ISO/TR 21582:2021
(Main)Pyrogenicity — Principles and methods for pyrogen testing of medical devices
Pyrogenicity — Principles and methods for pyrogen testing of medical devices
This document specifies the principles and methods for pyrogen testing of medical devices and their materials.
Pyrogénicité — Principes et méthodes d'essai pour la recherche des pyrogènes sur les dispositifs médicaux
Le présent document spécifie les principes et méthodes applicables aux essais de pyrogénicité des dispositifs médicaux et des matériaux entrant dans leur composition.
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TECHNICAL ISO/TR
REPORT 21582
First edition
2021-07
Pyrogenicity — Principles and
methods for pyrogen testing of
medical devices
Reference number
ISO/TR 21582:2021(E)
©
ISO 2021
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ISO/TR 21582:2021(E)
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ISO/TR 21582:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Characterization of pyrogen . 3
5.1 General . 3
5.2 Bacterial endotoxin . 3
5.3 Microbial components other than endotoxin . 4
5.4 Pro-inflammatory cytokines . 4
5.5 Chemical agents and other pyrogens . 4
5.6 Principle of febrile reaction . 5
6 Assessment of p yrogenicity. 5
6.1 General . 5
6.2 Bacterial endotoxin test (BET) . 6
6.2.1 General. 6
6.2.2 Principle of LAL reaction . 6
6.2.3 General procedure of BET . 6
6.2.4 Properties of the BET . 6
6.3 Rabbit pyrogen test . 7
6.3.1 General. 7
6.3.2 Principle of the rabbit test . 7
6.3.3 Procedure of the rabbit test. 7
6.3.4 Characteristic of the rabbit test. 8
6.4 Human cell-based pyrogen test (HCPT) . 8
6.4.1 General. 8
6.4.2 Principle of the HCPT . 8
6.4.3 Selection of human cells . 8
6.4.4 Selection of marker cytokine . 9
6.4.5 Procedure of HCPT . 9
6.4.6 Characteristic of the HCPT .10
6.4.7 Validation study .11
7 Conclusion .11
Bibliography .12
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ISO/TR 21582:2021(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 194, Biological and clinical evaluation of
medical devices.
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.
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ISO/TR 21582:2021(E)
Introduction
At present, safety assessments of medical devices are guided by the toxicological and other studies
recommended in the ISO 10993 series of standards.
Material-mediated pyrogenicity represents a systemic effect that is included in of ISO 10993-11:2017,
Annex G, but efforts have been taken to generally address pyrogenicity testing in this document.
A pyrogenic response is the adverse effect of a chemical agent or other substance, such as microbial
component to produce a febrile response. Tests for a pyrogenic response have been required to evaluate
the safety of products that have direct or indirect contact to blood circulation and the lymphatic system,
cerebrospinal fluid (CSF) and interact systemically with human body.
At present, the in vivo rabbit pyrogenicity test and the in vitro bacterial endotoxin test are available
as accepted methods for evaluating the pyrogenicity of medical devices and their materials. Basic
procedures, including sample preparation of each test article, are already established, internationally
harmonized, and mentioned in the related guidelines and pharmacopoeias.
Recently, an in vitro pyrogen test using human immune cells, the human cell-based pyrogen test (HCPT),
has been developed and applied for pyrogen testing of parenteral drugs. The concept of the application
of pyrogen testing for medical devices is being considered due to the direct or indirect exposure to
human blood cells (HCPT).
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TECHNICAL REPORT ISO/TR 21582:2021(E)
Pyrogenicity — Principles and methods for pyrogen testing
of medical devices
1 Scope
This document specifies the principles and methods for pyrogen testing of medical devices and their
materials.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https:// www .electropedia .org/
— ISO Online browsing platform: available at https:// www .iso .org/ obp
3.1
medical device
instrument, apparatus, implement, machine, appliance, implant, in vitro reagent or calibrator, software,
material or other similar or related article, intended by the manufacturer to be used, alone or in
combination, for human beings for one or more of the specific purpose(s) of
— diagnosis, prevention, monitoring, treatment or alleviation of disease;
— diagnosis, monitoring, treatment, alleviation of or compensation for an injury;
— investigation, replacement, modification, or support of the anatomy or of a physiological process;
— supporting or sustaining life;
— control of conception;
— disinfection of medical devices;
— providing information by means of in vitro examination of specimens derived from the human body;
and does not achieve its primary intended action by pharmacological, immunological or metabolic
means, in or on the human body, but which may be assisted in its function by such means.
Note 1 to entry: Products which may be considered to be medical devices in some jurisdictions but not in others
include:
— disinfection substances;
— aids for persons with disabilities;
— devices incorporating animal and/or human tissues;
— devices for in vitro fertilization or assisted reproduction technologies.
[SOURCE: GHTF/SG1/N071: 2012, 5.1]
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ISO/TR 21582:2021(E)
3.2
pyrogen
substance that causes fever
3.3
pyrogenicity
ability of a chemical agent or other substance to produce a febrile response
3.4
febrile response
temperature above the normal range due to an increase in the body’s temperature set point
Note 1 to entry: It is also referred to as fever or pyrexia.
3.5
oxidative phosphorylation
metabolic pathway in most aerobic organisms, which uses enzymes to oxidise nutrients to release
energy
4 Abbreviated terms
COX Enzyme cyclooxygenase
CpG Cytosine (C) next to guanine (G) in the DNA sequence, with the p indicating that C and G are con-
nected by a phosphodiester bond.methyl group to the 5 position of the cytosine pyrimdine ring
ELISA Enzyme llinked immunosorbent assay HCPT, e.g. monocyte activation test (MAT)
IKK IkB kinase, an enzyme complex involved in propagating cellular response to inflammation
IRAK Interleukin-1 receptor-associated kinase
LAL Limulus amebocyte lysate
LPS Lipopolysaccharide
MD-2 Molecule secreted glycoprotein that binds to extracellular domain of TLR4
MCP Macrophage chemotactic protein
MIP Macrophage inflammatory protein
NOD Nucleotide-binding oligomerization domain
PGE Prostaglandin E
2 2
RANTES Regulated on activation, normal T-expressed and Secreted
RNA Ribonucleic acid
SEA Staphylococcal enterotoxin A
Spe C Streptococcal pyrogenic exotoxin C
Spe F Streptococcal pyrogenic exotoxin F
TBK TANK binding kinase
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TLR Toll-like receptor
TNF Tumour necrosis factor
TSST Toxic shock syndrome toxin
5 Characterization of pyrogen
5.1 General
On the basis of pyrogen origin, febrile response can be divided into three groups:
a) material-mediated pyrogenicity caused by chemical agents;
b) endotoxin-mediated pyrogenicity;
c) pyrogenicity mediated by microbial components other than endotoxin.
Non-endotoxin-mediated pyrogenicity corresponds to a generic name of febrile responses originating
from a) and c) above. However, the latter can be clearly distinguished from material-mediated
pyrogenicity, because the febrile reaction is originated from microbial contamination.
TLRs are proteins that constitute an important part of the immune system against microbial infections,
closely relate to pyrogenicity of microbial components. Thirteen kinds of human TLRs from TLR1
to TLR13 and the agonists to some of them have been identified to date. Most pyrogens that can be
assessed in the field of medical devices can be bioactive substances derived from microorganisms
present as contaminants of the device manufacturing process or present in materials. Since the
components are TLR agonists and act as pyrogen to human, the knowledge for TLRs is very significant
for understanding pyrogens.
