EN ISO 7405:2008

SLOVENSKI STANDARD
01-marec-2009
1DGRPHãþD
SIST EN ISO 7405:2000
=RER]GUDYVWYR2YUHGQRWHQMHELRNRPSDWLELOQRVWLPHGLFLQVNLKSULSRPRþNRYY
]RER]GUDYVWYX,62
Dentistry - Evaluation of biocompatibility of medical devices used in dentistry (ISO
7405:2008)
Zahnheilkunde - Beurteilung der Biokompatibilität von in der Zahnheilkunde verwendeten
Medizinprodukten (ISO 7405:2008)
Art dentaire - Évaluation de la biocompatibilité des dispositifs médicaux utilisés en art
dentaire (ISO 7405:2008)
Ta slovenski standard je istoveten z: EN ISO 7405:2008
ICS:
11.060.01 Zobozdravstvo na splošno Dentistry in general
11.100.20 %LRORãNRRYUHGQRWHQMH Biological evaluation of
PHGLFLQVNLKSULSRPRþNRY medical devices
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 7405
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2008
ICS 11.060.10; 11.100 Supersedes EN ISO 7405:1997
English Version
Dentistry - Evaluation of biocompatibility of medical devices
used in dentistry (ISO 7405:2008)
Art dentaire - Évaluation de la biocompatibilité des Zahnheilkunde - Beurteilung der Biokompatibilität von in der
dispositifs médicaux utilisés en art dentaire (ISO Zahnheilkunde verwendeten Medizinprodukten (ISO
7405:2008) 7405:2008)
This European Standard was approved by CEN on 5 December 2008.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 7405:2008: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
This document (EN ISO 7405:2008) has been prepared by Technical Committee ISO/TC 106 "Dentistry" in
collaboration with Technical Committee CEN/TC 55 “Dentistry” the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by June 2009, and conflicting national standards shall be withdrawn at
the latest by June 2009.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 7405:1997.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 7405:2008 has been approved by CEN as a EN ISO 7405:2008 without any modification.

INTERNATIONAL ISO
STANDARD 7405
Second edition
2008-12-15
Dentistry — Evaluation of
biocompatibility of medical devices used
in dentistry
Art dentaire — Évaluation de la biocompatibilité des dispositifs
médicaux utilisés en art dentaire

Reference number
ISO 7405:2008(E)
©
ISO 2008
ISO 7405:2008(E)
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ii © ISO 2008 – All rights reserved

ISO 7405:2008(E)
Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Normative references.1
3 Terms and definitions .2
4 Categorization of medical devices .3
4.1 Categorization by nature of contact .3
4.2 Categorization by duration of contact.3
5 Biological evaluation process.4
5.1 General .4
5.2 Selection of tests and overall assessment.4
5.3 Selection of test methods.4
5.4 Types of test .5
5.5 Re-evaluation of biocompatibility.6
6 Test procedures specific to dental materials .6
6.1 Recommendations for sample preparation .6
6.2 Agar diffusion test.8
6.3 Filter diffusion test .10
6.4 Pulp and dentine usage test.13
6.5 Pulp capping test.17
6.6 Endodontic usage test.19
Annex A (informative) Types of test to be considered for evaluation of biocompatibility of medical
devices used in dentistry.23
Annex B (informative) Dentine barrier cytotoxicity test.25
Annex C (informative) Acute toxicity testing .32
Bibliography.33

ISO 7405:2008(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 7405 was prepared by Technical Committee ISO/TC 106, Dentistry.
This second edition cancels and replaces the first edition (ISO 7405:1997) which has been technically revised.
The following changes have been made:
a) addition of dentine barrier cytotoxicity test to Annex B;
b) improved description of test methods;
c) updated cross-references to ISO 10993 series.
iv © ISO 2008 – All rights reserved

