points selected, along with the explanation and justification for their selection, shall be recorded.

5.3.2

The local biological response to implanted materials depends both on the properties of the materials and on the response to the associated trauma of surgery. The tissue configuration in the vicinity of an implant changes with the time elapsed after surgery. During the first two weeks after implantation, the reaction due to the surgical procedure itself can be difficult to distinguish from the tissue reaction evoked by the implant. The time to reach a steady-state can be tissue- and device design-dependent. A justification for the time point selected for assessment in a specific tissue (i.e. muscle, bone) shall be documented. In muscle and connective tissue, depending on the species, the design of the device and the severity of the surgical trauma, a steady-state can be seen in the tissue-implant interface (including the microscopic maturation of immature fibrous tissue and associated remodelling) that can take 9 weeks to 12 weeks but can occur earlier. Implantation in bone tissue can need longer observation periods before a steady-state is reached.

5.3.3

For non-absorbable materials, the short-term responses are normally assessed from 1 week to 4 weeks and the long-term responses in tests exceeding 12 weeks. Additional intervals can be needed to characterize the response relative to the clinical use of the device.

5.3.4

For absorbable materials, the test period shall be related to the estimated degradation time of the test product at a clinically relevant implantation site. When determining the time points for sample evaluation, an estimation of the degradation time shall be made. This can be accomplished in vitro by real-time or accelerated degradation studies or in certain circumstances by mathematical modelling. In general, study duration should extend up to or beyond the point of complete absorption. The evaluation period for absorbable materials will depend in part on the degradation rate of the materials. Study intervals should span a significant portion of the degradation time frame for the implant, and shall include, as a minimum, the following time points:

1. a) Early time frame (where there is no or minimal degradation): For absorbable materials, usually a study interval of between 1 week to 4 weeks post-implantation should be used to assess the early tissue response.If a device completely absorbs within four weeks of implantation, a short-term implant study may be considered as an evaluation point; additional durations may be omitted from the implantation studies.
2. b) Mid time frame (when degradation is taking place): Subsequent study intervals for absorbable devices should be guided by the degradation profile of the specific absorbable material. The target interval should allow assessment of histological response when the tissue response is expected to be most pronounced (e.g. substantial structural disruption or fragmentation of the device is most likely to occur). Implants with longer-term degradation profiles can require multiple assessment time points, with intervals targeted in accordance with the expected pattern of degradation.When a device with multiple materials with differing absorption rates is implanted, implant intervals reflecting the degradation profile of those components should be included.
3. c) Late time frame (when the implant is essentially absorbed): This interval is targeted to observe when minimal amounts of the absorbable component remain at the implant site with a steady-state of the tissue reaction.Gross and microscopic evaluation after complete implant absorption is highly desirable. However, in the absence of complete absorption, the overall data collected should be sufficient to allow characterization of the local effects after implantation if:

   — the affected tissue’s response, structure and function have achieved an acceptable steady-state condition, and

   — the absorbable material or its degradation products are minimally visible during light microscopic evaluation.

NOTE

In vivo degradation can occur over a long period of time, sometimes more than one year. Additional animals to extend the observation period (intervals “to-be-determined” group) can be beneficial if the implant has not been completely absorbed within the expected investigational time period.

In those situations when the material is not fully absorbed within the late time frame, an appropriate scientific justification can be included for ending the study and, if feasible, the pathologist may approximate the percentage (%) of degradation in the report’s narrative for the final period during late degradation.

Long-term studies that span a significant portion of the degradation time frame for the implant are recommended. Implantation of in vitro pre-degraded material (for instance, up to 50 % loss of mass or 50 % loss of mechanical strength) can be considered on a case-by-case basis in order to more rapidly observe late stage events after implantation. However, these studies do not replace studies that characterize the real-time in vivo degradation profile of the absorbable device.

5.3.5

Characterization of an absorbable device’s degradation process is not necessarily applicable to the evaluation of the local effects of the same absorbable material when used in combination with, for example a drug as a carrier for drug release, as a scaffold for tissue-engineered medical products, or as a surface coating for non-absorbable implants. Since combinations of devices with drugs or cells can introduce new issues, the appropriate regulatory authorities should be consulted regarding study designs for absorbable combination products.

5.3.6

Although this document does not address the issues of systemic toxicity given in ISO 10993-11, it is recommended that the information required to meet this document can be obtained from an appropriately designed systemic toxicity studies using implantation.

5.3.7

For long-term studies, examples of generally accepted observation periods for non-absorbable materials are given in Table 1. Animals should be euthanized at each time point, in line with ISO 10993-2. Serial harvest under general anaesthesia with recovery can be acceptable under special circumstances, which shall be documented and justified.

5.4.1

Surgery shall be performed under general anaesthesia. If another type of anaesthesia is used, this shall be justified and shall be in accordance with ISO 10993-2. The specific insertion or implantation procedures for subcutaneous, intramuscular, bone, or neural implantation are described in Annex A, Annex B, Annex C and Annex D, respectively.

5.4.2

The number of implants per animal and the number of animals per observation period are described in Annex A, Annex B, Annex C and Annex D. If other implantation sites are chosen, the general scientific principles behind the test methods described in Annex A, Annex B, Annex C and Annex D shall still be adhered to and a justification be provided for establishing the number of implants per animal and the number of animals per observation period. A sufficient number of test and control samples shall be implanted to ensure that the final number of samples to be evaluated will give valid results.

5.4.3

The surgical technique can profoundly influence the result of any implantation procedure. Surgery shall be carried out under aseptic conditions and in a manner that minimizes trauma at the implant site. Remove the hair from the surgical area by clipping, shaving, other mechanical means or depilatories. Disinfect the exposed area of skin with an appropriate antiseptic. Ensure that the implants or wound surfaces do not come in contact with the hair. After surgery, close the wound using either sutures or wound clips, taking precautions to maintain aseptic conditions. Use of antibiotics should be justified.

5.4.4

The health of the animals shall be monitored, observed and recorded at regular intervals daily during the study. Following surgery, each animal shall be observed at a minimum daily during the test period and observed for any development of clinical signs that can necessitate veterinary intervention or early euthanasia. Any abnormal findings shall be recorded and discussed for their potential influence on the results obtained and described in the test reports.

5.4.5

Body mass measurements shall be taken and recorded. The frequency of the measurements should be planned at study-relevant intervals. The use of post-operative analgesics shall be in accordance with the requirements of ISO 10993-2.

5.4.6

At the end of the experimental period, euthanize the animals in accordance with current acceptable methods of euthanasia (see Reference [60]) and in accordance with the principles set out in ISO 10993-2.

5.5.1 General

Evaluate the biological response by documenting the clinical pathology data (if available), macroscopic and histopathological responses as a function of time. Compare the responses to the test sample to the responses obtained at the control sample or sham operated sites. Carry out comparison of the test and control implants at equivalent locations. Differences in the types of tissue responses should be clearly stated because similar total numerical scores do not inherently mean that the tissue responses are similar between implant types.

NOTE Examples of scoring systems that are given in Annex F and in the Bibliography can be informative and can be used by the pathologist to select appropriate tissue features and criteria to be scored by the assessment. However, the example schemes portrayed in Annex F alone can be insufficient to characterize pathological findings, especially those that indicate more thorough examination of the tissue response is warranted, and the schemes should not be considered as the only appropriate method for evaluation of tissue responses.

For each of the implant intervals, a sufficient number of test and control samples shall be evaluated as defined in Annex A, Annex B, Annex C and Annex D. If other implantation sites are chosen, the general scientific principles behind the test methods described in Annex A, Annex B, Annex C and Annex D shall still be adhered to and a justification be provided for establishing the number of implants to be evaluated. The test and control samples shall be evaluated using at least three different animals, if a sufficient number can be implanted in each animal. In rare instances, using only two animals can be permissible (i.e. for a wound healing study with multiple implant sites on each animal). In all cases, the number of samples per animal shall be documented and justified.