5.2 Bacterial endotoxin
Bacterial endotoxin, an important component of the outer membrane of Gram-negative bacteria, is the
most powerful pyrogen recognized by TLR4. Endotoxin is a modulator of the host immune response
and exhibits a variety of biological activities, for example, activation of macrophages, mitogenicity and
adjuvanticity, causing Schwartzman reactions in addition to pyrogenicity. From the clinical standpoint,
endotoxin causes sepsis, septic shock and multiple organ failure, which are systemic disorders with a
high mortality rate.
Endotoxin generally consists of a heteropolysaccharide part subdivided into an O-specific chain, a core
oligosaccharide, and a lipid component called lipid A that is a biologically active centre of endotoxin.
The potency of endotoxin is influenced by acylation and phosphorylation patterns, and the presence/
absence of polar-head group bound to phosphate residue in lipid A molecule. In addition, endotoxin has
species-specificity for the expression of its bioactivity.
In the natural world, Gram-negative bacteria are widely distributed in water (rivers and sea), air, soil, and
also human body. It is likely therefore that biomaterials made of natural substances are contaminated
with the bacteria and their components. Autoclaving, irradiation and gas sterilization during the
manufacturing process are able to kill the bacteria. However, microbial components, particularly
endotoxin cannot be inactivated by such ordinary sterilization methods, and once contaminated it is
quite difficult to remove the endotoxin during the manufacturing process. During the manufacturing
process, endotoxin contamination can be reduced or eliminated by depyrogenization (e.g. 250 °C for
[50][66]
30 min, use of chemicals to inactivate endotoxin such as polymyxin-B or by using endotoxin-free
water in the washing and manufacturing processes.
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5.3 Microbial components other than endotoxin
Microorganisms produce various bioactive substances other than endotoxins. Lipoteichoic acid, an
important component of the outer membrane of Gram-positive bacteria, represents a counterpart to
endotoxin and acts as a pyrogen recognized by TLR2 that interacts and forms a heterodimer with TLR1
or TLR6. Lipoproteins, lipopeptides, and lipoarabinomannan that are the cellular components of various
microorganisms are also known to act as TLR2 agonists. Although peptidoglycan that constructs
cell wall of Gram-positive and Gram-negative bacteria was considered as TLR2 agonist, it is recently
suggested that NOD1 and NOD2 proteins can play a role of mediating the expression of its bioactivity
rather than TLR2. In addition, viral double-stranded RNA, bacterial flagella, and bacterial and viral CpG
DNA have been identified as the agonists of TLR3, TLR5, and TLR9, respectively, and all of them acts
as pyrogens to human. Although pyrogenicity has not been reported for any kind of (1,3)-β -D-glucan
preparation, it can be noted that certain kinds of (1,3)-β -D-glucan can enhance endotoxin toxicity.
It has been reported that exotoxins and enterotoxins such as TSST-1, SEA, Spe F, and Spe C produced
by various pathogenic microorganisms cause febrile response in the human body by the toxin-specific
manner that can be different from TLR signal transduction. There was an outbreak of inflammation,
fever and peritonitis in some patients due to contamination of solution with peptidoglycan during
[52][76]
dialysis .
5.4 Pro-inflammatory cytokines
Since febrile responses induced by TLR agonists are mediated by pro-inflammatory cytokines such as
TNFα, IL-1β, IL-6, and INF-γ produced by human immune cells, the endogenous mediator itself naturally
acts as pyrogen. Each cytokine further activates immune cells through the cytokine network, because
receptors specific to the cytokines are located on the cell surface of monocytes and macrophages in
addition to TLRs.
5.5 Chemical agents and other pyrogens
Pyrogenicity of chemicals or natural substances other than microbial components is not well known.
In addition, over 1 000 new compounds are discovered or synthesized each year worldwide, but the
biological properties of each compound are not well understood. Most chemicals currently used as
biomaterials for medical devices, are safe and are non-pyrogenic to humans. However, it is possible that
some new biomaterials and chemicals can cause febrile reaction to human.
This possibility also holds true for non-autologous cellular products which can evoke immunological
recognition and activation of immune-competent cells.
As an example, chemicals that are known to induce febrile reaction in humans are listed below. These
chemicals can be divided mainly into three groups according to principle for inducing a febrile response:
a) agents that directly stimulate thermoregulatory centres of the brain and nervous system,
b) uncoupling agents of oxidative phosphorylation, and
c) pyrogens with mechanisms that are not well known.
The chemicals listed below are known to cause a febrile response in humans:
— prostaglandins;
— inducers (e.g. polyadenylic, polyuridylic, polybionosinic, and polyribocytidylic acids);
— substances disrupting the function of thermoregulatory centres (e.g. lysergic acid diethylamide,
cocaine, morphine);
— neurotransmitters (e.g. noradrenaline, serotonin);
— uncoupling agents of oxidative phosphorylation (e.g. 4, 6-dinitro-o-cresol, dinitrophenol, picric
acid);
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ISO/TR 21582:2021(E)
— N-phenyl-β -naphthylamine and aldo-α -naphthylamine (the febrile mechanism is unknown);
— metals such as nickel salts, in some instances.
[23] [61]
In addition to these chemicals, there is a possibility that microspheres and nanoparticles, including
[7] [23] [61]
implant-derived wear debris, can act as pyrogens. Microspheres, particles and nanoparticles
consisting of specific sizes could be phagocytosed by macrophages and activate macrophage-released
pro-inflammatory cytokines such as TNFα. TNFα is one of the endogenous pyrogens.
5.6 Principle of febrile reaction
TLRs are a class of single membrane-spanning non-catalytic receptors that recognize structurally
conserved molecules derived from microbes once they have breached physical barriers such as the skin
or intestinal tract mucosa and activate immune cells. They are believed to play a key role in the innate
immune system and are known to function as dimers. Although most TLRs appear to act as homodimers,
TLR2 forms heterodimers with TLR1 or TLR6, each dimer having different ligand specificity. TLRs can
also depend on other co-receptors for full ligand sensitivity, such as in the case of TLR4's recognition
of endotoxin, which requires a MD-2 molecule. CD14 and LPS binding protein are known to facilitate
the presentation of endotoxin to MD-2. When activated, TLRs recruit adapter molecules within the
cytoplasm of cells in order to propagate a signal. Four adapter molecules are known to be involved in
signalling. These proteins are known as MyD88, Tirap (also called Mal), Trif, and Tram. The adapters
activate other molecules within the cell, including certain protein kinases (IRAK1, IRAK4, TBK1, and
IKKi) that amplify the signal, and ultimately lead to the induction or suppression of genes (NF-κB, AP-1,
and IRP3) that orchestrate the inflammatory response.
Following activation by ligands of microbial origin, several reactions are possible. Immune cells can
produce cytokines that trigger inflammation. Particularly, IL-1β is closely associated with induction of
febrile reaction. IL-6 and TNFα were isolated later and found to be pyrogenic cytokines as well, although
[28],[29]
at much higher doses . The current understanding of the mechanism of fever in mammals is that
these proinflammatory cytokines result in the expression of the COX-2 which mediates PGE synthesis.
2
[30] [47] [48]
Mice deficient in COX-2 do not develop fever in response to LPS, IL-1 or IL-6 , and PGE triggers
2
an intracellular signalling cascade that changes the set point of body temperature. Thus, IL-1β, IL-6 and
TNFα are the mediators released by immune cells upon contact with pyrogens that are responsible for
triggering the fever reaction in the brain. Substance P is known to induce fever through the production
[17]
of TNF-α, IL-6 and PGE2 .
