ISO/TR 10993-55:2023

Biological evaluation of medical devices — Part 55: Interlaboratory study on cytotoxicity

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 evaluation of medical devices. The results of these tests were used for the revision of ISO 10993-5.[2] 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; according to ISO 10993-1[1], it is one of the very few tests which are proposed to be performed for every kind of device.

Évaluation biologique des dispositifs médicaux — Partie 55: Étude interlaboratoire sur la cytotoxicité

General Information

• Status: Published
• Publication Date: 05-Feb-2023
• ICS: 11.100.20 - Biological evaluation of medical devices
• Technical Committee: ISO/TC 194 - Biological and clinical evaluation of medical devices
• Drafting Committee: ISO/TC 194/WG 6 - Mutagenicity, carcinogenicity and reproductive toxicity
• Current Stage: 6060 - International Standard published
• Start Date: 06-Feb-2023
• Completion Date: 06-Feb-2023

Standards Content (Sample)

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

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ISO/DTR 10993-55:2022(E)
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Published in Switzerland.
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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

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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.
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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.
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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
1
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.

1
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.

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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.
50
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
50

2
®
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.
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ISO/DTR 10993-55:2022(E)
Laboratory LaboratoryIC
50

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
50
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
50
was an error in the 2009 version of ISO 10993-5:2009 and will be removed in the next versionedition.
Historical IC –-values are typical for a specific laboratory but cannot be compared between
50
laboratories.
7.1.27.1.3 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
50
using validated software, which is available in public, see Reference .[6]. The results of the eleven11
participants are summarized in Table 2 and illustrated in .Figure 1. Initially, the testing was conducted
using the following concentrations of the RM-A extract: 0,25 %, 0,5 %, 1,0 %, 2,0 %, 3,0 %,% and 4,0 %.
Unexpectedly, IC –-values were higher than expected from the colony formation assay, because the
50
neutral red assay is less sensitive probably due to the shorter exposure time. Therefore, the laboratories
repeated the test with the following concentrations of the RM-A extract: 5 %, 10 %, 20 %, 30 %, 40 %,%
Merged Cells
and 50 %.
Deleted Cells
Table 2 — IC -values of sample extracts in the NRU assay
50
Deleted Cells
Laboratory LaboratoryIC Deleted Cells
50

Deleted Cells
%
Deleted Cells
RM- RM- RM-C
Deleted Cells
A B
Deleted Cells
1 16,5 32,0 —
Deleted Cells
2 26,4 54,0 —
Deleted Cells
1 2 4 8 9 10 11
3 18,5 693,2 7—
Deleted Cells
R 16,5% 18, 24,3 15,1 11,7 6,7% 16,7%
15,6%
26, 2056, Deleted Cells
15,3
M- 5% % % %
4% —
% 6%
A Deleted Cells
Deleted Cells
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ISO/DTR 10993-55:2022(E)
5 20,6 79,6 —
R 32, 54, 93, 43,3 38,2 89,4 33,6%
Deleted Cells
93,3%
7915, 45,6
M- 0% 0% 2% 56,6% % % %
—
6% %
Deleted Cells
B
none none none none non no no no
none Deleted Cells
none2 none9
RM-C7 e ne ne ne
4,3 3,3 —
Deleted Cells
Deleted Cells
8 15,1 43,3 —
Deleted Cells
9 11,7 38,2 —
Deleted Cells
10 6,7 89,4 —
Deleted Cells
11 16,7 33,6 —
Deleted Cells
Results for RM-A varied from 6,7 % to 26,4 %, mean IC was (17,0 ± 5,5) %. Results for RM-B varied
50
Deleted Cells
from 32,0 % to 93,3 %, %, the mean IC was (59,9 ± 24,4) %.
50 Deleted Cells
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Deleted Cells
Deleted Cells
Deleted Cells
Deleted Cells
Deleted Cells


Key
X Laboratory number
Y IC
50
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Reference material A
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Reference material B
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ISO/DTR 10993-55:2022(E)
X laboratory number reference material A