If some implant sites are not available for evaluation or in the case of animal loss, in exceptional circumstances, the evaluating pathologist may determine that the number of sites is sufficient for an accurate overall evaluation, provided the available sites are uniform in their responses.

5.5.2 Macroscopic assessment

Each implant site shall be examined for alterations to the normal structure. The local tissue surrounding the implant should be examined and collected. If the test or control sample is encapsulated, the entire capsule shall be collected, unless otherwise justified. If not, at least 5 mm of tissue beyond any gross lesions at the sample application or implantation site shall be collected. The implant and surrounding tissue can be required to remain intact for histological assessments. Additionally, the regional draining lymph nodes^[40^][46^][76^][77^][78^] should be collected. Describe the gross observations (e.g. using descriptive, non-diagnostic terminology). Record the presence, form and location of the implant, including possible remnants of degradable materials. The use of colour macroscopic photography can be useful at the implant sites and where other abnormal gross findings occur. In addition to the inspection of the implant sites, whenever an animal has shown signs of ill health or reactions to the implant, a general necropsy should be conducted^[63^].

5.5.3 Implant retrieval and tissue sample collection

After the animal has been euthanized, excise each implant site together with sufficient unaffected surrounding tissue (minimum of 5 mm of tissue on all sides of the explanted test article) to enable evaluation of the local histopathological response. Care shall be taken so as not to induce collection artefacts of the implant-tissue interface on fresh unfixed tissues. If the candidate material is not evident at the site examined (absorbable materials), extend the explantation site to include several millimetres of normal tissue on all sides of the expected implant site. If an implant and its area of interest can be readily removed from its cavity or pocket during necropsy, then the fresh-tissue pocket findings (exudate, colour, other) shall be recorded. As removal of implants can affect orientation for trimming for complex devices and affects capsular thickness as the fibrosis contracts, further documentation can be needed during necropsy. Chemical fixation of the implant site containing the test or control material may be done at this stage. Chemical fixation in 10 % neutral buffered formalin (NBF) solution is suitable for most materials and stains. Fixation for 24 h to 72 h, or longer, is reasonable depending on tissue sample size. Once chemically fixed, hard materials, like metals or dense plastics can be carefully removed from the peri-implant capsule if there will not be substantial loss of interface information (i.e. material or tissue along the implant-tissue interface). The capsule marks the implant cavity. Some soft materials may be able to be trimmed and left in situ for processing and sectioning under paraffin microtomy. This is preferable to the removal of the implant if the materials are porous and there is tissue ingrowth over time. Consult a pathologist to determine if the tissue should not be separated from the test article because there is a concern that loss of interface information can impede histopathology interpretation; method development can be necessary to determine if a device can be removed or must be left in place for complete histological interpretation.

For degradable materials, regional draining lymphoid tissue should be collected, and histopathology of the draining lymphoid tissue can be performed to demonstrate potential migration of degradable materials or regional effects on immune responses. Consider collection of additional nondraining lymphoid tissues to use as control tissues.^[63^] Since evaluation of regional draining lymphoid tissue can be useful to demonstrate regional effects of non-degradable materials, regional lymphoid tissue can be retained for potential microscopic evaluation to detect migrating particulate matter or assess immune responses to novel test material.

NOTE 1

It is recognized that it is not always possible to locate the draining lymph nodes for all implant sites.

If indicated by ill health, clinical observation, or necropsy findings, or by experimental design to assess systemic toxicity, other organs shall be collected as appropriate.

Process the excised tissue samples according to appropriate procedures needed for histological evaluation, including fixation, trimming, embedding, microtomy and staining. If appropriate, record the location of the implant (to verify its identity), number of pieces that were embedded per paraffin block, their cutting geometry and the section thickness during microtomy.

When conventional techniques (paraffin embedding) are used, the tissue implant site can be opened before or after exposure to a fixative and the condition of the implant surface and tissue bed shall be reported. When the implant-tissue interface is to be studied for hard materials like metals or dense plastics, embedding of the intact tissue implant site with the implant in situ using hard plastics instead of paraffin is preferred; appropriate sectioning or grinding techniques are used for the preparation of histological sections.

When the tissues or implants cannot be sectioned in paraffin, other embedding or sectioning techniques (e.g. plastic embedding) can be necessary for tissue-implant interface evaluation. If embedding techniques alter the tissue-implant interface, any observations at the interface shall be documented.

NOTE 2

For “soft” implants (implants that can be cut with a razor blade) in soft tissues, processing of the tissue samples can be performed without removing the implant.

5.5.4 Microscopic assessment

There are many factors to consider when evaluating the microscopic response to an implanted material including the consistency of tissue sampling, sectioning, staining and imaging methods. Annex G provides information on aspects that can be considered when evaluating an implant site.

The scoring system used for the histological evaluation shall take into account the extent of the area affected using standard semiquantitative ordinal scoring or other evaluation criteria as deemed appropriate by the study pathologist. If semiquantitative scoring criteria are used, some examples can be found in Annex F. The location of the implant, number of implant sites and cutting geometry should be recorded. Due to the qualitative and context-based nature of microscopic data generation during a pathology evaluation, semiquantitative (ordinal) scoring criteria based on the implantation site and relevant cells or cell-specific parameters can be developed by the study pathologist. For medical devices or materials that are complex in nature (e.g. absorbables, nanomaterials), additional microscopic observations should be considered in the assessment.

The scoring criteria shall be reported and scientifically justified.

The biological response parameters, which shall be assessed and recorded, can include, but are not limited to the following:

1. a) any microscopically notable findings identified by the study pathologist;
2. b) a semiquantitative or quantitative assessment of the extent of capsule formation can be needed for implants with a fibrous capsule;
3. c) a semiquantitative approximation of the amount of inflammation present in various regions near the implant, with inflammatory cell types delineated as appropriate (e.g. polymorphonuclear cells, lymphocytes, plasma cells, eosinophils, macrophages and multinucleated giant cells);
4. d) the degree and distribution of necrosis;
5. e) other tissue alterations, such as vascularization, fatty infiltration, granuloma formation, mineralization, bone remodelling, new bone formation, presence of foreign material or other findings as deemed relevant by the study pathologist;
6. f) where possible, the locations and degradation characteristics of materials such as fragmentation, changes in material profiles, granularity, colour and size, and foreign particles;
7. g) the extent of tissue ingrowth into
   • 1) the open areas of the material for porous and absorbable implant materials, and
   • 2) the structure of the material for absorbable materials;
8. h) evaluation of the formation of the expected normal tissue at the site when the ultimate goal of an implant is to result in tissue remodelling or regeneration.

Histological responses, including any additional adverse or other noteworthy findings, shall be documented. The study pathologist shall have knowledge of adverse findings during the in-life phase of the study for each animal subject (see References [61] and [63]). Photomicrographs can be useful for documentation.

For degradable or absorbable materials, at the intermediate or nearly complete degradation levels, some residual material of the degradable implant can be present in the tissue samples examined. In addition, for evaluation of the restoration to normal structure, representative areas of the implant site can be evaluated, as indicated by marker or template.

For implants in bone, the interface between the tissue and the material is of special interest. Evaluate the area of bone contact and the amount of bone in the vicinity of the implant, as well as the presence of intervening non-calcified tissues. The presence of bone resorption or new bone formation shall be recorded.

Staining of tissue slides with Hematoxylin and Eosin is a gold standard, but consideration should be given to additional stains and analysis when further clarification is needed.

5.5.5 Evaluation of responses

Evaluate the local biological effects after implantation using the scoring system(s) as planned in the study protocol.

Examples of semiquantitative scoring systems are given in Annex F and in References [24], [26] and [28].

