ISO/TR 10993-55:2023

TECHNICAL ISO/TR
REPORT 10993-55
First edition
2023-02
Biological evaluation of medical
devices —
Part 55:
Interlaboratory study on cytotoxicity
Évaluation biologique des dispositifs médicaux —
Partie 55: Étude interlaboratoire sur la cytotoxicité
Reference number
© ISO 2023
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 below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Participants . 1
5 Materials and sample preparation .1
6 Test procedures . 2
7 Results . . 2
7.1 Neutral red uptake . 2
7.1.1 General . 2
7.1.2 Sodium lauryl sulfate as positive control . 2
7.1.3 Test samples . 3
7.2 Colony formation assay . 4
7.2.1 General . 4
7.2.2 Negative reference material . 5
7.2.3 Positive reference materials . 5
8 A ssessment of results . 7
Annex A (informative) Interlaboratory study protocol for the neutral red uptake
cytotoxicity test . 9
Annex B (informative) Interlaboratory study protocol for the colony formation cytotoxicity
test .17
Bibliography .23
iii
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.
A list of all parts in the ISO 10993 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
Introduction
The first edition of ISO 10993-5, published in 1992, allowed several different ways to assess cytotoxicity
of medical devices and gave an imprecise description of how to perform the tests. Qualitative assays
were accepted and only a small amount of guidance was given for the interpretation of the results.
Not surprisingly, the first interlaboratory study in 2000 resulted in quite low reproducibility of results.
Therefore, detailed protocols were included into the standard and in another study the practicability of
the protocols and reference materials were evaluated. The results of this second interlaboratory study
mainly influenced the revision of ISO 10993-5, which was published in 2009.
This document provides the historical report of the second interlaboratory study, conducted in 2006.
v
TECHNICAL REPORT ISO/TR 10993-55:2023(E)
Biological evaluation of medical devices —
Part 55:
Interlaboratory study on cytotoxicity
1 Scope
This document describes the results of an international interlaboratory study conducted in 2006 to
evaluate the performance of two different test protocols in terms of the cytotoxic effects in the biological
[2]
evaluation of medical devices. The results of these tests were used for the revision of ISO 10993-5.
Furthermore, the results of these tests were used to estimate the accuracy of these test systems with
living cells to define a threshold what is considered a cytotoxic effect.
NOTE The determination of cytotoxic effects has a high relevance in the biological evaluation of medical
[1]
devices; according to ISO 10993-1 , it is one of the very few tests which are proposed to be performed for every
kind of device.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Participants
1)
Twelve laboratories participated in this study . Eleven reports on a neutral red uptake (NRU) assay
and ten reports on a colony formation assay (CFA) were received. Four participants were commercial
test laboratories, four participants were internal test laboratories of medical device manufacturers and
four laboratories were in research institutes.
The laboratories were located in six different countries: one each in Austria, France and the Netherlands,
and three each in Germany, Japan and the United States.
5 Materials and sample preparation
The following materials were used for the study:
a) reference material-C [RM-C; Hatano Research Institute (HRI)]: high density polyethylene sheet;
b) RM-A (HRI): segmented polyurethane film containing 0,1 % zinc diethyldithiocarbamate (ZDEC);
1) The participating laboratories were: Deutsche Institute für Textil- und Faserforschung, Germany; Hatano
Research Institute, Food and Drug Safety Center, Japan; Medical University Vienna, Austria; National Institute of
Health Sciences, Japan; Envigo CRS GmbH, Germany; Terumo Corporation R&D, Japan; BD Technologies, United
States; NAMSA, United States; Gambro BCT, United States and three other laboratories.
c) RM-B (HRI): segmented polyurethane film containing 0,25 % zinc dibutyldithiocarbamate (ZDBC).
RM-C (HRI), RM-A (HRI) and RM-B (HRI) have been widely used as reference materials for cytotoxicity
tests of medical devices. The Food and Drug Safety Center of HRI has certified these materials. HRI
agreed to provide them for the interlaboratory study. Test samples were cut (2 mm × 15 mm) and
sterilized with ethylene oxide (EO) and were distributed from HRI to the participants. Extraction was
then performed in the participating laboratories according to the protocols.
6 Test procedures
Two test protocols were chosen by the working group developing the tests for cytotoxicity in vitro:
NRU and CFA. The NRU assay protocol is based on the protocol, which was used in a validation study
[4]
of Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). The
CFA protocol is based on the cytotoxicity test of the Japanese guidelines for basic biological tests of
[5]
medical materials and devices. These original protocols were modified to meet the requirements of
this specific study (see Annexes A and B). The protocols were sent to the participants together with the
test materials.
7 Results
7.1 Neutral red uptake
7.1.1 General
Eleven laboratories participated in this study. All test samples were extracted once as described in
Annex A. Each concentration of the dilution series was tested in six replicates. The mean values were
used to calculate the concentration producing 50 % inhibition of cell viability (IC ) values.
7.1.2 Sodium lauryl sulfate as positive control
The laboratories used different internal reference materials as positive controls. It was therefore
decided that all participants use the same common chemical substance as positive control and
® 2)
sodium lauryl sulfate (SLS, CAS Registry Number 151-21-3 ) was selected for this purpose. Table 1
summarizes the results.
Table 1 — IC -values of SLS in the NRU assay
Laboratory IC
µg/ml
1 34,0
2 83,0
3 62,4
4 77,0
5 85,9
6 75,6
7 67,8
8 47,8
9 49,8
10 62,1
11 22,0 ®
2) CAS Registry Number is a trademark of the American Chemical Society (ACS). This information is given for
the convenience of users of this document and does not constitute an endorsement by ISO of the product named.
Equivalent products may be used if they can be shown to lead to the same results.
The variation of the IC was from 22,0 µg/ml to 85,9 µg/ml, the mean IC was (60,7 ± 20,4) µg/ml.
50 50
In Annex A, an IC -value between 70 µg/ml and 116 µg/ml was requested as acceptance criterion. This
was an error in ISO 10993-5:2009 and will be removed in the next edition. Historical IC -values are
typical for a specific laboratory but cannot be compared between laboratories.
7.1.3 Test samples
The three different test samples RM-A, RM-B and RM-C were extracted as described in Annex A and
the extracts were diluted as defined. The cell viabilities at different extract concentrations were
determined as described in Annex A. The IC was determined from the concentration-response. This
was done by using validated software, which is av
...

