SIST EN ISO 3990:2023

SLOVENSKI STANDARD
01-oktober-2023
Zobozdravstvo - Vrednotenje protibakterijskega delovanja zobozdravstvenih
obnovitvenih materialov, zalivnih cementov, tesnilnih mas za fisure in
ortodontskih lepilnih ali zalivnih materialov (ISO 3990:2023)
Dentistry - Evaluation of antibacterial activity of dental restorative materials, luting
materials, fissure sealants and orthodontic bonding or luting materials (ISO 3990:2023)
Zahnheilkunde - Bewertung der antibakteriellen Wirkung von dentalen
Restaurationswerkstoffen, Befestigungszementen, Fissurenversieglern und
kieferorthopädischen Klebe- oder Befestigungswerkstoffen (ISO 3990:2023)
Médecine bucco-dentaire - Évaluation de l'activité antibactérienne des matériaux de
restauration dentaire, matériaux de scellement, produits de comblement des fissures et
matériaux de collage ou de scellement orthodontiques (ISO 3990:2023)
Ta slovenski standard je istoveten z: EN ISO 3990:2023
ICS:
11.060.10 Zobotehnični materiali Dental materials
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 3990
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2023
EUROPÄISCHE NORM
ICS 11.060.10
English Version
Dentistry - Evaluation of antibacterial activity of dental
restorative materials, luting materials, fissure sealants and
orthodontic bonding or luting materials (ISO 3990:2023)
Médecine bucco-dentaire - Évaluation de l'activité Zahnheilkunde - Bewertung der antibakteriellen
antibactérienne des matériaux de restauration Wirkung von dentalen Restaurationswerkstoffen,
dentaire, matériaux de scellement, produits de Befestigungszementen, Fissurenversieglern und
comblement des fissures et matériaux de collage ou de kieferorthopädischen Klebe- oder
scellement orthodontiques (ISO 3990:2023) Befestigungswerkstoffen (ISO 3990:2023)
This European Standard was approved by CEN on 1 July 2023.

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-CENELEC 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-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 3990:2023 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 3990:2023) 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 January 2024, and conflicting national standards shall
be withdrawn at the latest by January 2024.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
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,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 3990:2023 has been approved by CEN as EN ISO 3990:2023 without any modification.

INTERNATIONAL ISO
STANDARD 3990
First edition
2023-07
Dentistry — Evaluation of
antibacterial activity of dental
restorative materials, luting materials,
fissure sealants and orthodontic
bonding or luting materials
Médecine bucco-dentaire — Évaluation de l'activité antibactérienne
des matériaux de restauration dentaire, matériaux de scellement,
produits de comblement des fissures et matériaux de collage ou de
scellement orthodontiques
Reference number
ISO 3990:2023(E)
ISO 3990:2023(E)
© 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
ISO 3990:2023(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Requirements . 2
4.1 General . 2
4.2 Extract . . . 2
4.3 Direct contact . 2
5 Sample preparation and control material preparation . 3
5.1 General . 3
5.2 General requirements and recommendations for sample preparation . 3
5.3 Specific requirements and recommendations for light-curing materials . 4
5.4 Specific requirements and recommendations for chemically setting materials . 4
5.5 Specific requirements and recommendations for CAD/CAM milled or subtractive
manufactured materials . 5
5.6 Sterility of samples . 5
5.7 Preparation of liquid extracts of material . 5
5.7.1 Principles of extraction . 5
5.7.2 Extraction vehicle . 6
5.7.3 Extraction conditions . . 6
5.7.4 Consecutive elution cycles . 6
5.8 Preparation of materials for direct contact tests . 7
5.8.1 Form of samples . 7
5.8.2 Principles of direct contact tests . 7
6 Bacterial strains, nutrient broths and preparation of bacterial cultures .8
7 Test procedures . 8
7.1 General . 8
7.2 Test on extracts . . 9
7.2.1 Tests on extracts toward planktonic cultures of bacteria . 9
7.2.2 Test on extracts toward bacterial biofilms. 10
7.3 Test by direct contact . 11
7.3.1 Test by direct contact toward planktonic cultures of bacteria . 11
7.3.2 Test by direct contact toward bacterial biofilms .12
7.4 Determination of antibacterial effects . 13
7.4.1 General .13
7.4.2 A ssessment of the reduction of the bacterial ability to replicate .13
7.4.3 A ssessment of bacterial membrane damage . 14
7.4.4 A ssessment of the reduction in bacterial metabolic activity . 16
8 A ssessment of results .17
9 Final test report .17
Annex A (informative) Bacterial strains and corresponding nutrient broths .19
Bibliography .21
iii
ISO 3990:2023(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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use
of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed
patent rights in respect thereof. As of the date of publication of this document, ISO had not received
notice of (a) patent(s) which may be required to implement this document. However, implementers are
cautioned that this may not represent the latest information, which may be obtained from the patent
database available at www.iso.org/patents. ISO shall not be held responsible for identifying any or all
such patent rights.