ISO 7405:2008(E)
Introduction
This International Standard concerns the evaluation of the biocompatibility of medical devices used in
dentistry. It is to be used in conjunction with the ISO 10993 series of standards. This International Standard
contains special tests, for which ample experience exists in dentistry and which acknowledge the special
needs of dentistry.
Only test methods for which the members of the committee considered there was sufficient published data
have been included. In recommending test methods, the need to minimize the use of animals was given a
high priority. It is essential that the decision to undertake tests involving animals be reached only after a full
and careful review of the evidence indicating that a similar outcome cannot be achieved by other types of test.
In order to keep the number of animals required for tests to an absolute minimum, consistent with achieving
the objective indicated, it can be appropriate to conduct more than one type of test on the same animal at the
same time, e.g. pulp and dentin usage test and pulp capping test. However, in accordance with ISO 10993-2
these tests are performed both in an efficient and humane way. On all occasions when animal testing is
undertaken, such tests are conducted empathetically and according to standardized procedures as described
for each test.
This International Standard does not explicitly describe test methods for occupationally related risks.
Annexes B and C are included to encourage the development of in vitro and ex vivo test methods which will
further reduce the use of animals in the evaluation of the biocompatibility of medical devices used in dentistry.

INTERNATIONAL STANDARD ISO 7405:2008(E)

Dentistry — Evaluation of biocompatibility of medical devices
used in dentistry
1 Scope
This International Standard specifies test methods for the evaluation of biological effects of medical devices
used in dentistry. It includes testing of pharmacological agents that are an integral part of the device under
test.
This International Standard does not cover testing of materials and devices that do not come into direct or
indirect contact with the patient's body.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 1942, Dentistry — Vocabulary
ISO 6344-1, Coated abrasives — Grain size analysis — Part 1: Grain size distribution test
ISO 10993-1, Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk
management process
ISO 10993-2, Biological evaluation of medical devices — Part 2: Animal welfare requirements
ISO 10993-3, Biological evaluation of medical devices — Part 3: Tests for genotoxicity, carcinogenicity and
reproductive toxicity
ISO 10993-5, Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity
ISO 10993-6, Biological evaluation of medical devices — Part 6: Tests for local effects after implantation
1)
ISO 10993-10 , Biological evaluation of medical devices — Part 10: Tests for irritation and skin sensitization
ISO 10993-11, Biological evaluation of medical devices — Part 11: Tests for systemic toxicity
ISO 10993-12:2007, Biological evaluation of medical devices — Part 12: Sample preparation and reference
materials
ISO 14971, Medical devices — Application of risk management to medical devices

1) To be published.
ISO 7405:2008(E)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 1942, ISO 10993-1, ISO 10993-12
and the following apply.
3.1
medical device
any instrument, apparatus, appliance, software, material or other article, whether used alone or in combination,
together with any accessories, including the software necessary for its proper application intended by the
manufacturer to be used for medical purposes for human beings for the purpose of:
⎯ diagnosis, prevention, monitoring, treatment or alleviation of disease;
⎯ diagnosis, monitoring, treatment, alleviation of or compensation for an injury or handicap;
⎯ investigation, replacement or modification of the anatomy or of a physiological process;
⎯ control of conception;
and which does not achieve its principal intended action in or on the human body by pharmacological,
immunological or metabolic means, but which may be assisted in its function by such means
3.2
dental material
material and/or substance or combination of materials and/or substances specially formulated and prepared
for use in the practice of dentistry and/or associated procedures
3.3
final product
medical device in its “as-used” state
NOTE Many dental materials are used in a freshly mixed state, and evaluation of the materials in both freshly mixed
and set conditions should be considered.
3.4
positive control
positive control material
any well characterized material and/or substance that, when evaluated by a specific test method,
demonstrates the suitability of the test system to yield a reproducible, appropriately positive or reactive
response in the test system
3.5
negative control
negative control material
any well characterized material and/or substance that, when evaluated by a specific test method,
demonstrates the suitability of the test system to yield a reproducible, appropriately negative, non-reactive or
minimal response in the test system
NOTE In practice, negative controls include blanks, vehicles/solvents and reference materials.
3.6
reference material
material with one or more property values that are sufficiently reproducible and well established to enable use
of the material or substance for the calibration of an apparatus, the assessment of a measurement method or
for the assignment of values to materials
NOTE For the purpose of this document, a reference material is any well characterized material and/or substance
that, when tested by the procedure described, demonstrates the suitability of the procedure to yield a reproducible,
predictable response. The response may be negative or positive.
2 © ISO 2008 – All rights reserved