TLRs seem to be involved in the cytokine production and cellular activation as well as in the adhesion
and phagocytosis of microorganisms and other potential pyrogens.
Independent of TLR signalling pathway and subsequent cytokine production, body temperature could
be increased by agents that directly stimulate thermoregulatory centres. Also, uncoupling agents
of oxidative phosphorylation can increase body temperature as a result of activating the electron
transport chain in mitochondria.
6 Assessment of p yrogenicity
6.1 General
There are three methods used for pyrogenicity testing, which are described below. The in vivo rabbit
pyrogen test is the only test that directly measures the febrile response in the body as an end point,
in accordance with the definition of a pyrogen, which the other two methods do not. Instead the in
vitro methods detect pyrogens using different end points, such as cytokine production and protein
coagulation.
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ISO/TR 21582:2021(E)
6.2 Bacterial endotoxin test (BET)
6.2.1 General
The bacterial endotoxin test is harmonized with several pharmacopaeia. This test is to detect or
quantify bacterial endotoxin of Gram-negative bacterial origin using a lysate reagent that is an aqueous
extract of circulating amebocytes of the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).
The bacterial endotoxin test is technically divided into two methods; one is the gel-clot technique based
on gel formation by the reaction of the lysate reagent with endotoxins, and other is the photometric
technique originating from endotoxin-induced optical changes of the lysate reagent. The latter is further
subdivided into two methods: one is turbidimetric technique measuring the endotoxin concentrations
of sample solutions based on the measurement of turbidity change accompanying the gel formation, and
other is chromogenic technique estimating the endotoxin concentrations by measuring optical density
of the colour of chromophore released from a synthetic chromogenic substrate that is a substituent
for the final step of the enzymatic cascade reaction described below. Each photometric technique is
classified as either end point or kinetic method.
The bacterial endotoxin test can be used to monitor endotoxin contamination in manufacturing
processes of medical devices and the final products from the viewpoint of routine quality control.
Before starting use of the lysate reagent extracted from Tachypleus tridentatus, this test was termed
Limulus amoebocyte lysate (LAL) test in the past.
NOTE Other methods are available for the detection of bacterial endotoxins, for example, the fluorescent
method using recombinant Factor C, see Reference [6].
6.2.2 Principle of LAL reaction
The BET is an enzymatic cascade reaction that has the highest sensitivity to detect and quantify Gram-
negative bacterial endotoxins. First, factor C, endotoxin-sensitive serine protease zymogen present
in the lysate reagent, is activated by endotoxin. The activated factor C converts factor B from the
inactive form to the active form that further converts proclotting enzyme to clotting enzyme. Finally,
the clotting enzyme converts coagulogen to coagulin that leads to gel formation. In addition to factor
C, original lysate reagent contains factor G that is activated by (1,3)-β-D-glucans and subsequently
converts proclotting enzyme to clotting enzyme. Endotoxin-specific LAL reagent has been developed
by removing factor G or saturating its function.
Since the BET is based on an enzymatic reaction, it is influenced by the temperature and pH of sample
solution, and it is also enhanced or inhibited by various compounds such as protease, protease inhibitors,
metal ions, surfactants, chelates, salts and sugars if they are present in sufficient concentrations.
Therefore, tests for interfering factors can be performed to check the presence of inhibitors and
enhancers of the reaction in sample solution. The effect of the interfering factors can be avoided by the
dilution of the sample solution.
6.2.3 General procedure of BET
General methods for detection and quantification of endotoxin have been discussed in pharmacopoeias
in several countries and AAMI ST 72. The details are referred to in these documents.
It is noted that pyrogen-free water can be used as an extraction medium for preparing sample solution
unless otherwise specified. Endotoxin present in medical devices and their materials typically are
extracted at the ambient temperature.
6.2.4 Properties of the BET
The LAL assay is available as a simple and quick test with high-sensitively detect to endotoxin, indicating
the presence of Gram-negative bacterial contaminat
...
RAPPORT ISO/TR
TECHNIQUE 21582
Première édition
2021-07
Pyrogénicité — Principes et méthodes
d'essai pour la recherche des
pyrogènes sur les dispositifs médicaux
Pyrogenicity — Principles and methods for pyrogen testing of medical
devices
Numéro de référence
ISO/TR 21582:2021(F)
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ISO/TR 21582:2021(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2021
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Publié en Suisse
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ISO/TR 21582:2021(F)
Sommaire Page
Avant-propos .iv
Introduction .v
1 Domaine d'application .1
2 Références normatives .1
3 Termes et définitions . 1
4 Abréviations . 2
5 Caractérisation des pyrogènes .3
5.1 Généralités . 3
5.2 Endotoxine bactérienne . 3
5.3 Composants microbiens autres que l'endotoxine . 4
5.4 Cytokines pro-inflammatoires . 4
5.5 Agents chimiques et autres pyrogènes . 4
5.6 Principe de la réaction fébrile . 5
6 Évaluation de la pyrogénicité . .6
6.1 Généralités . 6
6.2 Essai de détection des endotoxines bactériennes (BET) . 6
6.2.1 Généralités . 6
6.2.2 Principe de la réaction LAL . . 6
6.2.3 Caractéristiques générales de l'essai BET. 7
6.2.4 Propriétés de l'essai BET . 7
6.3 Essai de pyrogénicité sur le lapin . 7
6.3.1 Généralités . 7
6.3.2 Principe de l'essai sur le lapin . 8
6.3.3 Mode opératoire de l'essai sur le lapin. 8
6.3.4 Caractéristiques de l'essai sur le lapin . 8
6.4 Essai de détection des pyrogènes à base de cellules humaines (HCPT) . 8
6.4.1 Généralités . 8
6.4.2 Principe de l'essai HCPT . 9
6.4.3 Sélection des cellules humaines . 9
6.4.4 Sélection du marqueur cytokine . 10
6.4.5 Mode opératoire de l'essai HCPT . 10
6.4.6 Caractéristiques de l'essai HCPT . 11
6.4.7 Étude de validation .12
7 Conclusion .12
Bibliographie .13
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ISO/TR 21582:2021(F)
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes
nationaux de normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est
en général confiée aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude
a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,
gouvernementales et non gouvernementales, en liaison avec l'ISO participent également aux travaux.
L'ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui
concerne la normalisation électrotechnique.
Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont
décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier de prendre note des différents
critères d'approbation requis pour les différents types de documents ISO. Le présent document
a été rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2
(voir www.iso.org/directives).
L'attention est attirée sur le fait que certains des éléments du présent document peuvent faire l'objet de
droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable
de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant
les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de
l'élaboration du document sont indiqués dans l'Introduction et/ou dans la liste des déclarations de
brevets reçues par l'ISO (voir www.iso.org/brevets).
Les appellations commerciales éventuellement mentionnées dans le présent document sont données
pour information, par souci de commodité, à l'intention des utilisateurs et ne sauraient constituer un
engagement.
Pour une explication de la nature volontaire des normes, la signification des termes et expressions
spécifiques de l'ISO liés à l'évaluation de la conformité, ou pour toute information au sujet de l'adhésion
de l'ISO aux principes de l'Organisation mondiale du commerce (OMC) concernant les obstacles
techniques au commerce (OTC), voir www.iso.org/avant-propos.
Le présent document a été élaboré par le comité technique ISO/TC 194, Évaluation biologique et clinique
des dispositifs médicaux.
Il convient que l'utilisateur adresse tout retour d'information ou toute question concernant le présent
document à l'organisme national de normalisation de son pays. Une liste exhaustive desdits organismes
se trouve à l'adresse www.iso.org/fr/members.html.