Examples of studies comprising quantitative variables and ordinal quantitative scoring variables are given in References [33] and [34]. Quantitative scoring systems (e.g. histomorphometry) are feasible only when paired with prior or parallel qualitative or semiquantitative scoring.

In addition, examples of other scoring systems are included in the Bibliography.

6.1 General

The test report shall have sufficient detail to allow an independent assessment of the results. When there is more than one device material, the pathologist should evaluate and report on each material individually. If evaluation of the response to individual materials in the device is not possible, the pathologist should evaluate and report on the response to the combined materials in the device test article. The International Standard used (including its year of publication) shall be reported with reference to the specific method used.

6.2 Test laboratory

The following information on the test laboratory shall be included in the test report:

1. a) name of the testing laboratory and the certifications of the laboratory;
2. b) date (of the test and final report), name and signature of the person(s) responsible for the report.

6.3 Implant samples

The following information on test and control implant samples shall be reported:

1. a) description of test and control materials, such as identification, surface condition, shape, size and mass (can be reported as weight, e.g. gram, kilogram) of the implants, as appropriate;
2. b) the rationale for choice of control sample and the geometrical characteristics of the material implanted shall be given.

6.4 Animals and implantation

The following information on animals and implantation method shall be reported:

1. a) species, strain, sex, age, mass and origin;
2. b) test conditions, including housing and diet;
3. c) all animal welfare observations during the study;
4. d) insertion techniques, including the surgical procedure, anaesthesia and post-surgical analgesia;
5. e) problems associated with implantation or explantation and all observations made during the study;
6. f) justification for the choice of method, implantation site and test periods.

6.5 Retrieval and histological procedure

The following information on retrieval and histological procedure shall be reported:

1. a) description of the retrieval technique, as well as the number of implants retrieved per animal and per observation period;
2. b) implant evaluation, including gross observations of implants, tissues and organs, and a description of the techniques employed for the fixation and preparation of the histological sections;
3. c) methods and results of histological evaluation of implant site and any organs showing alterations at necropsy, when indicated; a gross to microscopic correlation, or lack thereof, shall be recorded;
4. d) for absorbable materials, but not be limited to, a description of the degree of degradation, including material characteristics at explant (free particles, fibre formation, amorphous gel, crystallinity); Sample collection for potentially relevant additional assays, such as molar mass changes and mass loss, should be considered if the implant can be removed without damaging the implant-tissue interface.

6.6 Macroscopic and microscopic evaluation

The following information on macroscopic and microscopic evaluation shall be reported:

1. a) macroscopic observations that shall include the observations made on each implant as well as the macroscopic appearance of the tissue surrounding the implant; additionally, observation of the regional draining lymph nodes should be performed for absorbable materials and other implants when applicable;
2. b) results obtained from each microscopic examination of each site;
3. c) representative high-resolution photomicrographs of the tissue surrounding the implant site and images of any adverse findings as required.

6.7 Final evaluation

The report shall include a comparative evaluation of the local effects after implantation in terms of the biological responses to test and control materials, in context of any deviations noted from the procedure.

A.1 General

This test method is used for assessing the biological response of subcutaneous tissue to an implanted material.

Subcutaneous implantation study should be considered for devices implanted into subcutaneous tissue, or that have long-term contact to mucosal membranes. The study can be used to compare the effect of different surface textures or conditions of the same material, or to assess the effect of various treatments or modifications of a material.

A.2 Principle

The implant is inserted into the subcutaneous tissue of the test animal. The biological response to implants of test samples is compared with the biological response to implants of control samples. The control materials are those used in medical devices whose clinical acceptability and biocompatibility characteristics have been established.

A.3 Samples for testing

Common provisions for the preparation of test and control samples are described in 4.2. Implant sizes are based on the size of the test animal. The following shall be considered minimum dimensions.

1. a) When using discs, test samples of 10 mm to 12 mm in diameter and from 0,3 mm to 1,0 mm in thickness.

   NOTE

   The subcutaneous site, deep to the panniculus carnosa muscle, is particularly suitable for the evaluation of polymeric sheet material. Depending on stiffness, sheet material can become folded, and this can impact the interpretation of the tissue response relative to a flat sheet.

2. b) When using rods and cylinders, test samples shall be 1,5 mm to 2 mm in diameter, 5 mm to 10 mm in length and have rounded ends.
3. c) Non-solid samples (including powders, gels, liquids, creams and particulates) shall be implanted directly into the tissues. However, a location marker is recommended for absorbable materials (see 5.1.2). Volumes of 0,1 ml or masses of 0,1 g can be appropriate. Permanent location markers of some type can be placed near implant sites (e.g. when implanting absorbable materials, see 5.1.2).
4. d) Other dimensions that are anatomically compatible may be utilized, when conducting implantation tests as an example, with clinically relevant samples.

A.4 Test animals and implant sites

The implants shall be inserted in the dorsal subcutaneous tissue of an adult animal model of the most relevant species and strain for the device, study objectives and clinical indication, and a species in accordance with the provisions of ISO 10993-2.

Use at least three animals for each material and sufficient implant sites to yield a total of 10 test samples and 10 control samples for each material and implantation period. When implant sites are ≥1 cm in length, multiple tissue samples are taken from a single implant site and sections for histology shall be at least 1 cm apart.

A non-absorbable control sample should be evaluated at each time point.

If dealing with absorbable materials, comparison with a device or material at equivalent degradation time points can help with the interpretation of results.

A.5.1 General

Select one of the procedures described in A.5.2 and A.5.3.

NOTE

Subcutaneously implanted material can migrate after implantation.

A.5.2 Implantation alongside dorsal midline

Make an incision in the skin and make one or more subcutaneous pockets by blunt dissection. The base of the pocket shall be more than 10 mm from the line of incision. Place one implant in each pocket. The implants shall not be able to touch one another. Alternatively, both flanks may be used. The implants may be delivered by a trocar to the desired site or, when indicated, multiple small incisions can be made.

A.5.3 Implantation in the neck

In mice, make a 10 mm long incision above the sacrum and prepare a subcutaneous tunnel by blunt dissection towards the neck. Push one implant through the tunnel to position it at the neck^[31^][32^].

In rats, insert one implant of each of the control and candidate materials separately on each side of the neck. The implants shall not be able to touch one another. Alternatively, both flanks or hind legs can be used.

At some distance from the implant, close the tunnel with stitches of appropriate suture material to prevent the implant from moving.

A.6 Implantation period

To ensure a steady-state of biological tissue response, the implantation period(s) shall be selected as specified in 5.3.

A.7 Evaluation of biological response

The evaluation shall take into account the requirements specified in 5.5.

A.8 Test report

The presentation of the test results and final test report shall include the requirements specified in Clause 6.

B.1 General

This test method is used for assessing the biological response of muscle tissue to an implanted material.

B.2 Principle

The implant is inserted into the muscles of test animals. The biological response to implants of test samples is compared with the biological response to implants of control samples. The control materials are those used in medical devices whose clinical acceptability and biocompatibility characteristics have been established.

B.3 Samples for testing

Common provisions for preparation of test and control samples are described in 4.2. Implant sizes are based on the size of the muscle group chosen.

For rabbit paravertebral muscles, implants (e.g. rods) of a width of 1 mm to 3 mm with a length of approximately 10 mm are typically used. Alternatively, larger samples (e.g. disc) up to 10 mm in diameter and 3 mm in thickness can be surgically implanted.

Other dimensions that are anatomically compatible can be utilized, when conducting implantation tests as an example, with clinically relevant samples.

The samples shall have rounded edges.

B.4 Test animals and implant sites

Ensure that the muscles are of sufficient size to accommodate the implant samples. Use only one species per test. Insert the implants in the muscle of the animals under anaesthesia.

The paravertebral muscles of rabbits are suitable implant sites. Alternatively, for smaller samples, the gluteal muscles of rats or the quadriceps femoris or gluteal muscles of rabbits may be used.