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 106, Dentistry, in collaboration with
the European Committee for Standardization (CEN) Technical Committee CEN/TC 55, Dentistry, in
accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
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 3990:2023(E)
Introduction
Due to the general applicability of in vitro tests for antibacterial activity and their widespread use in
evaluating a large range of dental materials, it is the purpose of this document to define a scheme for
testing which requires decisions to be made in a series of steps rather than to specify a single test. This
should lead to the selection of the most appropriate test for a respective dental material to be evaluated.
Two categories of test are listed: extract test and direct contact test.
The choice of one or more of these categories depends upon the nature of the material to be evaluated,
the potential site of use and the nature of the use of the respective material. Extract tests are primarily
directed to substances leaching out from materials, whereas direct contact tests are directed to both,
effects from leachable substances and surface effects. The choice of test then determines the details of
the preparation of the samples to be tested, the preparation of the cultured bacteria or biofilms, and the
way in which the bacteria or biofilms are exposed to the samples or their extracts.
Both categories of tests are intended to be first conducted toward planktonic cultures of bacteria and
then, in case of positive results, toward bacterial biofilms.
This document proposes measurement of reduction of bacterial ability to replicate as the main method
to assess antibacterial effects. Additionally, bacterial membrane damage can be assessed in order to
further verify bacterial cell death and reductions in bacterial metabolic activity can be investigated as
another measure of bacterial viability.
There are several means of producing results in each of these test categories. The investigator should
be aware of the test categories and into which category a particular technique fits, in order to ensure
the comparability with other results on similar materials both at the intra- and interlaboratory level.
Examples of quantitative test protocols for assessing reduction of bacterial ability to replicate by colony
forming units (CFU) assay and for assessing bacterial membrane damage by flow cytometry and for
investigating reductions in bacterial metabolic activity by MTT assay are given in this document along
with guidance for the interpretation of the results.
v
INTERNATIONAL STANDARD ISO 3990:2023(E)
Dentistry — Evaluation of antibacterial activity of dental
restorative materials, luting materials, fissure sealants and
orthodontic bonding or luting materials
1 Scope
This document specifies test methods for the evaluation of dental restorative materials, luting
materials, fissure sealants and orthodontic bonding or luting materials that are claimed by their
respective manufacturers to exert “antibacterial” effects.
NOTE Materials for pulp capping (e.g. calcium hydroxide formulations), endodontic filling materials, dental
implants or implant systems, nightguards and additive manufactured (e.g. 3D-printed) materials are not covered
in this document.
This document does not cover tests on the effectiveness of sterilization or disinfection procedures. This
document cannot be used to demonstrate a lack of microbial contamination of medical devices used in
dentistry.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 4049, Dentistry — Polymer-based restorative materials
ISO 6344-3, Coated abrasives — Determination and designation of grain size distribution — Part 3:
Microgrit sizes P240 to P5000
ISO 7405, Dentistry — Evaluation of biocompatibility of medical devices used in dentistry
ISO 9917-1, Dentistry — Water-based cements — Part 1: Powder/liquid acid-base cements
ISO 9917-2, Dentistry — Water-based cements — Part 2: Resin-modified cements
ISO 10993-1, Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk
management process
ISO 10993-5, Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity
ISO 10993-12, Biological evaluation of medical devices — Part 12: Sample preparation and reference
materials
ISO 10993-18, Biological evaluation of medical devices — Part 18: Chemical characterization of medical
device materials within a risk management process
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 1942, ISO 7405, ISO 10993-1
and ISO 10993-5 apply.
ISO 3990:2023(E)
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/
3.1
dental restorative material
material or combination of materials specially formulated and prepared for use in dentistry and/or
associated procedures for restoring lost integrity of teeth or for replacing teeth
3.2
positive control material
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
[SOURCE: ISO 7405:2018, 3.3]
3.3
negative control material
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,
nonreactive or minimal response in the test system
Note 1 to entry: In practice, negative control materials include materials lacking the active component that is
responsible for antibacterial activity or materials used in clinical practice with no antibacterial activity.
[SOURCE: ISO 7405:2018, 3.4, modified — Note 1 to entry has been replaced.]
3.4
antibacterial material
material exhibiting antibacterial activity as compared to the negative control material (3.3)
4 Requirements
4.1 General
The material claiming to be antibacterial shall meet one of the requirements in 4.2 and 4.3.
4.2 Extract
For tests on extract, an antibacterial material shall exhibit a median reduction of bacterial ability to
replicate of at least 99,9 % (3 log steps) as compared to the negative control material when tested in
accordance with 7.1.
[1],[2],[3]
NOTE This requirement is in line with the definitions of the American Society of Microbiology .