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ISO/TR 21582:2021(F)
Introduction
Actuellement, la sécurité des dispositifs médicaux est évaluée sur la base des études toxicologiques et
autres travaux recommandés dans la série de normes ISO 10993.
La pyrogénicité due aux matériaux constitue un effet systémique qui est inclus dans l'Annexe G de
l'ISO 10993-11:2017; cependant, des mesures ont été prises afin que les essais de pyrogénicité soient
appréhendés d'une manière générale dans le présent document.
Une réponse pyrogénique correspond à l'effet indésirable d'un agent chimique ou d'une autre substance,
telle qu'un composant microbien, qui produit une réponse fébrile. Les essais sur la réponse pyrogénique
sont nécessaires pour évaluer la sécurité des produits qui se trouvent au contact direct ou indirect de la
circulation sanguine et du système lymphatique, du liquide cérébrospinal (LCS) et qui interagissent de
manière systémique avec le corps humain.
Actuellement, les méthodes acceptées pour évaluer la pyrogénicité des dispositifs médicaux et de leurs
matériaux sont l'essai de pyrogénicité in vivo sur le lapin et l'essai in vitro de détection des endotoxines
bactériennes. Les modes opératoires de base, y compris la préparation des échantillons de chaque
article d'essai, sont déjà établis, harmonisés au niveau international et mentionnés dans les lignes
directrices et les pharmacopées correspondantes.
Récemment, un essai de détection de pyrogènes in vitro utilisant des cellules immunitaires humaines,
appelé «essai de détection des pyrogènes à base de cellules humaines (HCPT)», a été développé et
appliqué pour détecter les pyrogènes dans les médicaments parentéraux. Le concept de l'application
de l'essai pour la recherche des pyrogènes sur les dispositifs médicaux est examiné en fonction de
l'exposition directe ou indirecte aux cellules sanguines humaines (HCPT).
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RAPPORT TECHNIQUE ISO/TR 21582:2021(F)
Pyrogénicité — Principes et méthodes d'essai pour la
recherche des pyrogènes sur les dispositifs médicaux
1 Domaine d'application
Le présent document spécifie les principes et méthodes applicables aux essais de pyrogénicité des
dispositifs médicaux et des matériaux entrant dans leur composition.
2 Références normatives
Le présent document ne contient aucune référence normative.
3 Termes et définitions
Pour les besoins du présent document, les termes et définitions suivants s'appliquent.
L'ISO et l'IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en
normalisation, consultables aux adresses suivantes:
— ISO Online browsing platform: disponible à l'adresse https:// www .iso .org/ obp
— IEC Electropedia: disponible à l'adresse https:// www .electropedia .org/
3.1
dispositif médical
instrument, appareil, outil, machine, dispositif, implant, réactif ou calibrateur in vitro, logiciel, matériel
ou autre article similaire ou associé, dont le fabricant prévoit qu'il soit utilisé, seul ou en association
chez l'être humain pour la (les) fin(s) spécifique(s) suivante(s):
— diagnostic, prévention, contrôle, traitement ou atténuation d'une maladie;
— diagnostic, contrôle, traitement, atténuation ou compensation d'une blessure;
— étude, remplacement, modification ou soutien de l’anatomie ou d’un processus physiologique;
— soutien ou maintien de la vie;
— maîtrise de la conception;
— désinfection des dispositifs médicaux;
— communication d'informations par un examen in vitro de spécimens (prélèvements) provenant du
corps humain;
et dont l'action principale voulue n'est pas obtenue par des moyens pharmacologiques ou
immunologiques ni par métabolisme, dans le corps humain ou à la surface de celui-ci, mais dont la
fonction peut être assistée par de tels moyens
Note 1 à l'article: Les produits pouvant être considérés comme des dispositifs médicaux dans certaines
juridictions, mais pas dans d’autres, incluent:
— les produits désinfectants;
— les aides pour les personnes handicapées;
— les dispositifs intégrant des tissus animaux et/ou humains;
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ISO/TR 21582:2021(F)
— les dispositifs pour les technologies de fécondation in vitro et de reproduction assistée.
[SOURCE: GHTF/SG1/N071: 2012, 5.1]
3.2
pyrogène
substance qui génère de la fièvre
3.3
pyrogénicité
capacité, de la part d'un agent chimique ou d'une autre substance, de produire une réponse fébrile
3.4
réponse fébrile
température supérieure à la normale due à une élévation du point d'équilibre thermique du corps
Note 1 à l'article: Elle est également appelée fièvre ou pyrexie.
3.5
phosphorylation oxydative
voie métabolique de la plupart des organismes aérobies, qui utilise des enzymes pour oxyder les
nutriments et libérer de l'énergie
4 Abréviations
COX Cyclooxygénase
CpG Cytosine (C) précédant une guanine (G) dans le segment d'ADN, le p indiquant que C et G sont
reliées par une liaison phosphodiester.groupe méthyle en position 5 du cycle pyrimidine de
la cytosine
ELISA Essai HCPT réalisé par dosage d'immunoabsorption par enzyme liée, par exemple, essai d'acti-
vation des monocytes (MAT)
IKK Kinase IkB, qui est un complexe enzymatique impliqué dans la propagation de la réponse
cellulaire à l'inflammation
IRAK Kinase associée au récepteur de l'interleukine-1
LAL Lysat d'amébocytes de limule
LPS Lipopolysaccharide
MD-2 Glycoprotéine sécrétée qui se lie au domaine extracellulaire du TLR4
MCP Protéine chimiotactique des macrophages
MIP Protéine inflammatoire des macrophages
NOD Domaine d'oligomérisation de liaison aux nucléotides
PGE Prostaglandine E
2 2
RANTES Regulated on Activation, Normal T-Expressed and Secreted
ARN Acide ribonucléique
SEA Entérotoxine staphylococcique A
Spe C Exotoxine pyrogène streptococcique C
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ISO/TR 21582:2021(F)
Spe F Exotoxine pyrogène streptococcique F
TBK Kinase de liaison à TANK
TLR Récepteur de type Toll
TNF Facteur de nécrose tumorale
TSST Toxine du syndrome de choc toxique
5 Caractérisation des pyrogènes
5.1 Généralités
En fonction de l'origine des pyrogènes, la réponse fébrile peut être classée en trois groupes:
a) pyrogénicité due aux matériaux, causée par des agents chimiques;
b) pyrogénicité due aux endotoxines;
c) pyrogénicité due à des composants microbiens autres que les endotoxines.
La pyrogénicité due à des composants autres que les endotoxines correspond à un nom générique de
réponses fébriles au sens des alinéas a) et c) ci-dessus. Toutefois, cette dernière peut être clairement
distinguée de la pyrogénicité due aux matériaux, car la réaction fébrile provient d'une contamination
microbienne.
Les TLR sont des protéines qui constituent une part importante du système immunitaire face aux
infections microbiennes et qui sont étroitement liées à la pyrogénicité des composants microbiens.
Treize types de TLR humains (TLR1 à TLR13) et les agonistes de certains d'entre eux ont été identifiés
à ce jour. La plupart des pyrogènes identifiables dans le domaine des dispositifs médicaux peuvent
être des substances bioactives issues de micro-organismes, présents en tant que contaminants lors
du processus de fabrication du dispositif ou présents dans les matériaux. Comme les composants sont
des agonistes des TLR et agissent comme des pyrogènes chez l'homme, la connaissance des TLR est
essentielle pour comprendre les pyrogènes.