Use at least three animals for each material and sufficient implant sites to yield a total of 10 test samples and 10 control samples for each material and each implantation period.

A non-absorbable control sample should be evaluated at each time point.

If dealing with absorbable materials, comparison with a device or material at equivalent degradation time points can help with the interpretation of results.

B.5 Implantation procedure

Implantation shall be by hypodermic needle or trocar. For larger implants, other appropriate surgical implantation techniques can be used. If the trocar method is used, it is recommended to verify complete ejection and appropriate placement of the test and control articles or to add additional implant sites to account for potential missed, incomplete or misplaced ejection.

Implant test samples into the body of the muscle with the long axis parallel to the muscle fibres.

For rabbit paravertebral muscles, implant sufficient samples of the test materials along one side of the spine, 25 mm to 50 mm from the midline and parallel to the spinal column, and about 25 mm apart from each other. Smaller spaces between implant sites can be considered if the integrity of the sites is not compromised at histological evaluation. In similar fashion, implant sufficient samples of the control material in the contralateral muscle of each animal.

B.6 Implantation period

To ensure a steady-state of biological tissue response, the implantation period(s) shall be selected as specified in 5.3.

B.7 Evaluation of biological response

The evaluation shall take into account the requirements specified in 5.5.

B.8 Test report

The presentation of the test results and final test report shall include the requirements specified in Clause 6.

C.1 General

This test method is used for assessing the biological response of bone tissue to an implanted material. The implantation site in cancellous (trabecular, “spongy”) or dense compact (lamellar) bone should be selected.

The study can be used to compare the effect of different surface textures or conditions of the same material, or to assess the effect of various treatments or modifications of a material.

C.2 Principle

The implant is inserted into the bone tissue of test animals. The biological response to implants of test samples is compared with the biological response to implants of control samples. The control materials are those used in medical devices whose clinical acceptability and biocompatibility characteristics have been established.

C.3.1 General

Common provisions for preparation of test and control samples are described in 4.2.

C.3.2 Shape of implant samples

Solid samples can be screw-shaped or threaded to provide initial stability of the implants in the bone. If preparation of a screw shape is impractical, a cylinder shape may be used.

Other sample forms (e.g. rods, pastes, granules) may be used depending on the nature of the materials and study objective.

C.3.3.1

Implant sizes are based on the size of the test animal and bone chosen. The following typical dimensions shall be considered for implants in some example cancellous and cortical bone locations.

C.3.3.2

The following typical dimensions shall be considered for implants in lateral condyle cancellous bone:

1. a) rabbits: cylindrical implants 2 mm to 6 mm in diameter and 6 mm to 10 mm in length;
2. b) dogs, sheep and goats: cylindrical implants of 4 mm to 10 mm in diameter and 12 mm in length;
3. c) rabbits, dogs, sheep, goats and pigs: 2 mm to 4,5 mm orthopaedic bone screw-type implants.

C.3.3.3

The following typical dimensions shall be considered for implants in mid-shaft cortical bone:

1. a) rabbits: cylindrical implants 2 mm in diameter and 6 mm in length;
2. b) dogs, sheep and goats: cylindrical implants of 4 mm in diameter and 12 mm in length;
3. c) rabbits, dogs, sheep, goats and pigs: 2 mm to 4,5 mm orthopaedic bone screw-type implants.

C.3.3.4

Other dimensions that are anatomically compatible may be utilized, when conducting implantation tests as an example, with clinically relevant samples.

In general, the test sample size should be chosen by taking into consideration the size of the bone used in order to prevent incidental bone breakage and subsequent euthanasia of the test animal over the course of the study.

C.4.1 Test animals

The implants shall be inserted into the bone of rodents, dogs, sheep, goats, pigs or rabbits. Select one species among these in line with the principles set out in ISO 10993-2. Species differences are important in bone physiology and should be assessed before implantation procedures are initiated. In addition, bone quality can vary between non-purpose-bred animals of the same species, and bone densitometry can be required to identify suitable test animals and to interpret the test results. Use of skeletally mature animals is recommended. If alternate animal models are used (e.g. for paediatric indications), the selection shall be justified and documented.

C.4.2 Implant sites

Equivalent anatomical sites shall be used for test and control samples. The test implants shall be contralateral to the control implants. Select the implant site to minimize the risk of mobility of the implant. At least 10 test samples and 10 control samples shall be evaluated for each material and implantation period. Tissue samples to be evaluated for a material shall originate from at least three animals.

A non-absorbable control sample should be evaluated at each time point.

If dealing with absorbable materials, comparison with a device or material at equivalent degradation time points can help with the interpretation of results.

The femur and tibia are typically utilized. Other sites can be suitable.

The number of implant sites shall be as follows.

1. a) In each rabbit there shall be a maximum of six implant sites:

   — three for test samples;

   — three for control samples.

2. b) In each dog, sheep, goat or pig, there shall be a maximum of 12 implant sites:

Do not insert more than 12 samples in any one animal.

The size, mass and age of the animal and the implant site chosen should ensure that the implant placement does not cause significant risk of pathological fracture of the test site. In younger animals, it is especially important to ensure that the epiphyseal area or other immature bone be avoided.

NOTE The number of implant sites per femur and tibia is limited by practical considerations of the animal model and anatomy.

C.5 Implantation procedure

Perform bone preparation using low drilling speed and intermittent drilling using profuse irrigation with physiological saline solution and suction, because overheating will result in local tissue necrosis.

It is important that the diameter of the implant and the implant bed in the bone match well enough to avoid ingrowth of fibrous tissue.

Expose the cortex of each femur or tibia and drill the appropriate number of holes to receive implants. For rabbits, prepare up to three holes; for larger animals prepare up to six holes. Ream to final diameter or tap screw thread before insertion. Insert cylinders by finger pressure to allow press fit. Tighten screw-shaped implants in place with an instrument capable of delivering a predetermined torque. Record the torque. Alternate implantation sites or procedures shall be justified or validated by literature.

C.6 Implantation period

To ensure a steady-state of biological tissue response, the implantation period(s) shall be selected as specified in 5.3.

C.7 Evaluation of biological response

The evaluation shall take into account the requirements specified in 5.5.

C.8 Test report

The presentation of the test results and final test report shall include the requirements specified in Clause 6.

D.1 General

This test method is used for assessing the biological response of brain tissue [central nervous system (CNS) and dura] to an implanted material. The implantation site in the brain, spinal cord, dura or cerebral spinal fluid (CSF) should be selected according to the end use of the material.

The material for a neuro-interventional device, which is in contact with the vessel wall but not with neural tissue directly, shall be evaluated in accordance with ISO 10993-4.

D.2 Principle

The implant is placed into or on neural tissue or dura, as clinically appropriate, in the test animals. The biological response to implants of test samples is compared with the biological response to implants of control samples. The control materials are those used in medical devices whose clinical acceptability and biocompatibility characteristics have been established.

D.3.1 General

Common provisions for preparation of test and control samples are described in 4.2.

A non-absorbable control sample should be evaluated at each time point.

If dealing with absorbable materials, comparison with a device or material at equivalent degradation time points can help with the interpretation of results.

D.3.2 Implant size and shape

Implant sizes are based on the animal species and the site chosen. The following typical dimensions shall be considered for rats and rabbits. The following are some example sizes of samples implanted in or on the brain parenchyma:

The thickness of the disc shall be justified in relation to the use of the material. For medical devices that are intended to contact primarily the parenchymal surface, the test article shall be implanted on the parenchymal surface. For medical devices intended to contact only the dura, the test article shall be implanted on the dural surface. A 4 mm to 6 mm diameter disc can be appropriate if two sites are to be implanted or larger diameter discs if a single site will be implanted, species dependent.