4.3 Direct contact
For tests by direct contact, an antibacterial material shall exhibit a median reduction of bacterial ability
to replicate at least 99 % (2 log steps) as compared to the negative control material when tested in
accordance with 7.2.
[4]
NOTE This requirement is in line with the definitions outlined in JIS Z 2801 .
ISO 3990:2023(E)
5 Sample preparation and control material preparation
5.1 General
The tests described in this document shall be performed on
a) an extract of the sample, and/or
b) the sample itself.
Assessment of antibacterial properties shall be made on the material prepared in accordance with the
manufacturer’s instructions. Before testing antibacterial properties of dental materials in accordance
with this document, the physical and chemical properties of the material (and extracts) shall be
assessed in accordance with ISO 10993-1 and ISO 10993-18. Before testing antibacterial properties of
polymer-based restorative materials, the physical behaviour of the material should be characterized
according to ISO 4049. Before testing antibacterial properties of cements, the physical behaviour of the
material should be characterized according to ISO 9917-1 or ISO 9917-2, respectively.
Negative and positive control materials shall be included in each assay. If appropriate and possible,
control materials should be prepared by the same procedure as the sample (see 5.2 to 5.5). In all cases,
control materials shall resemble the dimensions and other material properties such as roughness of the
test materials. For direct contact tests, test materials and control materials shall have a circular shape
with a diameter of 10 mm and a thickness of 1 mm to be used in 48-well plates (see 7.3).
[5]
For tests on extracts, 0,2 % chlorhexidine digluconate shall be used as a positive control. Additionally,
to the extracts from the negative control material, nutrient broth used for bacterial culture in the
respective set of experiments (see Annex A for examples) shall be used as further negative control to
ensure experimental validity.
For tests by direct contact, copper plates (circular shape; diameter 10 mm; purity ≥99 %; absence of
[6]
visible surface impurities) shall be used as a positive control. These plates shall be ground with a
P2000 paper in accordance with ISO 6344-3 in order to provide similar roughness as compared to the
samples.
Negative control materials shall not exhibit any antibacterial activity. Therefore, PTFE samples shall be
used that have the same size and dimensions as the test samples.
All test or control samples shall be stored in sterile water at (37 ± 1) °C after mixing/curing/milling
as described by the manufacturer for 24 h prior to testing, e.g. for allowing leaching of monomers
in polymers. After these initial 24 h, all test or control samples shall be tested at once and after
10 consecutive elution cycles (see 5.7.4) to provide an indication on long-term antibacterial activity.
[7]
If antibacterial activity is still observed after 10 elution cycles, a further test after 20 elution cycles
should follow in order to demonstrate a plateau (i.e. a persisting effect) of the antibacterial activity.
Chemical analysis of the extracts should be additionally performed according to ISO 10993-18.
5.2 General requirements and recommendations for sample preparation
Sample preparation shall be in accordance with ISO 7405, ISO 10993-12, ISO 4049, ISO 9917-1 and
ISO 9917-2.
For the preparation of 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. Sample preparation shall take into account the following factors:
a) temperature;
b) humidity;
ISO 3990:2023(E)
c) light exposure: samples of photosensitive materials shall 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;
e) oxygen exposure: for materials that produce an oxygen inhibition layer during hardening, both
ends of the mould shall be covered with transparent oxygen barrier materials (e.g. polyester/mylar
strips) during hardening;
f) samples shall be produced under aseptic conditions; in cases, where this is not possible, the samples
can be sterilized by the method appropriate to the material, if necessary and possible (see 5.7.3).
5.3 Specific requirements and recommendations for light-curing materials
In accordance with ISO 7405, the following factors shall be taken into account, considering the final use
of the light-curing material:
a) Material of sample mould: If possible, the material of the sample mould should be according to
ISO 4049, i.e. stainless steel moulds with a white backing (white filter paper) at the bottom of the
sample. In case this is not possible, reflection coefficients of materials used for sample moulds
should be as close as possible to that of the oral surface to which the material is applied in order to
simulate the clinical situation.
NOTE Suitable sample mould materials with reflection coefficients close to dental hard tissues can be
semi-translucent or white plastic materials such as polyethylene (PE) or polytetrafluoroethylene (PTFE).
b) Light exposure: Light-curing shall be done to simulate clinical usage as closely as possible. This
often requires curing from one side only but sometimes entails a two-sided cure. The cure method
is material and/or process specific. In the case of one-component materials, there shall be no
voids, clefts or air-bubbles present when viewed without magnification. To provide the same level
of curing as would be the case in clinical usage, follow the instructions for use of the material
manufacturer including the recommended powered polymerization activator, which shall include
the emission wavelength region(s), the irradiance and the exposure time. This information shall
be documented in the test report. Care shall be taken to ensure that the light source and operating
condition conform to the instructions for use of the material manufacturer.
c) Oxygen exposure: For materials that produce an oxygen inhibition layer during light-curing, both
ends of the mould shall be covered with transparent oxygen barrier materials (e.g. polyester/mylar
strips) during light-curing.
d) Sample surface treatment: If the material is recommended by the manufacturer for surface
finishing after curing, the sample surfaces shall be ground and polished using the recommended
clinical procedures. If there are no such instructions and if required for testing, the samples shall
be ground on both ends, with a P2000 paper in accordance with ISO 6344-3, after first being set
against the transparent oxygen barrier material.