ISO 7405:2008(E)
4 Categorization of medical devices
4.1 Categorization by nature of contact
4.1.1 General
For the purposes of this document, the classification of medical devices used in dentistry is derived from
ISO 10993-1. If a device or material can be placed in more than one category, the more rigorous testing
requirements shall apply. With multiple exposures the decision into which category a device is placed shall
take into account the potential cumulative effect, bearing in mind the period of time over which these
exposures occur.
NOTE In this context the term dentistry includes the oromaxillofacial environment.
4.1.2 Non-contact devices
These devices do not contact the patient's body directly or indirectly, and are not included in ISO 10993-1.
4.1.3 Surface-contacting devices
These devices include those that contact the surface of intact or breached or otherwise compromised skin, the
surface of intact or breached or otherwise compromised oral mucosa, and those that contact the external
surfaces of dental hard tissue, including enamel, dentine and cementum.
NOTE In some circumstances, dentine and cementum are considered as surfaces, e.g. after gingival recession.
4.1.4 External communicating devices
These devices include dental devices that penetrate and are in contact with oral mucosa, dental hard tissues,
dental pulp tissue or bone, or any combination of these, and are exposed to the oral environment.
NOTE This group also includes any kind of lining or base material to be used under a restoration.
4.1.5 Implant devices used in dentistry
These devices include dental implants and other dental devices that are partially or fully embedded in one or
more of the following:
a) soft tissue, e.g. subperiosteal implants and subdermal implants;
b) bone, e.g. endosteal implants and bone substitutes;
c) pulpodentinal system of the tooth, e.g. endodontic materials;
d) any combination of these, e.g. transosteal implants.
4.2 Categorization by duration of contact
4.2.1 General
For the purposes of this document, medical devices used in dentistry are classified by duration of contact as
described in ISO 10993-1 and listed in 4.2.2 to 4.2.4.
4.2.2 Limited exposure devices
Devices whose single or multiple use or contact is likely to be up to 24 h.
4.2.3 Prolonged exposure devices
Devices whose single, multiple or long-term use or contact is likely to exceed 24 h but not 30 d.
ISO 7405:2008(E)
4.2.4 Permanent contact devices
Devices whose single, multiple or long-term use or contact exceeds 30 d.
NOTE 1 The definition of the term “permanent” is meant to be applied solely for the use of this document. It is
consistent with the definition given in ISO 10993-1.
NOTE 2 With multiple exposures to the device, the decision into which category a device is placed should take into
account the potential cumulative effect, bearing in mind the period of time over which these exposures occur.
5 Biological evaluation process
5.1 General
Each medical device used in dentistry shall be subjected to a structured biological evaluation programme
within a risk management process (see ISO 10993-1). Guidance on the implementation of this programme is
provided in ISO 14971 and ISO 10993-1. The biological evaluation programme shall include the review of data
sets concerning the biological properties of each medical device used in dentistry. When this part of the
biological evaluation programme indicates that one or more data sets are incomplete and that further testing is
necessary, the tests should be selected from the methods described in the ISO 10993 series of standards or
in this International Standard, or in both. If tests that are not included in these International Standards are
selected, a statement shall be made that indicates that the tests described in these International Standards
have been considered and shall include a justification for the selection of other tests.
For combination products the final product should be evaluated according to this document in conjunction with
any applicable standards.
NOTE 1 In this context, combination products are dental devices of any kind that incorporate, or are intended to
incorporate, as an integral part, a substance that:
a) if used separately, would be a medicine or a biological product;
b) is liable to affect the patient’s body by an ancillary action.
An example would be a bone filling/augmentation device containing a growth factor (i.e. a biological product).
NOTE 2 For combination products, where the device and pharmacological components are packaged separately, it
may be informative to test the device components alone.
5.2 Selection of tests and overall assessment
The selection of tests and the overall assessment of the results shall be carried out by an expert who has the
appropriate chemical, physical and biological data concerning the device and who is aware of the intended
conditions of use.
5.3 Selection of test methods
The selection of test methods shall be based upon consideration of:
a) the intended use of the medical device;
b) the tissue(s) which the medical device may contact;
c) the duration of the contact.
If a test selected is not included in the International Standards, a justification for the choice of the methods
shall be included in the test report for each device. If more than one test method in the same category is
recommended by the standards, the selection of one test over the others should be justified.
4 © ISO 2008 – All rights reserved