D.4.1 Test animals

This protocol is for studies using rats or rabbits. If other species are used based on device considerations, modifications of the protocol can be necessary. Both sexes should be represented in equal numbers unless sufficient justification for a single sex is provided.

Animals that are healthy and have not been subjected to previous experimental procedures shall be used. Species and age are important factors to consider based on differences in neurophysiology and biological response and should be assessed before implantation procedures are initiated.

D.4.2 Implant sites

There should be a minimum of eight test sites and eight control sites for each material at each time point (split equally between male and female) to permit an evaluation of local neural effects. Equivalent anatomical sites shall be used for test and control samples. Careful selection of the implant site and surgical procedure is critical to minimize the risk of mechanical trauma. Only one hemisphere shall be implanted per animal, and shall include only one type of implant, either test or control. One site per hemisphere can be implanted in each rat and two sites can be implanted in each rabbit. When considering more than one implant site per brain hemisphere, consider that the effects of two implants separated by insufficient distance can cause a greater-than-additive tissue response at both sites.

With many bone, muscle and subcutaneous implant designs, test and control materials are typically implanted in the same animal. However, with materials placed in neural tissues, tissue responses are not always localized, but can affect a wide region and can even manifest across the brain hemisphere. In experimental animal models of brain injury, microglia can be induced to migrate along the myelinated sheaths of the corpus callosum to the contralateral hemisphere. Therefore, the implant-activated microglia can migrate across the corpus callosum and influence the biological response on the contralateral side. This can serve to exacerbate the response to a control implanted site, resulting in a shift in the baseline response. In addition, influence from the contralateral hemisphere injury can exacerbate the injury response at the test article implanted site. In the first case, one can shift the normal baseline response to a control material, resulting in a false-negative result for the test article. In the second case, the response occurring in the contralateral hemisphere can exacerbate the response to a test article leading to a false positive. Given the small number of animals required for testing, the ability to control for such factors and to decrease the variability of the data are critical. As such, it is useful to have separate test and control animals. Sex differences have been documented in responses that can be relevant to evaluation of response to implantation devices (see References [51], [52] and [54]). To control for potential variability due to sex differences, similar numbers of males and females of each species and strain should be used for the study.

D.5 Implantation procedure

Each animal should be weighed prior to implantation and periodically thereafter. Following species-appropriate analgesia and anaesthesia, surgically prepare the skull of the animal. Stable analgesia and anaesthesia shall be maintained during the full surgical procedure and for an identified time period after implantation.

Animals should be appropriately restrained during the operation procedure. Using aseptic surgical techniques, the skull is exposed and holes of sufficient similar diameter to insert the implant samples are made in the cranium. In addition, a small hole is prepared in the dura and the implant is gently introduced into the appropriate part of the brain.

The surgical technique profoundly influences the result of any brain implantation procedure as the severity of injury response (both glial and neuronal) is related to the level of physical trauma and can render a study uninterpretable (see References [53], [54] and [55]).

Stereotactic methods can allow for a significant level of control with regards to accurate placement and minimizing physical damage to the puncture location. An alternative control method for restraining the animals may be considered.

D.6 Implantation period

A one-week implantation period is necessary, as well as other appropriate time intervals, to adequately characterize the response. Neurodegenerative processes can be rapid and transient as cell death can occur in the first few days following administration, as shown with certain chemicals (see Reference [53]).

Longer implantation periods shall be considered in view of the clinical applications of the material.

D.7 Post-implantation observation

Animals should be initially housed individually and observed twice daily to ensure proper healing of the implant sites, return to normal eating and drinking behaviour and for any abnormal clinical signs due to the surgical procedure. The observation frequency is adjusted based on initial observations. If animals are treated with antibiotics, this needs to be stated as some compounds like minocycline can directly modulate the response of brain microglia and macrophages (see Reference [56]).

As damage to neural tissue can result in abnormal behaviour, clinical observations should be included in the evaluation of the effects of brain implants. Since an integrated assessment of functional and structural changes is important to neuropathological interpretation, the pathologist shall be provided with records of the clinical observations prior to performing the macroscopic and microscopic examinations.

A detailed (weekly) physical examination shall be performed on each animal to monitor general health. Observations shall be recorded and include all abnormal clinical signs, abnormal behaviour, or clinical systemic or central nervous system manifestations of the implant. To help with assessment of central nervous system disorder, a functional observation battery (FOB) or modified Irwin’s can be used. Clinical signs can include, but are not limited to, changes in skin, fur, eyes or mucous membranes, and occurrence of secretions and excretions or other evidence of autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Additionally, changes in gait, posture and response to handling, as well as the presence of clonic or tonic seizures, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilating, walking backwards) shall be recorded. For the behaviour and neurological signs, time of first observation and subsequent progression or resolution should be recorded. The initial finding of abnormal behaviour, neurological signs, ambulation, posture or reflexes shall initiate a daily observation schedule for the relevant signs.

Humane end points for early removal of an animal from the test should be set prior to the test. Once severe clinical effects have been identified, an attending or qualified laboratory animal veterinarian, or personnel trained to identify the clinical lesions, should be consulted for a clinical examination. The attending or qualified laboratory animal veterinarian, in concert with the study director or principal investigator, should determine whether test animals should be removed from the test and euthanized.

D.8 Evaluation of biological response

The evaluation shall take into account the requirements specified in 5.5. Manipulation of the implant or implant site in the CNS typically causes artefacts that can interfere with microscopic evaluation. Therefore, nervous tissue system [CNS and peripheral nervous system (PNS)] typically should be fully fixed to ensure appropriate tissue preservation prior to macroscopic examination. Macroscopic examination can be resumed on the following day or when the tissue will be trimmed for histologic processing.

All gross changes observed macroscopically, shall be further evaluated microscopically. Regional or whole body intravascular perfusion fixation with or without immersion fixation may be used when warranted to reduce artefacts that may occur in tissues fixed wholly by immersion fixation, but the decision regarding the appropriate fixation method should be made by the study director in collaboration with the study pathologist. Technical details for performing perfusion fixation during nonclinical studies can be found in References [71] and [72]. Additionally, for the particular test species, the most appropriate draining lymph node(s) or mucosal associated lymphoid tissue (MALT) shall be examined grossly, if possible, or the appropriate tissue region containing the lymphoid tissue may be collected and fixed appropriately for microscopic examination. Brain tissues from the animals in the control and treatment groups shall be examined. In addition, tissues from animals which died prematurely or were euthanized during the study should be grossly examined and a comprehensive set of tissues should be examined microscopically to determine the cause of death and assess tissue reactions to the implant.

The neuropathological evaluation should assess the tissue for gliosis and neurodegeneration using appropriate histological stains, biochemical indicators of damage or both. The use of a specific stain or damage indicator should be recorded and supported with appropriate peer-reviewed references describing the stains used in evaluating neurodegeneration or gliosis. The following stains and biomarkers given in Table D.1 are examples that can be used to assess the histopathologic effect of the implant (see References [57], [65], [70] and [71]).

Specific antibodies are not available for all species.

High resolution images from implant sites should be provided that are representative of the pathologist’s diagnosis or score observations and can demonstrate the morphological detail of the cellular response specific to the implant.

Specific cellular subpopulations shall be identified when warranted by appropriate staining. Images should include a scale bar indicating magnification.

The study pathologist should define the cellular criteria and characteristics used to identify tissue responses and determine whether or not they are adverse. An example of a scoring system for inflammatory changes in neural tissue is given in Table F.4. Importantly, the study pathologist has the discretion to select which cell changes are appropriate to score in assessing the tissue response to implants.

