ISO/TS 10993-20:2006

Biological evaluation of medical devices — Part 20: Principles and methods for immunotoxicology testing of medical devices

ISO/TS 10993-20:2006 presents an overview of immunotoxicology with particular reference to the potential immunotoxicity of medical devices. It gives guidance on methods for testing for immunotoxicity of various types of medical devices. ISO/TS 10993-20:2006 is based on several publications written by various groups of immunotoxicologists over the last few decades in which the development of immunotoxicology as a separate entity within toxicology took place.

Évaluation biologique des dispositifs médicaux — Partie 20: Principes et méthodes relatifs aux essais d'immunotoxicologie des dispositifs médicaux

General Information

• Status: Published
• Publication Date: 02-Aug-2006
• ICS: 11.100.20 - Biological evaluation of medical devices
• Technical Committee: ISO/TC 194 - Biological and clinical evaluation of medical devices
• Drafting Committee: ISO/TC 194/WG 7 - Systemic toxicity
• Current Stage: 9093 - International Standard confirmed
• Completion Date: 03-Sep-2021

Standards Content (Sample)

TECHNICAL ISO/TS
SPECIFICATION 10993-20
First edition
2006-08-01

Biological evaluation of medical
devices —
Part 20:
Principles and methods for
immunotoxicology testing of medical
devices
Évaluation biologique des dispositifs médicaux —
Partie 20: Principes et méthodes relatifs aux essais
d'immunotoxicologie des dispositifs médicaux




Reference number
ISO/TS 10993-20:2006(E)
©
ISO 2006

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ISO/TS 10993-20:2006(E)
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ISO/TS 10993-20:2006(E)
Contents Page
Foreword. iv
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Risk assessment and risk management.2
5 Identification of hazards . 2
6 Methods of assessment of immunotoxicity. 4
6.1 General. 4
6.2 Inflammation. 5
6.3 Immunosuppression. 5
6.4 Immunostimulation. 7
6.5 Hypersensitivity . 7
6.6 Auto-immunity. 7
7 Extrapolation of data provided by pre-clinical assays . 7
Annex A (informative) Current state of knowledge. 8
Annex B (informative) Clinical experience with medical devices . 12
Annex C (informative) Flow chart for immunotoxicity testing. 14
Bibliography . 15

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ISO/TS 10993-20:2006(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
In other circumstances, particularly when there is an urgent market requirement for such documents, a
technical committee may decide to publish other types of normative document:
— an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in
an ISO working group and is accepted for publication if it is approved by more than 50 % of the members of
the parent committee casting a vote;
— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical
committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a
vote.
An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a
further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is
confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an
International Standard or be withdrawn.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO/TS 10993-20 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical
devices.
ISO/TS 10993 consists of the following parts, under the general title Biological evaluation of medical devices:
⎯ Part 1: Evaluation and testing
⎯ Part 2: Animal welfare requirements
⎯ Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity
⎯ Part 4: Selection of tests for interactions with blood
⎯ Part 5: Tests for in vitro cytotoxicity
⎯ Part 6: Tests for local effects after implantation
⎯ Part 7: Ethylene oxide sterilization residuals
⎯ Part 9: Framework for identification and quantification of potential degradation products
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ISO/TS 10993-20:2006(E)
⎯ Part 10: Tests for irritation and delayed-type hypersensitivity
⎯ Part 11: Tests for systemic toxicity
⎯ Part 12: Sample preparation and reference materials
⎯ Part 13: Identification and quantification of degradation products from polymeric medical devices
⎯ Part 14: Identification and quantification of degradation products from ceramics
⎯ Part 15: Identification and quantification of degradation products from metals and alloys
⎯ Part 16: Toxicokinetic study design for degradation products and leachables
⎯ Part 17: Establishment of allowable limits for leachable substances
⎯ Part 18: Chemical characterization of materials
⎯ Part 19: Physico-chemical, morphological and topographical characterization of materials
⎯ Part 20: Principles and methods for immunotoxicology testing of medical devices
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ISO/TS 10993-20:2006(E)
Introduction
International and European Standards are the main focus for demonstration of the safety and compliance of
medical devices. There has been increasing attention over the past few years on the potential for medical
devices to cause changes in the immune system. It was felt necessary to provide guidance on how to address
adverse effects of medical devices on the immune system. As there are no standardized tests available, this
document provides a framework on how to approach the evaluation of immunotoxicity.
The intention of this document is:
⎯ to summarize the current state of knowledge in the area of immunotoxicology, including information on
methods of assessment of immunotoxicity and their predictive value;
⎯ to identify what the problems are and how they have been dealt with in the past.
For clinical indications of immune alterations due to medical devices, an extensive literature review has been
performed, primarily through Medline. The key areas which have been researched are:
⎯ immunosuppression;
⎯ immunostimulation;
⎯ hypersensitivity;
⎯ chronic inflammation;
⎯ autoimmunity.
These key words are linked with the following materials:
⎯ plastics and other polymers;
⎯ metals;
⎯ ceramics, glasses and composites;
⎯ biological materials.
NOTE See also Table 1 for possibilities of interaction of materials with the immune system.