5.2 Endotoxine bactérienne
L'endotoxine bactérienne, qui constitue la majeure partie de la membrane externe des bactéries à Gram
négatif, est le pyrogène le plus puissant reconnu par le TLR4. L'endotoxine est un modulateur de la
réponse immunitaire de l'hôte et présente une variété d'activités biologiques, telles que l'activation des
macrophages, la mitogénicité et l'adjuvanticité, provoquant des réactions de Shwartzman en plus de
la pyrogénicité. D'un point de vue clinique, l'endotoxine provoque un sepsis, un choc septique et une
défaillance multiorganique, qui sont des troubles systémiques avec un taux de mortalité élevé.
L'endotoxine se compose généralement d'une partie hétéropolysaccharidique qui se subdivise en une
chaîne O-spécifique, un cœur oligosaccharidique et un composant lipidique appelé lipide A, qui est un
centre biologiquement actif de l'endotoxine. La puissance de l'endotoxine est influencée par les degrés
d'acylation et de phosphorylation, ainsi que par la présence ou l'absence d'un groupe de tête polaire lié
à un résidu phosphate dans la molécule de lipide A. Enfin, l'endotoxine présente une spécificité d'espèce
en ce qui concerne l'expression de sa bioactivité.
Dans le monde de la nature, les bactéries à Gram négatif sont largement répandues dans l'eau (fleuves
et mer), l'air, le sol ainsi que le corps humain. Il est donc probable que les biomatériaux fabriqués à
partir de substances naturelles soient contaminés par des bactéries et leurs composants. L'autoclavage,
l'irradiation et la stérilisation au gaz, utilisés au cours du processus de fabrication, sont à même de
tuer les bactéries. Cependant, les composants microbiens, notamment l'endotoxine, ne peuvent pas
être inactivés par ces méthodes de stérilisation classiques, car, une fois qu'il y a eu contamination, il
est assez difficile d'éliminer l'endotoxine au cours du processus de fabrication. Lors du processus de
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ISO/TR 21582:2021(F)
fabrication, il est possible de réduire ou d'éliminer la contamination par endotoxine en procédant à une
dépyrogénisation (par exemple, 250 °C pendant 30 minutes), en utilisant des substances chimiques
[50],[66]
permettant d’inactiver les endotoxines, telles que la polymyxine B , ou en utilisant une eau
exempte d'endotoxines dans les processus de lavage et de fabrication.
5.3 Composants microbiens autres que l'endotoxine
Les micro-organismes produisent de nombreuses autres substances bioactives que des endotoxines.
L'acide lipoteichoïque, important composant de la membrane externe des bactéries à Gram positif,
représente une contrepartie de l'endotoxine et agit comme un pyrogène reconnu par le TLR2 qui
interagit et forme un hétérodimère avec le TLR1 ou le TLR6. Les lipoprotéines, les lipopeptides et les
lipoarabinomannanes qui sont les composants cellulaires de divers micro-organismes sont également
connus pour agir comme agonistes du TLR2. Bien que le peptidoglycane, qui constitue la paroi
cellulaire des bactéries à Gram positif et à Gram négatif, ait été considéré comme un agoniste du TLR2,
il a récemment été évoqué que les protéines NOD1 et NOD2 pouvaient jouer un rôle de médiateur dans
l'expression de sa bioactivité plutôt que le TLR2. D'autre part, l'ARN viral double brin, les flagelles
bactériens et l'ADN CpG bactérien et viral ont été identifiés comme étant, respectivement, des agonistes
du TLR3, TLR5 et TLR9, et tous agissent comme des pyrogènes pour l'homme. Bien qu'il n'y ait pas eu de
pyrogénicité rapportée pour un quelconque type de préparation de (1,3)-β -D-glucane, il est à noter que
certains types de (1,3)-β -D-glucane peuvent renforcer la toxicité des endotoxines.
Il a été observé que les exotoxines et les entérotoxines telles que TSST-1, SEA, Spe F et Spe C produites
par divers micro-organismes pathogènes provoquent une réponse fébrile du corps humain, d'une
manière spécifique de la toxine qui peut être différente de la transduction du signal du TLR. On a noté
des cas d'inflammation, de fièvre et de péritonite chez certains patients en raison de la contamination
[52],[76]
de la solution par le peptidoglycane en cours de dialyse .
5.4 Cytokines pro-inflammatoires
Étant donné que les réponses fébriles induites par les agonistes des TLR sont médiées par des cytokines
pro-inflammatoires (telles que le TNFα, l’IL-1β, l’IL-6 et l’INF-γ) produites par les cellules immunitaires
humaines, le médiateur endogène, lui-même, agit naturellement comme un pyrogène. Chaque cytokine
active à son tour les cellules immunitaires par le biais du réseau de cytokines, car les récepteurs
spécifiques des cytokines se trouvent à la surface des cellules des monocytes et des macrophages, en
plus des TLR.
5.5 Agents chimiques et autres pyrogènes
La pyrogénicité des substances chimiques ou des substances naturelles autres que les composants
microbiens n'est pas encore bien connue. En outre, plus de 1 000 nouveaux composés sont découverts
ou synthétisés chaque année dans le monde, mais les propriétés biologiques de chaque composé ne sont
pas bien comprises. La plupart des substances chimiques actuellement utilisées comme biomatériaux
pour les dispositifs médicaux sont sûres et non pyrogènes pour l'homme. Toutefois, il est possible que
certains nouveaux biomatériaux et substances chimiques puissent provoquer une réaction fébrile chez
l'homme.
Cette possibilité vaut également pour les produits cellulaires non autologues qui peuvent déclencher
une reconnaissance immunologique et une activation des cellules immunocompétentes.
À titre d'exemple, les substances chimiques connues pour induire une réaction fébrile chez l'homme sont
énumérées ci-dessous. Ces substances chimiques peuvent être divisées en trois groupes principaux,
selon le principe de déclenchement de la réponse fébrile:
a) agents stimulant directement les centres thermorégulateurs du cerveau et du système nerveux;
b) agents de découplage de la phosphorylation oxydative; et
c) pyrogènes dont les mécanismes ne sont pas bien connus.
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Les substances chimiques énumérées ci-dessous sont connues pour entraîner une réponse fébrile chez
l'homme:
— prostaglandines;
— inducteurs (par exemple, acides polyadénylique, polyuridylique, polybionosinique et
polyribocytidylique);
— substances empêchant le fonctionnement des centres thermorégulateurs (par exemple, diéthylamide
de l'acide lysergique, cocaïne, morphine);
— neurotransmetteurs (par exemple, noradrénaline, sérotonine);
— agents découplants de la phosphorylation oxydative (par exemple, 4,6-dinitro-o-crésol,
dinitrophénol, acide picrique);
— N-phenyl-β -naphtylamine et aldo-α -naphtylamine (mécanisme fébrile inconnu);
— métaux tels que les sels de nickel, dans certains cas.
[23] [61]
Hormis ces substances chimiques, il est possible que des microsphères et des nanoparticules ,
[7]
y compris des débris d'usure provenant d'implants , puissent agir comme des pyrogènes. Les
[23] [61]
microsphères, les particules et les nanoparticules de taille spécifique pourraient être phagocytées
par les macrophages et activer des cytokines pro-inflammatoires libérées par les macrophages, comme
le TNFα. Le TNFα est l'un des pyrogènes endogènes.