Y IC reference material B
50

Figure 1 — Comparison of IC -values of sample extracts in the NRU assay
50
7.2 Colony formation assay
7.2.1 General
The test samples RM-A, RM-B and RM-C were those materials, which were already recommended to be
used as reference materials in the Japanese Guidelines for the colony formation assay. For the study only
these materials were used by the laboratories, to assess the differences between individual laboratories.
The test samples were extracted as described in Annex B and the extracts were diluted as proposed. The
cell viabilities at different extract concentrations were determined by counting the colonies formed
[plating efficiency (PE)]. IC was calculated as the dose with 50 % PE which was calculated from the line
50
which passed through a dose with higher PE and a dose with lower PE than 50 %.
Ten of the twelve12 participants in the NRU study also participated in the CF assay and communicated
their results. All test samples were extracted once as described in .Annex B. Each concentration of the
dilution series was tested in triplicate. The mean values were used to calculate IC -values.
50
The plating efficiency of the controls in the different labslaboratories is listed in .Table 3.
Table 3 — Control PE of the CF assay in the participating labslaboratories
Laboratory Plating efficiency
Inserted Cells
%
1 68,0
1 3 4 5 7 9 10 12
Deleted Cells
2 62,8
Deleted Cells
3 75,7
Deleted Cells
4 101,5
Deleted Cells
5 92,2
Deleted Cells
7 71,2
Deleted Cells
8 108,7
Deleted Cells
9 85,0
Deleted Cells
62,8% 75,7% 101,5% 92,2% 71,2% 108,7% 85,0% 106,3%
68,0%10 75,0% Deleted Cells
Deleted Cells
12 106,3
Deleted Cells
The PE in the controls varied from 62,8 % to 108,7 %, mean value was (84,6 ± 15,8) %.
Deleted Cells
7.2.17.2.2 Negative reference material
Deleted Cells
Deleted Cells
The test sample RM-C is certified not to give any positive response in the test. Nevertheless, the extract
of RM-C was used in this study to detect the variation of results in this biological system. The results of
Deleted Cells
the 10 participants are summarized in Table 4 and illustrated in .Figure 2.
Deleted Cells
Table 4 — Plating efficiencies of RM-C in the CF assay
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ISO/DTR 10993-55:2022(E)
LaboratoryPlating efficiency
Concentration
%
of RM-C
%
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 12
0 100,0% 100,0% 100,0% 100,0% 100,0% 100,0% 100,0% 100,0% 100,0% 100,0%
25 112,0% 96,8% 100,0% 100,2% 93,3% 97,8% 85,3% 102,4% 98,7% 101,9%
50 94,0% 87,1% 81,5% 100,5% 103,1% 92,6% 101,8% 94,9% 79,6% 99,4%
75 110,0% 92,9% 96,0% 97,5% 92,9% 98,2% 96,6% 98,5% 85,3% 98,4%
100 106,0% 94,8% 110,1% 97,2% 95,1% 91,2% 92,0% 96,1% 77,8% 98,1%


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Key
X extract concentration in %
Y platting efficiency in % of the control
The linked image cannot be displayed. The file may have been moved, renamed, or deleted. Verify that the link points to the correct file and location.
Lab 1
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Lab 2
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Lab 3
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Lab 4
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Lab 5
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ISO/DTR 10993-55:2022(E)
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Lab 7
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Lab 8
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Lab 9
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Lab 12
X extract concentration, % laboratory 5

Y platting efficiency of the control, % laboratory 7

laboratory 1 laboratory 8

laboratory 2 laboratory 9

laboratory 3 laboratory 10

laboratory 4 laboratory 12

Figure 2 — Plating efficiencies of RM-C in the CF assay
7.2.27.2.3 Positive reference materials
Extracts of the test samples RM-A and RM-B were applied in the CF assay as indicated in .Annex B. The
results of the ten10 participants are summarized in Table 5 and Table 6 and illustrated in Figure 3 and
.Figure 4.
Table 5 — Plating efficiencies of RM-A in the CF assay
LaboratoryPlating efficiency
Concentration
%
of RM-A
%
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 12
0,00 100,0% 100,0% 100,0% 100,0% 100,0% 100,0% 100,0% 100,0% 100,0% 100,0%
0,25 100,0% 93,5% 90,6% 99,5% 0,0% 92,2% 71,8% 105,1% 100,9% 101,9%
0,50 101,0% 108,3% 0,7% 96,9% 0,0% 102,9% 15,6% 105,5% 58,6% 102,5%
1,00 0,0% 91,3% 0,0% 14,4% 0,0% 85,2% 0,0% 32,1% 0,4% 61,1%
2,00 0,0% 17,0% 0,0% 1,0% 0,0% 16,3% 0,0% 0,0% 0,0% 0,0%
4,00 0,0% 1,1% 0,0% 0,3% 0,0% 0,9% 0,0% 0,0% 0,0% 0,0%
8,00 0,0% 0,5% 0,0% 0,0% 0,0% 0,5% 0,0% 0,0% 0,0% 0,0%
Concentration of RM-A
%

Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 12
IC
0,75 1,56 0,36 0,78 n.d.— 1,51 0,35 0,88 0,55 1,13
50
The IC of RM-A concentrations ranged from 0,35 % to 1,56 %, mean IC was (0,87 ± 0,42) %.
50 50
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ISO/DTR 10993-55:2022(E)
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Key
X extract concentration in %
Y platting efficiency in % of the control
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Lab 1
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Lab 2
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Lab 3
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Lab 4
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Lab 5
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Lab 7
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Lab 8
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Lab 9
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Lab 10
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Lab 12
Split Cells
Figure — Plating efficiencies of RM-A in the CF assay
Inserted Cells
Inserted Cells
Table — Plating efficiencies of RM-B in the CF assay
Deleted Cells
Concent
Laboratoryextract concentration, % laboratory 5 Deleted Cells
ration

Deleted Cells
RM-B
[%]X
Deleted Cells
Y 1platting efficiency of the control, % 345la  boratory 7 8 9 1 1
Deleted Cells
0 2
2
Deleted Cells

Deleted Cells
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ISO/DTR 10993-55:2022(E)
1 1 1 1 1
0 0 0 0 0
0 0 0 0 0
0 100,0% 100,0% 100,0% 100,0% 100,0%
, , , , ,
0 0 0 0 0
% % % % %
1 1
8 7 9
0 0
8 0 0
1 4
20 0,0% 100,4% 96,9% 100,8% 52,1% , , ,
, ,
0 2 6
5 1
% % %
% %
099, 8 3 9 1 1
73,laboratory 1% 39,laboratory 8%
Deleted Cells
,8% 3, 4, 0, 0 0
40 84,1%
Deleted Cells
0 3 7 6 2, 3,

% % % % 2 1
Deleted Cells
% %
Deleted Cells
7 4 8 8 9
9 2 7 2 2
Deleted Cells
50 0,0% 88,2% 5,7% 27,6% 43,4% , , , , ,
6 6 9 8 2 Deleted Cells
% % % % %
Deleted Cells
0, 9 0, 6112
laboratory 2,0% 74,laboratory 9%
Deleted Cells
0 2, 0 5278
6 45,9
% 9 % ,,,,
Deleted Cells
0 %
% 2361

%%%%  Deleted Cells
0,00, 0, 0,00
0,0%laboratory 3 0,0%laboratory 10
Deleted Cells
0 , 0 0 0 , ,
80 49,9%
Deleted Cells
% 0% % % 06

% %%
Deleted Cells
0,0 7,laboratory 4% 0,0%laboratory 12 0, 00, 0, 0, 0,
Deleted Cells
% 0 ,0 0 0 0
100 0,0%
% 0% % % %
Deleted Cells

%
Deleted Cells
6 3 5 5 6
4 3 5 5 6 Deleted Cells
IC n.d. 79,95 43,43 46,04 23,41 , , , , ,
50
Deleted Cells
6 9 3 9 7
6 6 9 4 0 Deleted Cells
Deleted Cells
Figure 3 — Plating efficiencies of RM-A in the CF assay
Deleted Cells
Deleted Cells
Table 6 — Plating efficiencies of RM-B in the CF assay
Deleted Cells
Plating efficiency
Concentration
%
of RM-B
%
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 12
0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0
20 0,0 100,4 96,9 100,8 52,1 88,0 70,2 101,5 90,6 104,1
40 0,0 99,8 73,1 84,1 39,8 83,3 34,7 90,6 102,2 103,1
50 0,0 88,2 5,7 27,6 43,4 79,6 42,6 87,9 82,8 92,2
60 0,0 92,9 0,0 2,0 45,9 65,2 12,3 17,6 28,1 74,9
80 0,0 49,9 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,6
100 0,0 7,4 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0
Concentration of RM-B
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ISO/DTR 10993-55:2022(E)
%
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 12
IC
— 79,95 43,43 46,04 23,41 64,66 33,96 55,39 55,94 66,70
50
The IC of RM-B concentrations ranged from 23,41 % to 79,95 %, mean IC was (52,2 ± 16,5) %.
50 50
The linked image cannot be displayed. The file may have been moved, renamed, or deleted. Verify that the link points to the correct file and location.