The pathologist may select among numerous cell and tissue features in assessing responses to implants. Cellular criteria that can be scored when present include:

When warranted, the use of cell type-specific staining may be helpful to characterize the tissue response. The following characteristics in the tissue surrounding the implant shall be addressed when present:

1. a) disruption in the neuronal processes surrounding the implants;
2. b) zone of astrocytosis and connective tissue around the implant;
3. c) increase in the number of large blood vessels;
4. d) infiltrating leukocytes;
5. e) microglia activation – in a staging characterization;
6. f) capsule formation, presence of multinucleated giant cells and macrophages;
7. g) areas of mineralization or calcification;
8. h) changes in the ependymal layer and changes as above in the arachnoid granulations;
9. i) haemorrhage;
10. j) necrosis.

In addition to the implant sites, tissue adjacent to the implanted area (approximately 3 mm, ipsilateral) and any affected tissue away from the implanted area should also be examined. As warranted by the tissue response, parameters for evaluation include:

D.9 Test report

The presentation of the test results and final test report shall include the requirements specified in Clause 6.

E.1 General

This test method is used for assessing the biological response of peripheral nerve tissue to implanted device. The implantation site should be selected based on the intended clinical use of the device.

Examples of devices include, but are not limited to:

1. a) peripheral nerve stimulators (see also ISO 14708-3);
2. b) nerve guidance conduits (NGCs);
3. c) nerve protectant wraps;
4. d) nerve repair scaffolds.

For implantation studies with peripheral nerve stimulators, to assess implantation risks from device materials and designs, a passive implant is generally used in the study. The study design for a functional study or a stimulation safety study with the active implant such as active electrical nerve stimulator is not covered in this Annex. However, local tissue response to an implant from a biocompatibility perspective can be assessed in a functional or stimulation safety study, if adequately designed.

The test method described in this Annex is limited to the assessment of the local neural tissue response to the implanted device. If implantation and systemic toxicity evaluations are combined in a single study, the recommendations provided in ISO 10993-11 for systemic toxicity evaluation should also be fulfilled when designing such a study.

E.2 Principle

The device is implanted into or surrounding the neural tissue of test animals based on the intended use of the device. The biological response to implanted test samples is compared with the biological response to implanted control samples. In some cases, the tissue response can be compared with a sham surgical control.

E.3.1 General

Common provisions for preparation of test and control samples are described in 4.2.

A control sample should be evaluated at each time point. The control materials are those used in medical devices whose clinical acceptability and biocompatibility characteristics have been established. In some cases when a comparative control material is not available, high density polyethylene (HDPE) is a common reference material used for implantation studies because its clinical acceptability and biocompatibility characteristics have been well established. However, based on the device design and intended use, it cannot always be feasible to use HDPE as a reference control for peripheral nerve implantation studies. For example, for an implantation study with a cuff electrode that is wrapped around a nerve such as the sciatic or a cranial nerve (such as the vagus), the use of HDPE as a reference material can be problematic as wrapping the nerve with a hard plastic like HDPE would cause nerve damage. In such a case, an alternative reference material or a sham surgery control should be considered.

The selection of control(s) for the study should be justified.

E.3.2 Implant size and shape

It is recommended that the final, finished device be used for the implantation study in an appropriate animal model. The selection of animal model should be justified.

If the final, finished device cannot be implanted due to limitations with the selected animal model, a test coupon can be considered. The test coupon should undergo the same manufacturing and sterilization processes, have the same chemical, physical and surface properties, and the same ratio of component materials as the medical device in its final, finished form. The use of the test coupon should be justified.

E.4.1 Animal models

The animal model should be selected in accordance with the principles set out in ISO 10993-2 with due considerations to the intended clinical use, device characteristics, implant size, the number of implants per animal, the intended duration of the study with respect to the expected lifespan of the animals and any potential species differences in biological response. Rats, rabbits, dogs, sheep, goats and pigs are commonly used models for peripheral nerve implantation studies.

To control for potential variability due to sex differences, equal numbers of male and female animals should be used in the study. For example, studies have shown that there are sex-based differences in cardiovascular effects of vagus nerve stimulation characterized using electrocardiogram (ECG) morphology and heart rate variability (HRV) analyses (see Reference [66]). Justification should be provided if only a single sex is represented in the study or if both sexes are not represented equally.

Animals that are healthy and have not been subjected to previous experimental procedures should be used. Species, strain, sex, age, developmental stage, mass and microbiological status (i.e. specific pathogen free) are important factors to consider based on differences in neurophysiology and biological response.

E.4.2 Implant sites

There should be a minimum of eight test sites and eight control sites at each time point to permit an evaluation of local neural effects in small animal models (e.g. rats, rabbits). For large animal studies, a smaller number of test and control sites can be considered acceptable with appropriate justification. Equivalent anatomical sites should be used for test and control samples. Careful selection of the implant site and surgical procedure is critical to minimize the risk of mechanical trauma to the nerve and surrounding tissues.

Test and control samples can be implanted in the same animal (e.g. contralateral sciatic nerve implantation for cuff electrode stimulator in sheep). However, in some cases, it is not feasible to implant test and control samples in the same animal or have a sham surgery procedure in the same animal. For example, for an implantation study in a sciatic nerve transection model in a rat for assessment of a nerve wrap, a unilateral implantation in each animal is performed. For an implantation study of a vagus nerve stimulator, bilateral implantation can disrupt basic physiologic functions due to vagus nerve regulation of cardiac, digestive and respiratory functions among others and thus should be avoided.

E.5 Implantation procedure

Care should be taken when manipulating peripheral nerves during the implantation procedure because excessive handling can lead to dysfunction of the innervated region which can complicate the interpretation of study results (i.e. response to the implant versus surgical procedure). Traction, compression and contact with surgical instruments, such as suction or electrocautery devices, or exposure to air, blood or rinsing fluid can cause nerve injury that can or cannot be visible but can cause functional deficits after surgery (see Reference [67]).

E.6 Implantation period

The implantation period should be based on the device type (non-absorbables versus absorbables) and intended clinical exposure duration of the device.

For example, the time points for permanently implanted nonabsorbable devices typically include 30 d, 90 d and 180 d. However, a longer-term study can be necessary if the steady-state response has not been observed with the device by 180 d or if there are abnormal clinical or pathological observations in the study.

For absorbable materials, see 5.3.4.

E.7 Post-implantation observation

Animals should initially be housed individually until the incision heals and any skin closure device (suture or clips) is removed and observed twice daily to ensure proper healing of the implant sites, return to normal eating and drinking behaviour, and for any abnormal clinical signs due to the surgical procedure.

Animals shall be observed daily for general health throughout the study duration in accordance with ISO 10993-2.

A comprehensive (weekly) physical examination shall be performed on each animal to monitor general health. All observations shall be documented, including any abnormal clinical signs or behaviour. Depending on the device type, the animal model and the nerve that can be impacted, more specific additional assessments such as a neurologic examination can also be needed. For example, the injury to the sciatic nerve in animals can manifest as pressure sores, traumatic damage to the foot (induced sensorimotor deficits), or autotomy, which can manifest as loss of toenails, or toes (see Reference [68]). These complications shall be monitored, scored when appropriate and documented. Signs of sciatic nerve injury in dogs include an inability to flex the stifle and to flex or extend the hock and digits. There can be sensory defects (absent or delayed withdrawal, misplacement of paw) and eventually, atrophy of the gluteal, semimembranosus and semitendinosus muscles and all the muscles below the stifle. Thus, the animals shall be assessed for overall behaviour and mentation, and any abnormalities identified through physical or neurologic examination shall be pursued with a more thorough, localized examination.

For a long-term study (180 d) with non-absorbable devices, frequency of physical examinations can be decreased to monthly following 30 d after implantation, if adequate justification is provided.

Body mass should be measured at pre-treatment, weekly (first month), monthly thereafter and at termination.

Pre-determined criteria for early termination of animals shall be defined prior to initiating the study. Early termination or death shall be thoroughly investigated (e.g. necropsy, clinical pathology) to determine if it was related to the device.

E.8 Evaluation of biological response

The evaluation should take into account the requirements specified in 5.5.

