ISO/PRF TR 21582

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)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

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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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Any feedback or questions on this document should be directed to the user’s national standards body. A

At present, safety assessments of medical devices are guided by the toxicological and other studies

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,

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

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

This document specifies the principles and methods for pyrogen testing of medical devices and their

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

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

— diagnosis, monitoring, treatment, alleviation of or compensation for an injury;

— investigation, replacement, modification, or support of the anatomy or of a physiological process;

— 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

temperature above the normal range due to an increase in the body’s temperature set point

metabolic pathway in most aerobic organisms, which uses enzymes to oxidise nutrients to release

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

On the basis of pyrogen origin, febrile response can be divided into three groups:

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

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

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

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

30 min, use of chemicals to inactivate endotoxin such as polymyxin-B or by using endotoxin-free

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

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

peritonitis in some patients due to contamination of solution with peptidoglycan during dialysis .

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

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

This possibility holds also true for non-autologous cellular products which can evoke immunological

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,

— inducers (e.g. polyadenylic, polyuridylic, polybionosinic, and polyribocytidylic acids);

— substances disrupting the function of thermoregulatory centers (e.g. lysergic acid diethylamide,

— 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);

In addition to these chemicals, there is a possibility that microspheres and nanoparticles, including

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

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,

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

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

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

TLRs seem to be involved in the cytokine production and cellular activation as well as in the adhesion

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

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

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

NOTE Other methods are available for the detection of bacterial endotoxins, for example, the fluorescent

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

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

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