Key
X extract concentration in %
Y platting efficiency in % of the control
The linked image cannot be displayed. The file may have been moved, renamed, or deleted. Verify that the link points to the correct file and location.
Lab 1
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Lab 2
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Lab 3
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Lab 4
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Lab 5
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Lab 7
...

FINAL
TECHNICAL ISO/DTR
DRAFT
REPORT 10993-55
ISO/TC 194
Biological evaluation of medical
Secretariat: DIN
devices —
Voting begins on:
2022-10-14
Part 55:
Voting terminates on:
Interlaboratory study on cytotoxicity
2022-12-09
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/DTR 10993-55:2022(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. © ISO 2022

---------------------- Page: 1 ----------------------
ISO/DTR 10993-55:2022(E)
FINAL
TECHNICAL ISO/DTR
DRAFT
REPORT 10993-55
ISO/TC 194
Biological evaluation of medical
Secretariat: DIN
devices —
Voting begins on:
Part 55:
Voting terminates on:
Interlaboratory study on cytotoxicity
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022
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.
RECIPIENTS OF THIS DRAFT ARE INVITED TO
ISO copyright office
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
CP 401 • Ch. de Blandonnet 8
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
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DOCUMENTATION.
Phone: [phone]
IN ADDITION TO THEIR EVALUATION AS
Reference number
Email: [e-mail]
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/DTR 10993-55:2022(E)
Website: www.iso.org
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
Published in Switzerland
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
ii
  © ISO 2022 – All rights reserved
NATIONAL REGULATIONS. © ISO 2022