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TECHNICAL SPECIFICATION ISO/TS 10993-20:2006(E)

Biological evaluation of medical devices —
Part 20:
Principles and methods for immunotoxicology testing of
medical devices
1 Scope
This part of ISO 10993 presents an overview of immunotoxicology with particular reference to the potential
immunotoxicity of medical devices. It gives guidance on methods for testing for immunotoxicity of various
types of medical devices.
This part of ISO 10993 is based on several publications written by various groups of immunotoxicologists over
the last few decades in which the development of immunotoxicology as a separate entity within toxicology
took place.
The current state of knowledge with regard to immunotoxicity is described in Annex A. A summary of clinical
experience to date with immunotoxicology associated with medical devices is given in Annex B.
NOTE See also Bibliographic Reference [11].
2 Normative references
The following referenced documents are indispensable for the application 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 14971, Medical devices — Application of risk management to medical devices
ISO 10993-1, Biological evaluation of medical devices — Part 1: Evaluation and testing
ISO 10993-2, Biological evaluation of medical devices — Part 2: Animal welfare requirements
ISO 10993-6, Biological evaluation of medical devices — Part 6: Tests for local effects after implantation
ISO 10993-10, Biological evaluation of medical devices — Part 10: Tests for irritation and delayed-type
hypersensitivity
ISO 10993-11:2006, Biological evaluation of medical devices — Part 11: Test for systemic toxicity
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
immunotoxicology
study of the adverse health effects that result, directly or indirectly, from the interaction of xenobiotics with the
immune system
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ISO/TS 10993-20:2006(E)
3.2
medical device
any instrument, apparatus, appliance, material or other article, including software, whether used alone or in
combination, intended by the manufacturer to be used on human beings solely or principally for the purpose
of:
⎯ diagnosis, prevention, monitoring, treatment or alleviation of disease;
⎯ diagnosis, monitoring, treatment, alleviation of, or compensation for an injury or handicap;
⎯ investigation, replacement or modification of the anatomy or of a physiological process;
⎯ control of conception
and which does not achieve its principal intended action in or on the human body by pharmacological,
immunological or metabolic means, but which can be assisted in its function by such means.
NOTE 1 Devices are different from drugs and their biological evaluation requires a different approach.
NOTE 2 Use of the term "medical device" includes dental devices.
3.3
xenobiotic
substance foreign to the human body or living organisms
3.4
immunogenic
able to stimulate cells of the immune system resulting in an antigen specific immune response
4 Risk assessment and risk management
Risk assessment includes hazard identification, dose response assessment and exposure assessment, which
together allow characterization of the risk. Based on this risk characterization, risk management shall be
applied.
Because of the difficulties in predicting immunotoxicity of new chemicals and materials, effort and interest
need to be focused on the assessment and management of risks arising from known immunotoxic chemicals
contained in medical devices. Application of risk management to medical devices shall be performed in
accordance with ISO 14971. Possible immunotoxic hazards of the chemicals contained in the medical device
shall be identified first by an extensive literature search. Examples of such hazards are the production of
anaphylactic shock by chlorohexidine in medicines and by proteins in latex rubber. Subsequently the overall
risk management/reduction procedures shall be considered, together with the various possible actions that
could be taken to further reduce remaining risks such as indicating contra-indications on the label, product
recall, design-change and restrictions of use or application.
5 Identification of hazards
Immunological hazards should be identified by assessing exposure to medical device materials to identify the
presence of (potentially) immunotoxic agents. There are many sources from which information on
immunological hazards can be obtained. These sources include but are not limited to:
⎯ material characterization;
⎯ residue characterization;
⎯ characterization of the leachable materials;
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ISO/TS 10993-20:2006(E)
⎯ characterization of drugs and other substances added to the medical device;
⎯ characterization of exposure duration and route;
⎯ observations made during previous exposure to chemicals, drugs or materials;
⎯ toxicity testing.
Most immunological reactions identified to date relate to the additives to materials. Therefore exposure
assessment for these chemicals is important in order to identify the immunological hazard. Details of potential
outcomes with various materials from different types of medical devices are given in Table 1.
Table 1 — Potential responses of the immune system
Medical device categorization by Immune system responses
Nature of body contact Contact duration
A:limited
(u 24 h)
B:prolonged
Category Contact
(> 24 h to 30 d)
C:permanent
(> 30 d)
A × − − × × −
Skin B × × − × × −
C × × × × × ×
A × − × × × ×
Surface device
Mucosal membrane B × × × × × ×
C × × × × × ×
A × − × × × ×
Breached or
B × × × × × ×
compromised surface
C × × × × × ×
A × − − × × ×
Blood path, indirect
B × × × × × ×
External
C × × × × × ×
communicating
A × − × × × ×
device
Tissue, bone, dentin
communicating B × × × × × ×
implant devices
C × × × × × ×
A × − × × × ×
Tissue, bone and
Implant device B × × × × × ×
other body fluids
C × × × × × ×
NOTE This table is a framework for consideration of the potential interaction of materials from different types of
medical devices with various parts of the immune system, and is not a checklist for testing.