ISO 7405:2008(E)
5.4 Types of test
According to the categorization of the device, tests shall be considered for use as summarised in Table A.1.
This table indicates which types of test method shall be considered, but not that they are necessarily required
to be carried out. A decision not to carry out a type of test identified in Table A.1 shall be justified in the test
report on each device. The types of test listed are regarded as a framework for the evaluation of the
biocompatibility of medical devices used in dentistry. For most types of test, particular methods are identified,
although for some devices it is recognized that alternative methods not included in the International Standards
listed may be more appropriate.
For convenience, the types of test have been listed in three groups.
a) Group I
This group comprises in vitro tests of cytotoxicity. General guidance for in vitro cytotoxicity tests is presented
in ISO 10993-5 and shall be followed. Detailed test protocols for the agar or agarose diffusion and filter
diffusion methods, appropriate to dental materials, are included in this International Standard. The in vitro
cytotoxicity methods include:
1) agar diffusion test (see 6.2);
2) filter diffusion test (see 6.3);
3) direct contact or extract tests in accordance with ISO 10993-5;
4) dentine barrier cytotoxicity test (see Annex B);
5) tooth slice model.
NOTE 1 The order of listing does not indicate any preference for one method over another.
NOTE 2 This list does not indicate that all cytotoxicity tests mentioned have to be performed for each medical device
under consideration.
NOTE 3 The use of the dentine barrier cytotoxicity test is encouraged and a description of the test is presented in
Annex B. Another approach is the tooth slice model. References to this test are presented in the Bibliography.
b) Group II
This group comprises tests in accordance with the 10993 series of standards and particular tests, where
appropriate, are identified:
1) acute systemic toxicity — oral application — in accordance with ISO 10993-11;
2) acute systemic toxicity — application by inhalation — in accordance with ISO 10993-11;
3) subacute and subchronic systemic toxicity — oral application — in accordance with ISO 10993-11;
4) skin irritation and intracutaneous reactivity in accordance with ISO 10993-10;
5) delayed-type hypersensitivity in accordance with ISO 10993-10;
6) genotoxicity in accordance with ISO 10993-3;
7) local effects after implantation in accordance with ISO 10993-6.
NOTE 1 In order to allow use of the latest edition of the referenced document only, an undated cross-reference is
possible. An indication of the appropriate clause and subclause is only possible for dated references. Therefore, the user
of this International Standard is requested to check the referenced documents for the appropriate clause numbers.
ISO 7405:2008(E)
NOTE 2 Information regarding acute toxicity testing is presented in Annex C.
NOTE 3 In the evaluation of materials following local implantation involving mineralized tissues in accordance with
ISO 10993-6, examination of undemineralized sections, in addition to routine demineralized sections, is recommended.
c) Group III
This group comprises tests, specific for medical devices used in dentistry, not referred to in the 10993 series
of standards:
1) pulp and dentine usage test (see 6.4);
2) pulp capping test (see 6.5);
3) endodontic usage test (see 6.6).
NOTE Dental implant system usage test in accordance with ISO/TS 22911 can also be considered, if applicable.
5.5 Re-evaluation of biocompatibility
In accordance with ISO 10993-1, a device shall be considered for re-evaluation of its biocompatibility as
described in 5.4 when revisions or modifications to the formula, quality and/or performance specifications are
made.
6 Test procedures specific to dental materials
6.1 Recommendations for sample preparation
6.1.1 General
These recommendations have been designed for in vitro testing, but can also be used for other purposes, if
suitable.
6.1.2 General recommendations for sample preparation
For the preparation of test samples, consult the respective product standards and/or the manufacturer’s
instructions, and follow those descriptions as closely as possible. Justify any deviation from the
manufacturer's instructions. A detailed description of the sample preparation shall be included in the test
report. Take the following (e.g. environmental) factors into account, considering the final use of the device:
a) temperature;
b) humidity;
c) light exposure: samples of photosensitive materials should be produced under the condition that
ambient light does not activate them;
d) material of sample mould: ensure that the material of the sample mould and eventual lubricant used do
not interfere with the setting process of the material;
NOTE Suitable materials can be semitranslucent or white plastic materials such as polyethylene or
polytetrafluoroethylene (PTFE).
e) oxygen exposure: for materials that produce an oxygen inhibited layer during hardening ensure that the
sample mould is properly sealed during hardening;
6 © ISO 2008 – All rights reserved