ISO/TC 194
Date:  2022-09-01
ISO/DTR 10993--55:2022(E)
ISO/TC 194/WG 5
Secretariat: DIN
Biological evaluation of medical devices — Part 55: Interlaboratory study on
cytotoxicity
Copyright notice
ThisFirst edition
Date: 2022-09-30
ISO/DTR 10993-55:2022(E)
© ISO document is a working draft 2022
All rights reserved. Unless otherwise specified, or committee draft and is copyright-protected by
ISO. While required in the reproductioncontext of working drafts or committee drafts in any form
for use by participants in the ISO standards development process is permitted without prior
permission from ISO, neither its implementation, no part of this document nor any extract from
itpublication may be reproduced, stored or utilized otherwise in any form or transmitted in any
form for any other purposeby any means, electronic or mechanical, including photocopying, or
posting on the internet or an intranet, without prior written permission from ISO. Permission can
be requested from either ISO at the address below or ISO's member body in the country of the
requester.
ISO Copyright Office
CP 401 • CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.orgRequests for permission to reproduce this document for the purpose of selling it
should be addressed as shown below or to ISO's member body in the country of the requester:
[Indicate the full address, telephone number, fax number, telex number, and electronic mail address,
as appropriate, of the Copyright Manager of the ISO member body responsible for the secretariat of
the TC or SC within the framework of which the working document has been prepared.]
Reproduction for sales purposes may be subject to royalty payments or a licensing agreement.
Violators may be prosecuted.
Published in Switzerland.
ii
ISO/DTR 10993-55:2022(E)
Contents Page
Foreword . iii
Introduction . iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Participants . 1
5 Materials and Sample preparation . 2
6 Test procedures . 2
7 Results . 2
7.1 Neutral Red Uptake . 2
7.1.1 Sodium Lauryl Sulfate as positive control . 2
7.1.2 Test samples . 3
7.2 Colony Formation Assay . 3
7.2.1 Negative reference material . 4
7.2.2 Positive reference materials . 5
8 Assessment of results . 7
Annex A (informative) Interlaboratory study protocol for the neutral red uptake (NRU)
cytotoxicity test . 9
Annex B (informative) Interlaboratory study protocol for the colony formation cytotoxicity
test . 17
Bibliography. 24

iii
ISO/DTR 10993-55:2022(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.
A list of all parts in the ISO 10993 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
ISO/DTR 10993-55:2022(E)
Introduction
The first versionedition of ISO 10993-5, published in 1992, allowed several different ways to assess
cytotoxicity of medical devices and gave an imprecise description of how to perform the tests. Qualitative
assays were accepted and only a small amount of guidance was given for the interpretation of the results.
Not surprisingly, the first interlaboratory study by the members of working group 5 (WG 5) of ISO/TC
194 in 2000 resulted in quite low reproducibility of results. It was therefore the consensus of WG 5 to
includeTherefore, detailed protocols were included into the standard and to evaluate in another study
the practicability of the protocols and reference materials proposed by the working group members.were
evaluated. The results of this second interlaboratory study mainly influenced the revision of the
standardISO 10993-5, which was published in 2009.
This technical report isdocument provides the historical report of the second interlaboratory study,
conducted in 2006.
v
TECHNICAL REPORT ISO/DTR 10993-55:2022(E)