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ISO/DTR 10993-55:2022(E)
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 . 6
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
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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
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ISO/DTR 10993-55:2022(E)
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
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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
[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 10 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: 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 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.
1
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ISO/DTR 10993-55:2022(E)
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 CF. 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 CF
assay 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.
50
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
50
Laboratory IC
50
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 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.
2
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ISO/DTR 10993-55:2022(E)
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
50
was an error in ISO 10993-5:2009 and will be removed in the next edition. Historical IC -values are
50
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
50
was done by using validated software, which is available in public, see Reference [6]. The results of
the 11 participants are summarized in Table 2 and illustrated in Figure 1. Initially, the testing was
conducted using the following concentrations of the RM-A extract: 0,25 %, 0,5 %, 1,0 %, 2,0 %, 3,0 %
and 4,0 %. Unexpectedly, IC -values were higher than expected from the colony formation assay,
50
because the neutral red assay is less sensitive probably due to the shorter exposure time. Therefore, the
laboratories repeated the test with the following concentrations of the RM-A extract: 5 %, 10 %, 20 %,
30 %, 40 % and 50 %.
Table 2 — IC -values of sample extracts in the NRU assay
50
Laboratory IC
50
%
RM-A RM-B RM-C
1 16,5 32,0 —
2 26,4 54,0 —
3 18,5 93,2 —
4 15,3 56,6 —
5 20,6 79,6 —
6 15,6 45,6 —
7 24,3 93,3 —
8 15,1 43,3 —
9 11,7 38,2 —
10 6,7 89,4 —
11 16,7 33,6 —
Results for RM-A varied from 6,7 % to 26,4 %, mean IC was (17,0 ± 5,5) %. Results for RM-B varied
50
from 32,0 % to 93,3 %, the mean IC was (59,9 ± 24,4) %.
50
3
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ISO/DTR 10993-55:2022(E)
Key
X laboratory number reference material A
Y IC reference material B
50
Figure 1 — Comparison of IC -values of sample extracts in the NRU assay
50
7.2 Colony formation assay
7.2.1 General
The test samples RM-A, RM-B and RM-C were those materials, which were already recommended to be
used as reference materials in the Japanese Guidelines for the colony formation assay. For the study only
these materials were used by the laboratories, to assess the differences between individual laboratories.
The test samples were extracted as described in Annex B and the extracts were diluted as proposed.
The cell viabilities at different extract concentrations were determined by counting the colonies formed
[plating efficiency (PE)]. IC was calculated as the dose with 50 % PE which was calculated from the
50
line which passed through a dose with higher PE and a dose with lower PE than 50 %.
Ten of the 12 participants in the NRU study also participated in the CF assay and communicated their
results. All test samples were extracted once as described in Annex B. Each concentration of the dilution
series was tested in triplicate. The mean values were used to calculate IC -values.
50
The plating efficiency of the controls in the different laboratories is listed in Table 3.
Table 3 — Control PE of the CF assay in the participating laboratories
Laboratory Plating efficiency
%
1 68,0
2 62,8
3 75,7
4 101,5
5 92,2
7 71,2
8 108,7
9 85,0
10 75,0
4
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ISO/DTR 10993-55:2022(E)
TTabablele 3 3 ((ccoonnttiinnueuedd))
Laboratory Plating efficiency
%
12 106,3
The PE in the controls varied from 62,8 % to 108,7 %, mean value was (84,6 ± 15,8) %.
7.2.2 Negative reference material
The test sample RM-C is certified not to give any positive response in the test. Nevertheless, the extract
of RM-C was used in this study to detect the variation of results in this biological system. The results of
the 10 participants are summarized in Table 4 and illustrated in Figure 2.
Table 4 — Plating efficiencies of RM-C in the CF assay
Plating efficiency
Concentration
of RM-C
%
%
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 12
0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0
25 112,0 96,8 100,0 100,2 93,3 97,8 85,3 102,4 98,7 101,9
50 94,0 87,1 81,5 100,5 103,1 92,6 101,8 94,9 79,6 99,4
75 110,0 92,9 96,0 97,5 92,9 98,2 96,6 98,5 85,3 98,4
100 106,0 94,8 110,1 97,2 95,1 91,2 92,0 96,1 77,8 98,1
Key
X extract concentration, % laboratory 5
Y platting efficiency of the control, % laboratory 7