ISO 7405:2008(E)
f) sterilization: samples should either be produced under aseptic conditions or be sterilized by the method
appropriate to the material, if necessary and possible; ensure that sterilization does not affect the material
(e.g. sterilization shall not elute substances from material);
g) ratio of sample surface area versus cell layer surface or cell culture medium: document the ratio of
sample surface area versus cell layer surface or cell culture medium; justify the selection of shape and
sample surface area and the applied ratio of sample surface area versus cell layer surface or cell culture
medium;
h) extracts: if extracts are required for a test procedure, prepare extract samples in accordance with
ISO 10993-12:2007, Clause 10.
6.1.3 Specific recommendations for light curing materials
Take the following factors into account, considering the final use of the light curing material:
a) material of sample mould: the reflection coefficient of materials used for sample moulds should be as
close as possible to that of dentin in order to simulate the clinical situation;
NOTE Suitable materials can be semitranslucent or white plastic materials such as polyethylene or PTFE.
b) light exposure: light curing should be done to simulate clinical usage as closely as possible. The
manufacturer's instructions for use should be followed to provide the same level of curing as would be the
case in actual usage. This will often require curing from one side only but will sometimes entail a
two-sided cure. The cure method is material and/or process specific. Where fully cured test samples are
required for testing, it is important to ensure that the test samples are homogeneous after removal from
the mould. In the case of one-component materials, there should be no voids, clefts or air-bubbles
present when viewed without magnification. Reference should be made to the light source used (light
intensity, curing time, spectral distribution of curing light and type of curing light should be documented).
Care shall be taken to ensure that the light source is recommended for the materials to be tested and that
it is in a satisfactory operating condition;
c) oxygen exposure: for materials that produce an oxygen inhibited layer during light curing, both ends of
the mould should be covered with transparent oxygen barrier materials (e.g. a polyester film) during light
curing. If the material is recommended by the manufacturer for surface finishing after curing, the sample
surfaces should be ground and polished using the recommended clinical procedures. If there are no such
instructions and if required for testing, the samples should be ground on both ends, with a P2 000 paper
in accordance with ISO 6344-1, after first being set against the transparent oxygen barrier material.
6.1.4 Specific recommendations for chemically setting materials
Take the following factors into account, considering the final use of the chemically setting materials:
a) mixing: mix sufficient material to ensure that the preparation of each test sample is completed from one
batch. Prepare a fresh mix for each test sample. The mixing shall be performed in accordance with the
respective product standards, if applicable;
b) oxygen exposure: for materials that produce an oxygen inhibited layer during chemical curing, both ends
of the mould should be covered with oxygen barrier materials (e.g. a polyester film) during curing. If the
material is recommended by the manufacturer for surface finishing after curing, the sample surfaces
should be ground and polished using the recommended clinical procedures. If there are no such
instructions and if required for testing, the samples should be ground on both ends, with a P2 000 paper
in accordance with ISO 6344-1, after first being set against the oxygen barrier material.
ISO 7405:2008(E)
6.2 Agar diffusion test
6.2.1 Objective
This test is designed to demonstrate the nonspecific cytotoxicity of test materials after diffusion through agar
or agarose. This test method is not appropriate for leachables that do not diffuse through agar or agarose.
6.2.2 Cell line
Use an established fibroblast or epithelial cell line, which is readily available [e.g. from the American Type
2)
Culture Collection (ATCC), see http://www.atcc.org] . Specify in the report the identification number of the
cell line, if applicable, the description and designation of the cell line used and a justification for its selection.
6.2.3 Culture medium, reagents and equipment
Use the culture medium specified for the selected cell line. Sterilize by filtration. For the preparation of the
agar, prepare a double-concentration of the culture medium. Sterilize by filtration. Prepare either 3 % agar or
3 % agarose. Sterilize by autoclaving.
Prepare the vital stain by diluting a stock solution of 1 % aqueous neutral red solution (record source) 1:100
3)
with 0,01 mol/l phosphate-buffered saline solutions [e.g. Dulbecco’s phosphate-buffered saline solution ]
immediately before use. Store neutral red solutions protected from the light. Use 6-well tissue culture plates
(35 mm in diameter) or Petri dishes of 50 mm to 100 mm in nominal diameter suitable for tissue culture.
6.2.4 Sample preparation
Prepare the samples in accordance with 6.1. The test shall be performed on either an extract of the material
and/or the material itself, according to the guidance in ISO 10993-5.
a) For solid materials, prepare circular test samples of approximately 5 mm diameter, with a flat surface to
ensure adequate contact with the agar overlay.
b) For setting materials, insert the freshly mixed material into rings of internal diameter 5 mm and height
2 mm. The material of the ring shall be stated in the test report. When testing materials in the freshly
mixed state, place the rings on the agar prior to inserting the material. When testing after various setting
periods, fill the rings so that the material is flush with the rim and allow it to set at (37 ± 2) °C and a
relative humidity of (90 ± 10) % until ready for testing.
c) For fluid test samples or extracts, imbibe 0,01 ml of the fluid on a borosilicate microglass filter disc of
5 mm diameter, placed on the agar.
NOTE 1 Suitable inert materials are glass or PTFE.
NOTE 2 Suitable discs can be prepared from prefilters.
6.2.5 Controls
Use positive controls, negative controls and reference materials.