Biological evaluation of medical devices — Part 55:
Interlaboratory study on cytotoxicity
1 Scope
This document describes the results of an international interlaboratory study conducted in 2006 to
evaluate the performance of two different test protocols in terms of the cytotoxic effects in the biological
[2 ]
evaluation of medical devices. The results of these tests were used for the revision of ISO 10993-5 [. ].
Furthermore, the results of these tests were used to estimate the accuracy of these test systems with
living cells to define a threshold what is considered a cytotoxic effect.
NOTE The determination of cytotoxic effects has a high relevance in the biological evaluation of medical devices;
[1]
according to ISO 10993-1 it is one of the very few tests which are proposed to be performed for every kind of
device.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminologicalterminology databases for use in standardization at the following
addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Participants
Twelve laboratories participated in this study mainly from entities represented in ISO/TC 194, which is
responsible for this document. Eleven reports on a neutral red uptake (NRU) assay were received and
ten10 reports on a colony formation (CF) assay were received. Four participants were commercial test
laboratories, four participants were internal test laboratories of medical device manufacturers and four
laboratories were in research institutes.
The laboratories were located in six different countries.

The participating laboratories include: Deutsche Institute für Textil- und Faserforschung, Germany; Hatano Research
Institute, Food and Drug Safety Center, Japan; Medical University Vienna, Austria; National Institute of Health Sciences,
Japan; Envigo CRS GmbH, Germany; Terumo Corporation R&D, Japan; BD Technologies, United States; NAMSA, United
States; Gambro BCT, United States and three other laboratories.

ISO/DTR 10993-55:2022(E)
: one each in Austria, France and the Netherlands, and three each in Germany, Japan and the United
States.
5 Materials and sample preparation
The following materials were used for the study:
a) reference material-C [RM-C; Hatano Research Institute (HRI):)]: high density polyethylene sheet.;
b) RM-A (HRI): segmented polyurethane film containing 0,1 % zinc diethyldithiocarbamate (ZDEC));
c) RM-B (HRI): segmented polyurethane film containing 0,25 % zinc dibutyldithiocarbamate (ZDBC)).
RM-C (HRI), RM-A (HRI) and RM-B (HRI) have been widely used as reference materials for cytotoxicity
tests of medical devices. The Food and Drug Safety Center of the Hatano Research Institute (HRI) (Ochiai
729-5, Hadanoshi, Kanagawa 257-8523, Japan) has certified these materials and offers them for sale.
HRI agreed to provide them for the interlaboratory study. Test samples were cut (2 mm x × 15 mm) and
sterilized with ethylene oxide (EO) and were distributed from HRI to the participants. Extraction was
then performed in the participating laboratories according to the protocols.
6 Test procedures
Two test protocols were chosen by the working group developing the tests for cytotoxicity in vitro:
neutral red uptake (NRU) and colony formation (CF). The NRU assay protocol is based on the protocol,
which was used in a validation study of Interagency Coordinating Committee on the Validation of
[4]
Alternative Methods (ICCVAM) .). The CF assay protocol is based on the cytotoxicity test of the Japanese
[5]
guidelines for basic biological tests of medical materials and devices. These original protocols were
modified to meet the requirements of this specific study (see Annexes A and ).B). The protocols were sent
to the participants together with the test materials.
7 Results
7.1 Neutral red uptake
7.1.1 General
Eleven laboratories participated in this study. All test samples were extracted once as described in
.Annex A. Each concentration of the dilution series was tested in six replicates. The mean values were
used to calculate the concentration producing a 50 % inhibition of cell viability (IC ) values.
7.1.17.1.2 Sodium lauryl sulfate as positive control
The laboratories were usingused different internal reference materials as positive controls. It was
therefore decided that all participants use the same common chemical substance as positive control and
® 2
sodium lauryl sulfate (SLS, CAS #Registry Number 151-21-3 ) was selected for this purpose. is a
summary ofTable 1 summarizes the results.
Table 1 — IC -values of SLS in the NRU assay ®
CAS Registry Number is a trademark of CAS corporation. This information is given for the convenience of users of this
document and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they
can be shown to lead to the same results.
ISO/DTR 10993-55:2022(E)
Laboratory LaboratoryIC
µg/ml
1 34,0
2 83,0
1 2 5 6 7 8 9 10 11
3 62,4 Deleted Cells
34,0 83,0 85,9 75,6 67,8 47,8 49,8 62,1 22,0 Deleted Cells
IC
62,4 77,0
Deleted Cells
µg/ml
Deleted Cells
5 85,9
Deleted Cells
6 75,6
Deleted Cells
7 67,8
Deleted Cells
8 47,8
Deleted Cells
9 49,8
Deleted Cells
10 62,1
Deleted Cells
11 22,0
The variation of the IC was from 22,0 µg/ml to 85,9 µg/ml, the mean IC was (60,7 ± ± 20,4) µg/ml.
50 50
In Annex A, an IC –-value between 70 µg/ml and 116 µg/ml was requested as acceptance criterion. This
was an error in the 2009 version of ISO 10
...