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Irritation/
acute inflammation
Chronic inflammation
Immunosuppression
Immunostimulation
Hypersensitivity
Autoimmunity

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ISO/TS 10993-20:2006(E)
Effects on the immune system (immunotoxicity) occur due to an encounter of immunologically competent cells
with foreign substances that are toxic and kill the cells, or result from foreign substances that interact with the
early events of the immune response and alter subsequent responses. Prediction of the likelihood of
immunotoxicity is difficult but can be based on known events in immunology.
First of all, for a substance to stimulate the immune response, it must be recognized as foreign to the host.
The likelihood of being immunogenic is greatest with proteins, then polysaccharides, then nucleic acids and
then lipids. Small molecular weight substances are generally not immunogenic. However, these substances
may become immunogenic by binding to host proteins and altering the structure of the protein. These
substances are usually referred to as haptens.
It is possible that polymeric materials, ceramic materials, and metallic materials may have leachable, wear or
degradation products that bind to host proteins. Materials of biological origin, such as collagens, natural latex
proteins, albumins and animal tissues are known to stimulate the immune response, and efforts must be taken
to make these materials non-immunogenic. In order for large substances (size > 1 000 000 daltons) to be
immunogenic, they must be broken down and delivered as smaller substances.
The foregoing are examples of substances and materials which may have immunogenic potential and thus
should be considered for their adverse effects on the immune system.
Body contact: every body contact listed in ISO 10993-1 is capable of manifesting an inappropriate immune
response (immunotoxicity). Skin and mucous membranes are particularly likely to develop Type I and Type IV
reactions. Other routes are likely to give systemic responses including Type I and Type IV reactions.
Duration of contact: in general, the longer the material is in contact with the body, the greater the likelihood of
immunogenic substances forming. However, some chemicals will act rapidly and immune responses from
materials in contact with the body for less than 24 hours can be immunogenic.
6 Methods of assessment of immunotoxicity
6.1 General
Immunotoxicity testing can be carried out using in vivo and in vitro assays. In contrast to in vivo
immunotoxicity testing, possibilities for in vitro testing are limited as the models lack the complexity of the
intact immune system. The value of in vitro methods in assisting extrapolation of animal data to man (by
elucidating mechanisms of toxicity) is further limited because they are not yet sufficiently developed and
standardized. However, they can be useful as mechanistic studies.
An important focus of immunotoxicology is the detection and evaluation of undesired effects of substances by
means of tests on rodents. When animal tests are considered, to satisfy the provisions of ISO 10993-2 all
reasonable and practically available replacement, reduction and refinement alternatives should be identified
and implemented. Although there are validated laboratory tests, in many cases the biological significance and
predictive value of immunotoxicity tests require careful consideration. The potential for effects on the immune
system can be indicated by alterations in lymphoid organ weight or histology, changes in total or differential
peripheral leukocyte counts, depressed cellularity of lymphoid tissues, increased susceptibility to infections by
opportunistic organisms or neoplasia. The prime concern within the area of immunotoxicology is therefore to
identify such changes and assess their significance with regard to human health.
In the context of immunotoxicity two kinds of assays can be distinguished: non-functional and functional. Non-
functional assays have a descriptive character in that they measure, in morphological and/or quantitative
terms, alterations in the extent of lymphoid tissue, the number of lymphoid cells and levels of immunoglobulins