2) This information is given for the convenience of users of this International Standard 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.
3) Dulbecco is a trade name. This information is given for the convenience of users of this International Standard 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.
8 © ISO 2008 – All rights reserved

ISO 7405:2008(E)
6.2.6 Test procedure
Culture the cells until they reach the end of the log growth phase. Pipette the proper volume (e.g. 10 ml for a
100 mm Petri dish) of cell suspension (2,5 × 10 cells/ml) into a sufficient number of Petri dishes and incubate
at (37 ± 2) °C in a water-saturated atmosphere with 5 % (volume fraction) carbon dioxide for 24 h. If different
cell culturing conditions are used, justification shall be provided.
Heat the sterile agar or agarose to 100 °C in a water bath and allow it to cool to 48 °C. Mix one part of agar or
agarose with one part of double-concentrated, freshly prepared culture medium and heat to 48 °C. Aspirate
the liquid culture medium from each Petri dish and replace with 10 ml of freshly prepared agar or
agarose/culture medium mixture.
Allow the agar or agarose/culture medium mixture to solidify at room temperature (approximately 30 min). Add
10 ml neutral red solution and keep dark for 15 min to 20 min. Aspirate excess neutral red solution.
Protect the culture from light in the presence of neutral red, as the cells can be damaged.
Apply to each dish an appropriate number of samples of test material and controls, with an adequate distance
(> 20 mm) between adjacent samples, if applicable. Incubate at (37 ± 2) °C in a water-saturated atmosphere
with 5 % (volume fraction) carbon dioxide for 24 h. Examine each test material at least in quadruplicate
(i.e. two dishes per test material).
6.2.7 Parameters of assessment
Assess the decolorization zone around the test materials and controls using an inverted microscope with a
calibrated screen, and determine a decolorization index and a lysis index for each test sample in accordance
with the criteria specified in Tables 1 and 2.
Table 1 — Decolorization index
Decolorization index Description
0 No detectable decolorization zone around or under specimen
1 Decolorization zone limited to area under specimen
2 Decolorization zone extends less than 0,5 cm beyond specimen
3 Decolorization zone extends 0,5 cm to 1,0 cm beyond specimen
Decolorization zone extends further than 1,0 cm beyond specimen but does not
involve entire dish
5 Decolorization zone involves entire dish
Table 2 — Lysis index
Lysis index Description of decolorized zone
0 No observable cytotoxicity
1 < 20 % of the decolorized zone affected
20 % to < 40 % of the decolorized zone affected
3 40 % to < 60 % of the decolorized zone affected
4 60 % to 80 % of the decolorized zone affected
> 80 % of the decolorized zone affected
ISO 7405:2008(E)
Calculate the median decolorization index and lysis index separately for each test material. If the index values
for the four replicates of the test substance differ by more than 2 units in the range 0 to 3, repeat the test. With
indices of 4 and 5, no repetition is necessary. When extracts are tested, subtract the median index of the
extraction medium alone from the median index of the extraction medium containing test substance to obtain
the index for the test substance alone. If the median index for the extraction medium serving as a control is
> 1, repeat the test using a different extraction medium.
NOTE For a valid test, an intact cell layer should be found under the negative control.
6.2.8 Assessment of results
Take into account all information gathered in the test in assessing the test results, particularly any differences
in results between the experimental and control groups. The cell response is based on the median
decolorization index and lysis index of at least four replicate tests. The cell response shall be graded
separately for each parameter, in accordance with Table 3.
Table 3 — Cell response (graded separately for decolorization index and lysis index)
and interpretation of cytotoxicity
Scale Cell response Interpretation
0 0 Non cytotoxic
1 1 Mildly cytotoxic
2 2 to 3 Moderately cytotoxic
3 4 to 5 Severely cytotoxic
Include the results of the assessment in the test report.
NOTE It should be borne in mind that the interpretation of data from cell culture tests must take the limitations of this
test system into account; i.e. a material that is cytotoxic is not per se unsuitable, but the data must be interpreted for each
specific application.
6.2.9 Test report
Submit the results in a test report that includes a complete record of all procedures followed, all results
obtained and any other data necessary for the assessment of results. Include details of the preparation and
methods of application of the test material, together with the lot number of the material when appropriate.
6.3 Filter diffusion test
6.3.1 Objective
This test is designed to demonstrate the nonspecific cytotoxicity of test materials after diffusion through a
cellulose acetate filter.
6.3.2 Cell line
Use an established fibroblast or epithelial cell line, which is readily available [e.g. from the American Type
Culture Collection (ATCC), see http://www.atcc.org]. Specify in the report the identification number of the cell
line, if applicable, the description and designation of the cell line used, and a justification for its selection.
10 © ISO 2008 – All rights reserved