5.6 Principe de la réaction fébrile
Les TLR sont une classe de récepteurs transmembranaires simples, non catalytiques, qui reconnaissent
les structures moléculaires conservées des microbes, une fois que ceux-ci ont franchi des barrières
physiques, telles que la peau ou la muqueuse du tractus intestinal, et activent les cellules immunitaires.
Ils sont censés jouer un rôle clé dans le système immunitaire inné et sont connus pour fonctionner en
tant que dimères. Bien que la plupart des TLR semblent fonctionner comme des homodimères, TLR2
forme des hétérodimères avec le TLR1 ou TLR6, chaque dimère présentant une spécificité différente
par rapport aux ligands. Les TLR peuvent également dépendre d'autres corécepteurs pour atteindre
une sensibilité totale aux ligands, comme dans le cas de la reconnaissance de l'endotoxine par le TLR4,
qui nécessite une molécule MD-2. Le CD14 et la protéine de liaison au LPS sont connus pour faciliter
la présentation de l'endotoxine à MD-2. Lorsqu'ils sont activés, les TLR recrutent des molécules
adaptatrices dans le cytoplasme des cellules afin de propager un signal. Quatre molécules adaptatrices
sont connues pour être impliquées dans la signalisation. Ces protéines sont connues sous le nom
de MyD88, Tirap (également appelée Mal), Trif et Tram. Les adaptateurs activent d'autres molécules au
sein de la cellule, notamment certaines protéines kinases (IRAK1, IRAK4, TBK1 et IKKi) qui amplifient
le signal et conduisent finalement à l'induction ou à la suppression de gènes (NF-κB, AP-1 et IRP3) qui
orchestrent la réponse inflammatoire.
Après activation par des ligands d'origine microbienne, plusieurs réactions sont possibles. Les cellules
immunitaires peuvent produire des cytokines qui déclenchent une inflammation. En particulier,
l'IL-1β est étroitement associée à l'induction de la réaction fébrile. L'IL-6 et le TNFα ont été isolés
plus tardivement et se sont révélés être également des cytokines pyrogéniques, bien qu'à des doses
[28],[29]
beaucoup plus élevées . Au stade actuel de la compréhension du mécanisme de la fièvre chez les
mammifères, il semble que ces cytokines pro-inflammatoires entraînent l'expression de la COX-2 qui
[30]
induit la synthèse de la PGE . Les souris présentant une déficience en COX-2 ne développent pas
2
[47] [48]
de fièvre en réponse au LPS, l’IL-1 ou l’IL-6 , et la PGE déclenche une cascade de signalisation
2
intracellulaire qui modifie le point d'équilibre thermique du corps. Ainsi, l’IL-1β, l’IL-6 et le TNFα sont
les médiateurs libérés par les cellules immunitaires au contact des pyrogènes qui sont responsables du
déclenchement de la réaction fébrile dans le cerveau. La substance P est connue pour induire la fièvre
[17]
par la production de TNF-α, IL-6 et PGE .
2
Les TLR semblent être impliqués dans la production de cytokines et l'activation cellulaire, ainsi que
dans l'adhésion et la phagocytose de micro-organismes et autres pyrogènes potentiels.
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Indépendamment de la voie de signalisation des TLR et de la production ultérieure de cytokines, il
se pourrait que des agents stimulant directement les centres de thermorégulation augmentent la
température corporelle. De même, les agents de découplage de la phosphorylation oxydative peuvent
faire monter la température corporelle en raison de l'activation de la chaîne de transport des électrons
dans les mitochondries.
6 Évaluation de la pyrogénicité
6.1 Généralités
Il existe trois méthodes utilisées pour les essais de pyrogénicité, qui sont décrites ci-dessous. L'essai de
pyrogénicité in vivo sur le lapin est le seul essai qui mesure directement la réponse fébrile de l'organisme
en tant que point final, conformément à la définition d'un pyrogène, ce qui n’est pas le cas pour les deux
autres méthodes. À l'inverse, les méthodes in vitro détectent les pyrogènes à l’aide de différents points
finaux, tels que la production de cytokines ou la coagulation des protéines.
6.2 Essai de détection des endotoxines bactériennes (BET)
6.2.1 Généralités
L'essai de détection des endotoxines bactériennes est harmonisé avec plusieurs pharmacopées. Cet
essai consiste à détecter ou à quantifier les endotoxines bactériennes issues de bactéries à Gram
négatif à l'aide d'un réactif de lysat, qui est un extrait aqueux d'amébocytes circulants de limule
(Limulus polyphemus ou Tachypleus tridentatus). L'essai de détection de
...
TECHNICAL ISO/TR
REPORT 21582
First edition
Pyrogenicity — Principle and method
for pyrogen testing of medical devices
PROOF/ÉPREUVE
Reference number
ISO/TR 21582:2021(E)
©
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ISO/TR 21582:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
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Published in Switzerland
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ISO/TR 21582:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Characterization of pyrogen . 3
5.1 General . 3
5.2 Bacterial endotoxin . 3
5.3 Microbial components other than endotoxin . 4
5.4 Pro-inflammatory cytokines . 4
5.5 Chemical agents and other pyrogens . 4
5.6 Principle of febrile reaction . 5
6 Assessment of p yrogenicity. 5
6.1 General . 5
6.2 Bacterial endotoxin test . 6
6.2.1 General. 6
6.2.2 Principle of LAL reaction . 6
6.2.3 General procedure of BET . 6
6.2.4 Properties of the BET . 6
6.3 Rabbit pyrogen test . 7
6.3.1 General. 7
6.3.2 Principle of the rabbit test . 7
6.3.3 Procedure of the rabbit test. 7
6.3.4 Characteristic of the rabbit test. 8
6.4 Human cell-based pyrogen test . 8
6.4.1 General. 8
6.4.2 Principle of the HCPT . 8
6.4.3 Selection of human cells . 8
6.4.4 Selection of marker cytokine . 9
6.4.5 Procedure of HCPT . 9
6.4.6 Characteristic of the HCPT .10
6.4.7 Validation study .11
7 Conclusion .11
Bibliography .12
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ISO/TR 21582:2021(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee [or Project Committee] ISO/TC 194, Biological
and clinical evaluation of medical devices.
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.
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ISO/TR 21582:2021(E)
Introduction
At present, safety assessments of medical devices are guided by the toxicological and other studies
recommended in the ISO 10993 series of standards.
Material-mediated pyrogenicity represents a systemic effect that is included in of ISO 10993-11:2017,
Annex G, but efforts have been taken to generally address pyrogenicity testing in this document.
A pyrogenic response is the adverse effect of a chemical agent or other substance, such as microbial
component to produce a febrile response. Tests for a pyrogenic response have been required to evaluate
the safety of products that have direct or indirect contact to blood circulation and the lymphatic system,
cerebrospinal fluid (CSF) and interact systemically with human body.
At present, the in vivo rabbit pyrogenicity test and the in vitro bacterial endotoxin test are available
as accepted methods for evaluating the pyrogenicity of medical devices and their materials. Basic
procedures, including sample preparation of each test article, are already established, internationally
harmonized, and mentioned in the related guidelines and pharmacopoeias.
Recently, an in vitro pyrogen test using human immune cells, the human cell-based pyrogen test (HCPT),
has been developed and applied for pyrogen testing of parenteral drugs. The concept of the application
of pyrogen testing for medical devices is being considered due to the direct or indirect exposure to
human blood cells (HCPT).