or other markers of immune function. In contrast, functional assays determine activities of cells and/or organs,
such as proliferative responses of lymphocytes to mitogens or specific antigens, cytotoxic activity and specific
antibody formation (e.g. in response to sheep erythrocytes).
A new development in this area is the application of “-omics” for the detection of alterations in the expression
of genes involved in immune functions.
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ISO/TS 10993-20:2006(E)
The evaluation of immunotoxicological hazards should be planned in accordance with the flow chart given in
Annex C. Examples of tests for and indicators of immune responses are given in Table 2.
Although there are specific materials that are known or suspected to be immunotoxic, immunotoxicity testing
related to immunosuppression or immunostimulation shall initially be limited to those assays carried out in the
phase of general toxicity testing. Only those agents that show evidence of causing immunosuppression or
immunostimulation shall be subjected to further investigation. Sub-acute tests are useful for obtaining general
indications of potential immunosuppression or immunostimulation. If they are performed, they shall be carried
out in accordance with ISO 10993-11.
6.2 Inflammation
Agents can interact with components of the non-specific arm of the immune system, i.e. granulocytes,
macrophages and other cell types that are capable of producing and releasing inflammatory mediators. It
should be noted that after implantation of a foreign body, a local inflammatory response is quite common. The
duration and degree of the response determines whether it indicates an adverse effect. The most direct and
adequate method of assessing the degree of induction of inflammation after exposure to agents is
histopathology of the injection or implantation site of the agent. Chronic inflammation associated with
immunotoxicity is a lesion which is predominant in lymphocytic cells as opposed to the foreign body reaction
which is composed of macrophages and foreign body giant cells at the tissue/material interface. Initial tests for
local inflammation are described in ISO 10993-6. Other useful tests include serum assays for C-reactive
protein and acute phase protein.
6.3 Immunosuppression
For the detection of immunosuppression a tiered approach is warranted in order to reflect the complexity of
the immune system with its variety of functions and components. This tiered approach comprises a first tier of
immunosuppression testing using non-functional assays, followed by a second tier, that includes functional
assays. This tiered approach does not provide the most sensitive approach available as functional assays are
more sensitive than non-functional assays. The rationale for including less sensitive indicators as a first tier
and more sensitive indicators as a second tier is not because it best assesses the immune system, but rather
because it reduces the need for additional test animals.
In the first tier, indications for immunosuppression are induced alterations in, for instance, weight of immune
organs, in cell numbers and/or cell populations and in immunoglobulins.
In the second tier, more specific immune function assays can then be utilized, such as determination of the
influence of the agent on NK cell activity and/or on immune function during active immunization, for instance,
the assay of antigen-specific antibody production after sensitization. In some guidelines some of these assays
are already included in the first tier (antibody response to T-cell dependent antigens such as sheep red blood
cells).
The real consequence of immunosuppression can probably be best determined by assessing effects on
resistance against infection in bacterial, viral and/or parasitic animal models, and/or effects on resistance
against tumours. The importance of these types of assays is that they assess the immune system as a
complete and functional entity. However, since it is not practical to evaluate all immunologically relevant
parameters in a single toxicity or immunosuppression study, the most important predictive parameters need to