ISO 7405:2008(E)
6.3.3 Culture medium, reagents and equipment
Prepare culture medium and agar or agarose for use as an overlay as described in 6.2.3. Prepare solutions
either for succinate dehydrogenase staining or for nonspecific hydrolase staining.
For succinate dehydrogenase staining, prepare the following stock solutions:
a) succinate solution, 13,6 g sodium succinate in 100 ml of 0,2 mol/l phosphate buffer, pH 7,6;
b) nitro blue tetrazolium chloride solution, 100 mg nitro blue tetrazolium chloride in 100 ml of 0,2 mol/l
phosphate buffer, pH 7,6;
c) phenazine methosulfate solution, 4 mg phenazine methosulfate in 10 ml fresh distilled water.
Prepare a staining solution of 1 ml succinate solution, 9 ml nitro blue tetrazolium chloride solution and 1 ml
phenazine methosulfate solution.
For nonspecific hydrolase staining, prepare a stock solution of fluorescein diacetate consisting of 5 mg
fluorescein diacetate in 1 ml acetone. For use, add 20 µl of stock solution to 100 ml phosphate-buffered saline
solution (e.g. Dulbecco’s phosphate-buffered saline solution). Use Petri dishes of 60 mm nominal diameter,
suitable for tissue culture.
Use filters, composed of a mixture of cellulose acetate and cellulose nitrate, 47 mm diameter, 0,45 µm pore
4)
size .
6.3.4 Sample preparation
Prepare the samples in accordance with 6.1. The test shall be performed on either an extract of the material
or the material itself, according to the guidance in ISO 10993-5.
a) For solid materials, prepare circular test samples of approximately 5 mm diameter, with a flat surface to
ensure adequate contact with the filter. The mass of the test samples shall not exceed 3,5 g.
b) For setting materials, insert the freshly mixed material into rings of internal diameter 5 mm and height
2 mm. When testing materials in the freshly mixed state, place
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