laboratory 1 laboratory 8
laboratory 2 laboratory 9
laboratory 3 laboratory 10
laboratory 4 laboratory 12
Figure 2 — Plating efficiencies of RM-C in the CF assay
5
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ISO/DTR 10993-55:2022(E)
7.2.3 Positive reference materials
Extracts of the test samples RM-A and RM-B were applied in the CF assay as indicated in Annex B. The
results of the 10 participants are summarized in Table 5 and Table 6 and illustrated in Figure 3 and
Figure 4.
Table 5 — Plating efficiencies of RM-A in the CF assay
Plating efficiency
Concentration
of RM-A
%
%
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 12
0,00 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0
0,25 100,0 93,5 90,6 99,5 0,0 92,2 71,8 105,1 100,9 101,9
0,50 101,0 108,3 0,7 96,9 0,0 102,9 15,6 105,5 58,6 102,5
1,00 0,0 91,3 0,0 14,4 0,0 85,2 0,0 32,1 0,4 61,1
2,00 0,0 17,0 0,0 1,0 0,0 16,3 0,0 0,0 0,0 0,0
4,00 0,0 1,1 0,0 0,3 0,0 0,9 0,0 0,0 0,0 0,0
8,00 0,0 0,5 0,0 0,0 0,0 0,5 0,0 0,0 0,0 0,0
Concentration of RM-A
%
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 12
IC 0,75 1,56 0,36 0,78 — 1,51 0,35 0,88 0,55 1,13
50
The IC of RM-A concentrations ranged from 0,35 % to 1,56 %, mean IC was (0,87 ± 0,42) %.
50 50
Key
X extract concentration, % laboratory 5
Y platting efficiency of the control, % laboratory 7
laboratory 1 laboratory 8
laboratory 2 laboratory 9
laboratory 3 laboratory 10
laboratory 4 laboratory 12
Figure 3 — Plating efficiencies of RM-A in the CF assay
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ISO/DTR 10993-55:2022(E)
Table 6 — Plating efficiencies of RM-B in the CF assay
Plating efficiency
Concentration of
RM-B
%
%
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 12
0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0
20 0,0 100,4 96,9 100,8 52,1 88,0 70,2 101,5 90,6 104,1
40 0,0 99,8 73,1 84,1 39,8 83,3 34,7 90,6 102,2 103,1
50 0,0 88,2 5,7 27,6 43,4 79,6 42,6 87,9 82,8 92,2
60 0,0 92,9 0,0 2,0 45,9 65,2 12,3 17,6 28,1 74,9
80 0,0 49,9 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,6
100 0,0 7,4 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0
Concentration of RM-B
%
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 7 Lab 8 Lab 9 Lab 10 Lab 12
IC — 79,95 43,43 46,04 23,41 64,66 33,96 55,39 55,94 66,70
50
The IC of RM-B concentrations ranged from 23,41 % to 79,95 %, mean IC was (52,2 ± 16,5) %.
50 50
Key
X extract concentration in % laboratory 5
Y platting efficiency in % of the control laboratory 7
laboratory 1 laboratory 8
laboratory 2 laboratory 9
laboratory 3 laboratory 10
laboratory 4 laboratory 12
Figure 4 — Plating efficiencies of RM-B in the CF assay
8 Assessment of r esults
Two different tests were used in this study to determine cytotoxicity of three different test samples,
which are recommended as reference materials in the standard. The study demonstrated that both tests
are suitable for this purpose. Despite a considerable variation of the results between the participating
laboratories, the assessment resulted in the same conclusions for nearly all participants.
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ISO/DTR 10993-55:2022(E)
The negative reference material RM-C was non-cytotoxic in both tests and for all laboratories. The
strongest variation from this result was a plating efficiency for the 100 % extract in one laboratory,
which was reduced to 77,8 %. As the non-toxic effect had already been certified, all extract
concentrations ought to have resulted in a 100 % plating efficiency. Therefore, it was considered that
the variations found here in the CF test for RM-C reflect the measurement uncertainty of this kind of
test on extracts with living cells and a threshold value was introduced in ISO 10993-5:2009; only a
reduction of cell viability of more than 30 % is now considered as a cytotoxic effect.
All laboratories found a severe cytotoxicity caused by RM-A in both test systems. IC values were in a
50
quite close range and were clearly lower in the CF test than in the NRU test. This is expected because
contact duration to the extract is one day in NRU but six days in CF. All groups met the validation criteria
for the CF test (IC < 7 %).
50
RM-B showed a much broader variation of the results. For all participants, the material was significantly
cytotoxic [100 % extracts had viabilities of 0 % to 14 % for NRU (data not shown) and 0 % to 7 % for the
CF test (see Table 6)], but the cytotoxic effect was much lower than for RM-A. IC values were similar in
50
both tests, but variation of the results was broader.
The study demonstrated that the proposed testing protocols for the colony formation assay and the
neutral red uptake assay are suitable to assess cytotoxic effects of medical devices after extraction.
These protocols were therefore included in ISO 10993-5:2009. The testing of the 100 % extract gives a
secured finding of extractable components with a cytotoxic potential, the possible grade of cytotoxicity
can be estimated by testing a concentration series.
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ISO/DTR 10993-55:2022(E)
Annex A
(informative)

