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)
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 .................................................................................................................................................................. 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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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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ISO/TR 21582:2021(E)
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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ISO/TR 21582:2021(E)
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

— 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

[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.

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

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
...

ISO/PRF TR 21582

Pyrogenicity -- Principle and method for pyrogen testing of medical devices

Pyrogenicity -- Principle and method for pyrogen testing of medical devices

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