be identified and a practical approach chosen to assess immunosuppression for a particular agent.
As the general malaise of an individual also affects the immune system, immunosuppression is considered to
exist when immune alterations are detected at dose levels inducing no overt general toxicity. Therefore,
immunosuppression testing is best performed in the context of general toxicity testing, since general toxicity
testing uses a range of doses of an agent and evaluates all major organ systems.
[1]
For the detection of general toxicity of chemicals after sub-acute exposure OECD 407 was recently adapted
to include several immunotoxicological parameters for the determination of an immunotoxic effect of the
compound under investigation.
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ISO/TS 10993-20:2006(E)
Table 2 — Examples of tests for and indicators of the evaluation of immune responses
Non-functional assays
Immune responses Functional assays
a
Soluble mediators Phenotyping Other
Implant/systemic Cell surface Organ weight
Tissue/Inflammatory N.A.
ISO 10993-6 and ISO 10993-11 markers analysis
Immunoassays (e.g. ELISA) for
antibody responses to antigen
b
plus adjuvant
Plaque-forming cells
Complement
(including C3a and
Cell surface
Lymphocyte proliferation
Humoral response
C5a anaphylatoxins)
markers
Antibody dependent cellular
Immune complexes
cytotoxicity
Passive cutaneous anaphylaxis
Direct anaphylaxis
Cellular Responses
Guinea pig maximization test
Mouse local lymph node assay
Cytokine patterns Cell surface
T-cells Mouse ear swelling test indicative of T cell markers (helper and
subset (Th1, Th2) cytotoxic T cells)
Lymphocyte proliferation
Mixed lymphocyte reaction
Cell surface
NK cells Tumour cytotoxicity N.A.
markers
Cytokines (IL1,
Phagocytosis
Macrophages and
TNFα, IL6, TGFβ, MHC markers
other monocytes
Antigen presentation
IL10, γ-interferon)
Cell surface
Dendritic cells Antigen presentation to T-cells N.A.
markers
Vascular endothelial
Activation
cells
Granulocytes Chemokines,
Degranulation
(Basophils, bioactive amines,
N.A. Cytochemistry
Eosinophils, inflammatory
Phagocytosis
Neutrophils) cytokines, enzymes
Resistance to bacteria viruses
Host resistance N.A. N.A.
and tumours
Allergy, skin rash,
urticaria, oedema,
Clinical symptoms N.A. N.A. N.A.
lymphadenopathy,
inflammation
a
Animal models of some human auto-immune diseases are available. However, routine testing for induction of auto-immune
diseases by materials/devices is not recommended.
b
Most commonly used tests. Functional assays are generally more important than tests for soluble mediators or phenotyping.
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ISO/TS 10993-20:2006(E)
6.4 Immunostimulation
Immunostimulation does not, in most cases, lead to diminished resistance to infectious diseases; in contrast,
immunostimulation can have consequences in terms of exacerbation of existing allergic or auto-immune
phenomena.
Assays that are used for detection of immunosuppression are generally also suitable for detection of
immunostimulation. The consequences of exposure to those agents that have been shown to stimulate the
immune system non-specifically can be best studied using animal models in which allergy or auto-immunity is
induced. As is the case with host resistance models, allergy and auto-immunity models are generally quite
cumbersome. There are no validated animal models for testing allergy and auto-immunity, which allow
extrapolation of animal data to humans.
Besides the immunostimulation properties of the material itself, immunostimulation activity of contaminations,
such as pyrogens, shall also be considered, as stated in Annex F of ISO 10993-11:2006.
6.5 Hypersensitivity
Agents can be recognized by the immune system on the basis of their antigenic properties. As such, these
agents can act as allergens, inducing hypersensitivity. The most common forms of hypersensitivity are
delayed-type hypersensitivity (Type IV) and immediate-type hypersensitivity (Type I). There is no good
predictive test for Type I hypersensitivity.
Delayed-type hypersensitivity comprises antigen-specific cellular inflammatory responses. Tests for this are
given in ISO 10993-10.
IgE mediates immediate-type hypersensitivity. Detection of spec
...