Gross findings should be further evaluated microscopically. Regional or whole body intravascular perfusion fixation with or without immersion fixation may be used when warranted to reduce artefacts that may occur in tissues fixed wholly by immersion fixation, but the decision regarding the appropriate fixation method should be made by the study pathologist. Technical details for performing perfusion fixation during nonclinical studies can be found in References [71] and [72].

The nerve implant site(s) and the segments proximal and distal to the implants shall be evaluated. For nerves implanted with absorbable materials, collection of the associated ganglia should also be considered to evaluate the potential effects of axonal transport or migration of degradation products. Peripheral nerve injury can result in cell type-specific injury (e.g. axonal degeneration, altered Schwann cell phenotypes) that can be detected using certain biomarkers appropriate to assessing peripheral nerves, such as neurofilament protein (NFP) for axonal integrity and matrix metalloproteinase 9 (MMP9) or S100 to assess Schwann cell morphology.

In addition, the organ(s) or tissue(s) in contact with the device should be evaluated grossly and microscopically. If the innervated tissue is muscle, it should be weighed at necropsy. The results from these assessments should be compared between the test and control groups.

In addition, tissues from animals which died or were euthanized prematurely during the study should be grossly examined and any abnormalities identified examined microscopically.

E.9 Test report

The presentation of the test results and final test report shall include the requirements specified in Clause 6.

F.1 General

The scoring systems presented in this annex have historically been used primarily for biocompatibility studies in muscle and subcutaneous tissues. Aspects of this scheme have been adapted to other tissues such as bone, brain and skin. As this scheme is informative, it should not be considered as the only appropriate scheme for evaluation of tissue responses nor applicable to all tissues (e.g. when evaluating wound healing studies) without adaptation.

For examples of semiquantitative and quantitative scoring systems, see References [24], [26], [28], [29], [32], [33], [34], [38], [45], [47] and [69].

For each histological characteristic evaluated (such as capsule formation, inflammation, presence of polymorphonuclear cells, multinucleated giant cells, plasma cells or degradation of material), the semiquantitative scoring system used shall be described in the evaluation report. In addition to the scoring of the reaction components, the extent of the whole reaction should also be evaluated.

The evaluation system as specified in Tables F.1 and F.2 may be converted to an implant evaluation system as specified in Table F.3. An example of a histological evaluation system for neural tissue responses is provided in Table F.4.

In this semiquantitative scoring scheme, inflammatory cell infiltrates and necrosis are scored using the scoring scheme in Table F.1. Neovascularization, fibrosis and fatty infiltration are scored using the scoring scheme in Table F.2. In the example given in Table F.3, due to the greater importance of inflammatory cell infiltrates and necrosis, these parameters (see Table F.1) are multiplied by a factor of 2 to provide a weighted value as compared to neovascularization, fibrosis and fatty infiltration parameters (see Table F.2). The values are totalled and then an average score for test and control implant sites is calculated. The average score for the control implant sites is subtracted from the test implant site average to determine a reactivity grade based on the scale in Table F.3.

The study report should comment on each cell type and neovascularization response. For each cell type where there is a significant difference in value between the treatment and control sites, the study report should explain the relevance of the difference.

An example of a scoring system used for biological evaluation of absorbable materials is described in Reference [28].

F.2 Conclusion

Under the conditions of this study, the test sample was considered to demonstrate

to the tissue as compared to the comparative or reference control sample within Table F.3. The example given in Table F.3 shows a “mean difference” of six which corresponds to a “slight relative reactivity” score according to the reactivity ranking given in this clause. Therefore, the conclusion for the example in Table F.3 would be “slight relative reactivity”. The conclusion should also discuss the amount of tissue necrosis, foreign debris and number of implants examined.

F.3 Comments

Additional comments may be added to provide a description of observations about the tissue response or changes to the implant material, if applicable.

G.1 General

Local effect studies focus upon the implant-tissue interface and the peri-implant tissues in a complex biological test system (i.e. animal model). Thus, the tissue responses to the physical and chemical properties of an implant at the implant-tissue interface and within the peri-implant tissues can be evaluated. The pathologist can be able to identify particles or foreign debris associated with the surgical procedure and the tissue response, from implant manufacturing. Particles from the implant can be identifiable in regional draining lymphoid tissue or in distant lymphoid tissues (e.g. spleen), providing more confidence of their origin from the implant. In some cases, specific chemical constituents remaining in the tissue can be detectable with appropriate staining or other bioanalytical methods. However, confounding factors can mask or complicate such evaluations. Factors affecting the evaluation of the tissue responses to an implant following a surgery that mimics the clinical use of an implant include but are not limited to:

1. a) complex surgical approaches to the implant location;
2. b) implant procedures typically associated with variable and frequently mild-to-moderate surgical trauma;
3. c) low-to-moderate repeatability of the surgical procedure (can lead to variability in local tissue responses between implant sites and animals);
4. d) the presence of non-implant site alterations (abdominal and thoracic surgical adhesions when penetrating body cavities);
5. e) general surgery-related complications (abdominal wall closure dehiscence, incision line complications, abdominal and thoracic adhesions when body cavities are penetrated, endogenous tissue damage responses [e.g. damaged hair follicles leading to foreign body granulomas]);
6. f) the use of ancillary devices necessary for the implantation procedure such as the use of electrocautery (can delay or change healing at the implant site, contribute to the inflammation seen at implant sites);
7. g) introduction of non-implant foreign bodies (sources include laparotomy pads and gauze sponges used during the surgical procedure);
8. h) tissue drying from prolonged air exposure or insufflation gases; and
9. i) a higher risk of surgical site bacterial contamination due to loss of aseptic technique and prolonged surgery times.

The suggested tissue response and scoring system (see also Annex F) can be used in intended simulated clinical (preclinical or efficacy) use studies, but the system can require significant modification to be appropriate for the surgical implant procedure and the tissues implanted. The interpretation of results from such a study should take into consideration aforementioned factors that can negatively affect the tissue responses at the implant-tissue interface and within the peri-implant tissues.

Criteria to be scored and the relevant scoring schemes for each tissue and implant should be determined by the study pathologist based on the study design (objectives, characteristics of the implant, site of implantation, etc.) and features observed in the tissue response^[62^][63^][73^][74^][75^].

When selecting an amount or size of an implant, the general nature and composition of the sample being implanted should be considered (i.e. solid versus gel versus porous implants, absorbable versus permanent implants). The amount or size of the implant should be appropriate for the animal model and location selected. An exaggerated amount or size of a sample within an implant site can adversely affect the tissue responses observed leading to an inaccurate conclusion about the biocompatibility of an implant. For example, a sample that absorbs through hydrolysis is implanted in such an amount that fluid cannot penetrate to the centre region of the implant unlike what will be encountered in the clinical use. The lack of hydrolysis in the central region of the implant leads to encapsulation or isolation of the non-absorbed or degraded portion of the implant inaccurately reflecting poor local biocompatibility of the implant. An exaggerated implantation of a permanent implant for a selected tissue location and animal can result in delayed implant site surgical healing due to tissue compression (i.e. local ischemia, pressure necrosis, migration from gravity pulling dorsally located implants ventrally). An exaggerated amount or size of an implant typically is seen when the evaluation of local effects of an implant is combined with a systemic effect study. In these studies, exaggerated dosing is required in order to evaluate the systemic safety of an implant. The selected dose and exaggeration factor (see ISO 10993-11) shall be justified and documented. When exaggerated doses are used dividing the total dose among multiple implant sites and additional tissues should be considered.

Local effect studies are one aspect of determining the overall biocompatibility of an implant and should not be considered in isolation but in combination with all available in vitro and in vivo assessments.