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TECHNICAL REPORT ISO/TR 21582:2021(E)
Pyrogenicity — Principle and method for pyrogen testing
of medical devices
1 Scope
This document specifies the principles and methods for pyrogen testing of medical devices and their
materials.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https:// www .electropedia .org/
— ISO Online browsing platform: available at https:// www .iso .org/ obp
3.1
medical device
instrument, apparatus, implement, machine, appliance, implant, in vitro reagent or calibrator, software,
material or other similar or related article, intended by the manufacturer to be used, alone or in
combination, for human beings for one or more of the specific purpose(s) of
— diagnosis, prevention, monitoring, treatment or alleviation of disease;
— diagnosis, monitoring, treatment, alleviation of or compensation for an injury;
— investigation, replacement, modification, or support of the anatomy or of a physiological process;
— supporting or sustaining life;
— control of conception;
— disinfection of medical devices;
— providing information by means of in vitro examination of specimens derived from the human body;
and does not achieve its primary intended action by pharmacological, immunological or metabolic
means, in or on the human body, but which may be assisted in its function by such means
Note 1 to entry: Products which may be considered to be medical devices in some jurisdictions but not in others
include:
— disinfection substances;
— aids for persons with disabilities;
— devices incorporating animal and/or human tissues;
— devices for in vitro fertilization or assisted reproduction technologies.
[SOURCE: GHTF/SG1/N071: 2012, 5.1]
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3.2
pyrogen
substance that causes fever
3.3
pyrogenicity
ability of a chemical agent or other substance to produce a febrile response
3.4
febrile response
temperature above the normal range due to an increase in the body’s temperature set point
Note 1 to entry: It is also referred to as fever or pyrexia.
3.5
oxidative phosphorylation
metabolic pathway in most aerobic organisms, which uses enzymes to oxidise nutrients to release
energy
4 Abbreviated terms
COX Enzyme cyclooxygenase
CpG Cytosine (C) next to guanine (G) in the DNA sequence, with the p indicating that C and G are con-
nected by a phosphodiester bond.methyl group to the 5 position of the cytosine pyrimdine ring
ELISA Enzyme llinked immunosorbent assay HCPT, e.g. monocyte activation test (MAT)
IKK IkB kinase, an enzyme complex involved in propagating cellular response to inflammation
IRAK Interleukin-1 receptor-associated kinase
LAL Limulus amebocyte lysate
LPS Lipopolysaccharide
MD-2 Molecule secreted glycoprotein that binds to extracellular domain of TLR4
MCP Macrophage chemotactic protein
MIP Macrophage inflammatory protein
NOD Nucleotide-binding oligomerization domain
PGE Prostaglandin E
2 2
RANTES Regulated on activation, normal T-expressed and Secreted
RNA Ribonucleic acid
SEA Staphylococcal enterotoxin A
Spe C Streptococcal pyrogenic exotoxin C
Spe F Streptococcal pyrogenic exotoxin F
TBK TANK binding kinase
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TLR Toll-like receptor
TNF Tumour necrosis factor
TSST Toxic shock syndrome toxin
5 Characterization of pyrogen
5.1 General
On the basis of pyrogen origin, febrile response can be divided into three groups:
a) material-mediated pyrogenicity caused by chemical agents;
b) endotoxin-mediated pyrogenicity;
c) pyrogenicity mediated by microbial components other than endotoxin.
Non-endotoxin-mediated pyrogenicity corresponds to a generic name of febrile responses originating
from a) and c) above. However, the latter can be clearly distinguished from material-mediated
pyrogenicity, because the febrile reaction is originated from microbial contamination.
TLRs are proteins that constitute an important part of the immune system against microbial infections,
closely relate to pyrogenicity of microbial components. Thirteen kinds of human TLRs from TLR1
to TLR13 and the agonists to some of them have been identified to date. Most pyrogens that can be
assessed in the field of medical devices can be bioactive substances derived from microorganisms
present as contaminants of the device manufacturing process or present in materials. Since the
components are TLR agonists and act as pyrogen to human, the knowledge for TLRs is very significant
for understanding pyrogens.
5.2 Bacterial endotoxin
Bacterial endotoxin, an important component of the outer membrane of Gram-negative bacteria, is the
most powerful pyrogen recognized by TLR4. Endotoxin is a modulator of the host immune response
and exhibits a variety of biological activities, for example, activation of macrophages, mitogenicity and
adjuvanticity, causing Schwartzman reactions in addition to pyrogenicity. From the clinical standpoint,
endotoxin causes sepsis, septic shock and multiple organ failure, which are systemic disorders with a
high mortality rate.
Endotoxin generally consists of a heteropolysaccharide part subdivided into an O-specific chain, a core
oligosaccharide, and a lipid component called lipid A that is a biologically active centre of endotoxin.
The potency of endotoxin is influenced by acylation and phosphorylation patterns, and the presence/
absence of polar-head group bound to phosphate residue in lipid A molecule. In addition, endotoxin has
species-specificity for the expression of its bioactivity.
In the natural world, Gram-negative bacteria are widely distributed in water (rivers and sea), air, soil, and
also human body. It is likely therefore that biomaterials made of natural substances are contaminated
with the bacteria and their components. Autoclaving, irradiation and gas sterilization during the
manufacturing process are able to kill the bacteria. However, microbial components, particularly
endotoxin cannot be inactivated by such ordinary sterilization methods, and once contaminated it is
quite difficult to remove the endotoxin during the manufacturing process. During the manufacturing
process, endotoxin contamination can be reduced or eliminated by depyrogenization (e.g. 250 °C for
[50][66]
30 min, use of chemicals to inactivate endotoxin such as polymyxin-B or by using endotoxin-free
water in the washing and manufacturing processes.
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5.3 Microbial components other than endotoxin
Microorganisms produce various bioactive substances other than endotoxin as its cellular component.
Lipoteichoic acid, an important component of the outer membrane of Gram-positive bacteria,
represents a counterpart to endotoxin and acts as a pyrogen recognized by TLR2 that interacts and
forms a heterodimer with TLR1 or TLR6. Lipoproteins, lipopeptides, and lipoarabinomannan that are
the cellular components of various microorganisms are also known to act as TLR2 agonists. Although
peptidoglycan constructing cell wall of Gram-positive and Gram-negative bacteria was considered
as TLR2 agonist, it is recently suggested that NOD1 and NOD2 proteins can play a role of mediating
the expression of its bioactivity rather than TLR2. In addition, viral double-stranded RNA, bacterial
flagella, and bacterial and viral CpG DNA have been identified as the agonists of TLR3, TLR5, and TLR9,
respectively, and all of them acts as pyrogens to human. Although pyrogenicity has not been reported
for any kind of (1,3)-β -D-glucan preparation, it can be noted that certain kinds of (1,3)-β -D-glucan can
enhance endotoxin toxicity.
It has been reported that exotoxins and enterotoxins such as TSST-1, SEA, Spe F, and Spe C produced by
various pathogenic microorganisms cause febrile response in human body by the toxin-specific manner
that can be different from TLR signal transduction. There was an outbreak of inflammation, fever and
[52][76]
peritonitis in some patients due to contamination of solution with peptidoglycan during dialysis .
5.4 Pro-inflammatory cytokines
Since febrile responses induced by TLR agonists are mediated by pro-inflammatory cytokines such as
TNFα, IL-1β, IL-6, and INF-γ produced by human immune cells, the endogenous mediator itself naturally
acts as pyrogen. Each cytokine further activates immune cells through the cytokine network, because
receptors specific to the cytokines are located on the cell surface of monocytes and macrophages in
addition to TLRs.
5.5 Chemical agents and other pyrogens
Pyrogenicity of chemicals or natural substances other than microbial components is not well known.