Interlaboratory study protocol for the neutral red uptake
cytotoxicity test
A.1 General aspects
The test protocol is based on Annex C of Reference [4].
The following protocol describes only those parts of Annex C of Reference [4], relevant for the
implementation of the interlaboratory test. For the general parts (background, rationale as well as
general cell culture procedures like the preparation of a stock) refer to the original document.
A.2 Experimental procedure
A.2.1 Basic procedure
The BALB/c 3T3 cells were seeded into 96-well plates and maintained in culture for 24 h (approximately
one doubling period) to form a semi-confluent monolayer (see Reference [4] for more information on
cell maintenance and culture procedures). Cells were then exposed to the test compound over a range
of eight concentrations. After 24 h exposure, NRU was determined for each treatment concentration
and compared to that determined in control cultures. For each treatment (i.e. concentration of the test
chemical) the percent inhibition of growth was calculated. The IC (i.e. the concentration producing
50
50 % reduction of NR uptake) was calculated from the concentration-response and expressed as μg/ml.
A.2.2 Test limitations
Test limitations as they were described in Reference [4] for the neutral red assay were considered for
this test.
A.2.3 Material
A.2.3.1 Cell lines
BALB/c 3T3 cells, clone 31 (e.g. ECACC # 86110401, European Collection of Cell Cultures, Salisbury,
Wiltshire SP4 OJG, UK; CCL-163, American Type Culture Collection [ATCC], Manassas, VA, USA)
A.2.3.2 Technical equipment
The following technical equipment was used:
— incubator, 37 °C, humidified, 5 % CO /air;
2
NOTE 1 The original document asked for 7,5 % CO /air because cells are very sensitive to pH-changes but
2
was changed to use 5 %, which is more common in most laboratories and to add HEPES (4-(2-Hydroxyethyl)
Piperazine-1-Ethanesulfonic Acid) for better buffering additionally.
— laminar flow clean bench (standard: “biological hazard”);
— water bath, 37 °C;
NOTE 2 Incubation of solutions is technically feasible in an incubator.
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ISO/DTR 10993-55:2022(E)
— inverse phase contrast microscope
— laboratory burner
— centrifuge (optionally: equipped with microtitre plate rotor);
— laboratory balance;
— 96-Well plate photometer equipped with 540 nm filter;
— shaker for microtitre plates;
— cell counter or hemacytometer;
— pipetting aid;
— pipettes, 8-channel-pipettes, dilution block;
— cryotubes;
2 2
— tissue culture flasks (80 cm , 25 cm );
— 96-well tissue culture microtitre plates.
A.2.3.3 Chemicals, media and sera
— Dulbecco’s Modification of Eagle’s Medium (DMEM) without L-Glutamine
— L-Glutamine 200 mM
— Newborn calf serum (NBCS)
ATTENTION — Fetal calf serum (FCS) was not used. FCS causes a strongly reduced optical density
(OD) due to the formation of vacuoles in the cells.
Due to lot variability of NBCS, each lot was first checked for growth stimulating properties with 3T3
cells (20 h to 25 h doubling time) and then reserved in sufficient quantity.
— Trypsin/ethylenediaminetetraacetic acid (EDTA) solution
2+ 2+
— Phosphate buffered saline (PBS) without Ca and Mg (for trypsinization)
— HEPES
2+ 2+
— PBS with Ca and Mg (for rinsing)
— Penicillin/streptomycin solution
— Neutral red (NR)
— Dimethyl sulfoxide (DMSO), analytical grade
— Ethanol (ETOH), analytical grade
— Glacial acetic acid, analytical grade
— Distilled H O or any purified water suitable for cell culture
2
A.2.3.4 Preparations
All solutions (except NR stock solution, NR medium and NR desorbing solution), glassware, etc., were
sterile and all procedures were carried out under aseptic conditions and in the sterile environment of a
laminar flow cabinet (biological hazard standard).
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ISO/DTR 10993-55:2022(E)
A.2.3.5 Media
A.2.3.5.1 General
DMEM (buffered with sodium bicarbonate) supplemented with (final concentrations in DMEM are
quoted):
a) for freezing
— 20 % NBCS
— 7 % to 10 % DMSO
b) for routine culture
— 10 % NBCS
— 4 mM Glutamine or Glutamax
— 100 IU Penicillin
— 100 μg/ml Streptomycin
— 20 mM HEPES
c) for treatment with test samples
— 5 % NBCS
— 4 mM Glutamine or Glutamax
— 100 IU Penicillin
— 100 μg/ml Streptomycin
— 20 mM HEPES
NOTE The serum concentration of treatment medium was reduced to 5 %, since serum proteins can mask
the toxicity of the test substance. Serum was not totally excluded because cell growth is markedly reduced in its
absence.
Complete media was kept at 4 °C and stored for less than two weeks.
A.2.3.5.2 Neutral red stock solution 0,4 %
The neutral red stock
...