G.2 Tissue section preparation

Soft implants and implants that absorb or degrade that are initially soft should be left in place for tissue trimming and paraffin-based tissue processing and microtomy. Exposure to the tissue processing agents used for paraffin-based processing can remove or dissolve some polymeric implant material leaving an implant void lined by an intact implant-tissue interface which are more than adequate for microscopic evaluation. To reduce artefacts at the implant-tissue interface of hard non-porous implants, like metals and hard plastics, these implants should be removed if possible, from the implant sites after tissue fixation at tissue trimming and prior to paraffin-based tissue processing and microtomy or left in situ and prepared after embedment in hard plastics or by tissue grinding. If the tissue-implant interface is not evaluated at necropsy, then this interface should be evaluated and documented at tissue trimming (i.e. an additional macroscopic evaluation can need to be conducted at tissue trimming to properly document the macroscopic findings). This should be done on a case-to-case basis as loss of tissue at the interface during device removal can prevent complete understanding of changes at the interface.

Soft or hard resin-based tissue processing should be used for:

G.3 Implant-tissue interface and peri-implant tissue evaluation

Suggested tissue response scoring systems are presented in Annex F for general tissue implantations and central nervous system. These scoring systems can be used to standardize how results are documented and to support the study-specific pathologic interpretations. The tissue response scoring systems are suggested, not mandatory, and, Annex F, is not appropriate for all tissue types and test samples because types of residential cells and the process through which a tissue heals can be different. If the tissue response scoring systems Annex F are not appropriate for the tissues implanted or the implant being evaluated, the pathologist shall document any modifications to the scoring system, based upon their experiences with medical device evaluations and scientific literature.

The suggested system in Annex F uses a semiquantitative scoring system (see Reference [69]). The suggested scoring system in Annex F, uses a two-part ordinal scoring scale. The first part focuses upon the inflammatory cell types and approximate number of these cells present at the implant-tissue interface and within the peri-implant tissues along with any article-related induced necrosis (not any of the necrosis that can be associated with implantation or surgical trauma). These cell types and number as well as the presence of necrosis are central in determining the biocompatibility of the implant; therefore, are emphasized by multiplying the score by two. The multiplication of components in the scoring scheme emphasizes the biologically important cellular component involved in determining the biocompatibility of an implant. The second part of the scoring scale primarily evaluates the healing of the tissues in response to the implant which is necessary for an implant to be considered biocompatible. Even though the healing response is involved in and contributes to the overall biocompatibility of an implant, the inflammatory cells and the presence of implant-related necrosis involved in the tissue response to an implant primarily determines if an implant is biocompatible.

The tissue response scoring portion of the suggested system in Annex F compares the average tissue response scores to the average scores of a control sample and puts the tissue responses to an implant into perspective from a clinical standpoint. It is recommended that the control sample matches the chemical composition and structure of the test sample as much as reasonably possible. The selected control sample should be a commercially available and marketed implant (not recalled in any country) with a well-established history of safe clinical use and a well-established and understood biocompatibility profile. A reference control implant such as high-density polyethylene (HDPE- USP reference control plastic) can be used when no adequate commercially marketed implant is available or exists; however, the physical differences between the implant and the HDPE must be considered when interpreting results (e.g. a porous implant can score greater than the solid HDPE due to tissue ingrowth, a gel implant is be associated with more macrophages than HDPE). The suggested ranking of the average score of an implant in Annex F is based upon what level of biocompatibility is acceptable in a clinical setting versus what is acceptable level of biocompatibility in an isolated test system.

The study pathologist can use a severity grade to reflect intensity of tissue responses at the implant-tissue interface and peri-implant tissues. Nonetheless, it is recommended to provide a comprehensive definition for the utilized grading. Terms like minimal, mild, moderate and severe vary between pathologists making comparisons between pathologists or studies difficult-to-impossible to assess and which frequently leads to requests for clarification from regulatory agencies. Traditional or classical diagnostic pathology terminology (e.g. granulomatous inflammation, foreign body response or granuloma) can be used if warranted. Globally standardized toxicological pathology nomenclature developed through the International Harmonization of Nomenclature and Diagnostic criteria (INHAND) initiative was not directly designed for application in evaluating local biocompatibility of an implant. Nonetheless, this harmonized and widely accepted terminology can potentially serve as descriptors in evaluation of implants where toxicological effects are evaluated while also aiding the study pathologist in accurately recording general tissue reactions and recognizing common background findings in implanted tissues. The INHAND terminology can be accessed online via the goRENI1

1 Available at https://www.goreni.org/gr3_nom_inhand_publ.php

2 Available at https://www.toxpath.org/inhand.asp

In addition to scoring tissue responses to implants, the physical attributes of the implant such as fragmentation and particulate generation (and any associated migration thereof), evidence of dyes or solvents leaching from the implant, and for absorbable or degradable implants, an estimate of the amount of implant remaining extracellular in location at different study intervals should appear as comments following tables containing the individual implant site tissue response scores or as a standalone section in the pathology report. Assessing the original native form of the implant can enhance the characterization of morphological features associated with the implant or device.

Using any tissue response scoring system does not alleviate the pathologists’ responsibilities of justifying the use of a scoring system and interpreting results.

G.4 Images

Representative test and control implant site microscopic images for illustrative purposes, as well as selected microscopic images illustrating certain implant site findings, can be useful for documentation of microscopic findings but do not supersede written descriptions or implant site tissue response scoring results. The inclusion of illustrative representative and selected (when warranted) microscopic images of test and control implant sites at each study interval in the pathology section of a local effects study report is expected by the study sponsor and the regulatory agency (for some regulatory agencies such images are a requirement); therefore, it should be routinely included. Generating additional information or evaluations beyond what is present in the pathology report using illustrative images should not be done.

G.5 Lymph nodes

For medical devices or materials that are non-absorbable in composition, regional draining lymph nodes should be collected, evaluated macroscopically and then fixed for possible future microscopic evaluation. Any macroscopic alterations of these lymph nodes constitute a reason for microscopic evaluation of the lymph nodes. For absorbable or degradable medical devices or materials regional draining lymph nodes should be collected and microscopically evaluated.

The macroscopic identification of regional draining lymphoid tissues can be problematic because of their small size (sometimes even if degradants are present) and the presence of abundant adipose tissue that makes it difficult to find the lymphoid tissues. The end user is also cautioned that the draining lymphoid tissue should be selected based on published information for the species and location of the implant.

NOTE Various techniques such as the use of lymphoid tissue-revealing solution can be exploited to assist in identifying lymphoid tissues at the time of trimming when abundant adipose tissue is present.

G.6 Evaluating the absorption or degradation of an implant

Evaluating and scoring the absorption of an absorbable or degradable implant is not an exact process. In general, a histopathology assessment of degradation is semiquantitative. If a more quantitative assessment is warranted, other methods may be used depending on the nature of the material. These additional methods may include digitalization or semi-automated or automated image analysis. When performed via histopathology (subjective, ordinal scoring), the absorption or degradation is often estimated using a single 5 μm section through a much larger implant, and the assigned score is an estimation of the amount remaining either extracellularly or intracellularly, or both, compared to initially implanted article. Intracellular or suspected intracellular material at the site of implantation if present should be described minimally noting the cell type(s) involved, the type of cytoplasmic change induced and any evidence of migration of the involved cells.

Absorption or degradation of an implant can vary significantly between implant sites within the same animal and between animals due to a number of factors including, but not limited to:

Degradation assessment should employ a semiquantitative grading system, such as a scale from 0 to 4 compared to the initially implanted article at day 0. If feasible, the pathologist may estimate the percentage of degradation in the report's narrative for the final period.

Table G.1 and Table G.2 provide two examples of a scoring scheme. These are provided as examples and should not be presumed to suggest a requirement. Since the plane of section, the number of sections and other variables can influence the appearance of residual article in a tissue specimen, the end user is cautioned of the subjective nature of such an estimate.

Bibliography