In addition, over 1 000 new compounds are discovered or synthesized each year world wide, but the
biological properties of each compound are not well understood. Most chemicals currently used as
biomaterials for medical devices, are safe and are non-pyrogenic to humans. However, it can be possible
that some new biomaterials and chemicals can cause febrile reaction to human.
This possibility holds also true for non-autologous cellular products which can evoke immunological
recognition and activation of immune-competent cells.
As an example, chemicals that are known to induce febrile reaction to human are listed below. These
chemicals can be divided mainly into three groups according to principle for inducing a febrile response,
a) agents that directly stimulate thermoregulatory centers of the brain and nervous system,
b) uncoupling agents of oxidative phosphorylation, and
c) pyrogens with mechanisms that are not well known.
The chemicals listed below are known to cause a febrile response in humans:
— prostaglandins;
— inducers (e.g. polyadenylic, polyuridylic, polybionosinic, and polyribocytidylic acids);
— substances disrupting the function of thermoregulatory centers (e.g. lysergic acid diethylamide,
cocaine, morphine);
— neurotransmitters (e.g. noradrenaline, serotonin);
— uncoupling agents of oxidative phosphorylation (e.g. 4, 6-dinitro-o-cresol, dinitrophenol, picric
acid);
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— N-phenyl-β -naphthylamine and aldo-α -naphthylamine (the febrile mechanism is unknown);
— metals such as nickel salts, in some instances.
[23] [61]
In addition to these chemicals, there is a possibility that microspheres and nanoparticles, including
[7] [23] [61]
implant-derived wear debris, can act as pyrogens. Microspheres, particles and nanoparticles
consisting of specific sizes could be phagocytosed by macrophages and activate macrophage-released
pro-inflammatory cytokines such as TNFα. TNFα is one of the endogenous pyrogens.
5.6 Principle of febrile reaction
TLRs are a class of single membrane-spanning non-catalytic receptors that recognize structurally
conserved molecules derived from microbes once they have breached physical barriers such as the skin
or intestinal tract mucosa and activate immune cells. They are believed to play a key role in the innate
immune system and are known to function as dimers. Although most TLRs appear to act as homodimers,
TLR2 forms heterodimers with TLR1 or TLR6, each dimer having different ligand specificity. TLRs can
also depend on other co-receptors for full ligand sensitivity, such as in the case of TLR4's recognition
of endotoxin, which requires a MD-2 molecule. CD14 and LPS binding protein are known to facilitate
the presentation of endotoxin to MD-2. When activated, TLRs recruit adapter molecules within the
cytoplasm of cells in order to propagate a signal. Four adapter molecules are known to be involved in
signalling. These proteins are known as MyD88, Tirap (also called Mal), Trif, and Tram. The adapters
activate other molecules within the cell, including certain protein kinases (IRAK1, IRAK4, TBK1, and
IKKi) that amplify the signal, and ultimately lead to the induction or suppression of genes (NF-κB, AP-1,
and IRP3) that orchestrate the inflammatory response.
Following activation by ligands of microbial origin, several reactions are possible. Immune cells can
produce cytokines that trigger inflammation. Particularly, IL-1β is closely associated with induction of
febrile reaction. IL-6 and TNFα were isolated later and found to be pyrogenic cytokines as well, although
[28] [29]
at much higher doses , and. The current understanding of the mechanism of fever in the mammal
is that these proinflammatory cytokines result in the expression of the COX-2 which mediates PGE
2
[30] [47] [48]
synthesis. Mice deficient in COX-2 do not develop fever in response to LPS, IL-1 or IL-6 , and
PGE triggers an intracellular signalling cascade that changes the set point of the body temperature.
2
Thus, IL-1β, IL-6 and TNFα are the mediators released by immune cells upon contact with pyrogens
that are responsible for triggering the fever reaction in the brain. Substance P is known to induce fever
through the production of TNF-α, IL-6 and PGE2, see Reference [17].
TLRs seem to be involved in the cytokine production and cellular activation as well as in the adhesion
and phagocytosis of microorganisms and other potential pyrogens.
Independent of TLR signalling pathway and subsequent cytokine production, body temperature could
be increased by agents that directly stimulate thermoregulatory centers. Also, uncoupling agents
of oxidative phosphorylation can increase body temperature as a result of activating the electron
transport chain in mitochondria.
6 Assessment of p yrogenicity
6.1 General
There are three methods used for pyrogenicity testing, which are described below. The in vivo rabbit
pyrogen test is the only test that directly measures the febrile response in the body as an end point,
in accordance with the definition of a pyrogen, which the other two methods do not. Instead the in
vitro methods detect pyrogens using different end points, such as cytokine production and protein
coagulation.
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6.2 Bacterial endotoxin test
6.2.1 General
The bacterial endotoxin test is harmonized with several pharmacopaeia. This test is to detect or
quantify bacterial endotoxin of Gram-negative bacterial origin using a lysate reagent that is an aqueous
extract of circulating amebocytes of the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).
Bacterial endotoxin test is technically divided into two methods; one is the gel-clot technique based
on gel formation by the reaction of the lysate reagent with endotoxins, and other is the photometric
technique originating from endotoxin-induced optical changes of the lysate reagent. The latter is further
subdivided into two methods: one is turbidimetric technique measuring the endotoxin concentrations
of sample solutions based on the measurement of turbidity change accompanying the gel formation, and
other is chromogenic technique estimating the endotoxin concentrations by measuring optical density
of the colour of chromophore released from a synthetic chromogenic substrate that is a substituent
for the final step of the enzymatic cascade reaction described below. Each photometric technique is
classified as either end point or kinetic method.
The bacterial endotoxin test can be used to monitor endotoxin contamination in manufacturing
process of medical devices and the final products from the viewpoint of routine quality control. Before
starting use of the lysate reagent extracted from Tachypleus tridentatus, this test was termed Limulus
amoebocyte lysate (LAL) test in the past.
NOTE Other methods are available for the detection of bacterial endotoxins, for example, the fluorescent
method using recombinant Factor C, see Reference [6].
6.2.2 Principle of LAL reaction
The BET is an enzymatic cascade reaction that has the highest sensitivity to detect and quantify Gram-
negative bacterial endotoxins. First, factor C, endotoxin-sensitive serine protease zymogen present
in the lysate reagent, is activated by endotoxin. The activated factor C converts factor B from the
inactive form to the active form that further converts proclotting enzyme to clotting enzyme. Finally,
the clotting enzyme converts coagulogen to coagulin that leads to gel formation. In addition to factor
C, original lysate reagent contains factor G that is activated by (1,3)-β-D-glucans and subsequently
converts proclotting enzyme to clotting enzyme. Endotoxin-specific LAL reagent has been developed
by removing factor G or saturating its function.
Since the BET is based on an enzymatic reaction, it is influenced by the temperature and pH of sample
solution, and it is also enhanced or inhibited by various compounds such as protease, protease inhibitors,
metal ions, surfactants, chelates, salts and sugars if they are present in sufficient concentrations.
Therefore, tests for interfering factors can be performed to check the presence of inhibitors and
enhancers of the reaction in sample solution. The effect of the interfering factors can be avoided by the
dilution of the sample solution.
6.2.3 General procedure of BET
General methods for detection and quantification of endotoxin have been discussed in pharmacopoeias
in several countries and AAMI ST 72. The details are referred to in these documents.
It is noted that pyrogen-free water can be used as an extraction medium for preparing sample solution
unless otherwise specified. Endotoxin present in medical devices and their materials typically are
extracted at the ambient te
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