5.4 Specific requirements and recommendations for chemically setting materials
In accordance with ISO 7405, ISO 9917-1 and ISO 9917-2, the following factors shall be taken into
account, considering the final use of the chemically setting material:
a) Mixing: Mix sufficient material to ensure that the preparation of each sample is completed from
one batch. Prepare a fresh mix for each sample. The mixing shall be performed in accordance with
the respective product standards, if applicable.
ISO 3990:2023(E)
b) Oxygen exposure: For materials that produce an oxygen inhibition layer during chemical curing,
both ends of the mould shall be covered with oxygen barrier materials (e.g. polyester/mylar strips)
during curing.
c) Sample surface treatment: If the material is recommended by the manufacturer for surface
finishing after curing, the sample surfaces shall be ground and polished using the recommended
clinical procedures. If there are no such instructions and if required for testing, the samples shall
be ground on both ends, with a P2000 paper in accordance with ISO 6344-3, after first being set
against the transparent oxygen barrier material.
5.5 Specific requirements and recommendations for CAD/CAM milled or subtractive
manufactured materials
The following factors shall be taken into account, considering the final use of the CAD/CAM milled or
subtractive manufactured material: sample surface treatment: if the material is recommended by the
manufacturer for surface finishing after CAD/CAM milling or subtractive manufacturing, the sample
surfaces shall be ground and polished using the recommended clinical procedures. If there are no such
instructions and if required for testing, the samples shall be ground on both ends, with a P2000 paper
in accordance with ISO 6344-3.
5.6 Sterility of samples
Sterility of the samples shall be taken into account.
Samples from dental materials that are supplied sterile shall be handled aseptically throughout the test
procedure.
Samples from dental materials that are normally supplied non-sterile but are sterilized before use shall
be sterilized by the method recommended by the manufacturer and handled aseptically throughout
the test procedure. The effect of sterilization methods or agents on the dental material should be
considered in defining the preparation of the sample prior to use in the test system.
Samples from dental materials not required to be sterile in use shall be used as supplied and handled
aseptically throughout the test procedure. It can be justifiable to decontaminate the test material in
order to avoid cross-contamination of the bacterial culture; however, the decontamination process shall
not alter the properties of the test material. An immersion in 70 % ethanol for 1 min is recommended
– unless specified otherwise – to reduce cross-contamination, followed by immersion in sterile water
for 1 min. Other methods for decontamination can be used if their efficacy has been proven and if it has
been verified that they do not change material properties.
If non-sterile samples are used, they should be checked for bacterial cross-contamination because the
contamination can lead to a false assessment of antibacterial properties.
5.7 Preparation of liquid extracts of material
5.7.1 Principles of extraction
Preparation of extracts shall be performed after a 24 h-storage in sterile water at (37 ± 1) °C following
mixing/curing as described by the manufacturer and after 10 consecutive elution cycles (see 5.7.4) to
[7]
provide an indication on long-term antibacterial activity .
If antibacterial activity is still observed for the extract after the 10th elution cycle, a further test on an
extract after 20 elution cycles should follow in order to demonstrate a plateau (i.e. a persisting effect) of
the antibacterial activity.
th th
Chemical analysis of the extracts should be additionally performed after the 10 and 20 elution cycle
according to ISO 10993-18.
ISO 3990:2023(E)
Extracting conditions should attempt to simulate or exaggerate the clinical use conditions so as to
determine the potential antibacterial activity without causing significant changes in the sample, such
as fusion, melting or any alteration of the chemical structure, unless this is expected during clinical
application. Due to the nature of certain materials (e.g. biodegradable materials), alteration of the
chemical structure can occur during the extraction procedure.
NOTE The concentration of any endogenous or extraneous substances in the extract, and hence the amount
exposed to the test bacteria, depends on the interfacial area, the extraction volume, pH, chemical solubility,
diffusion rate, osmolarity, agitation, temperature, time and other factors.
5.7.2 Extraction vehicle
The choice of the extraction vehicle(s) taking into account the chemical characteristics of the sample
shall be justified and documented. One or more of the following vehicles shall be used:
a) nutrient broth used for bacterial culture in the respective set of experiments (see Annex A for
examples);
b) phosphate-buffered saline (PBS);
c) serum (for extraction of lipids).
The choice of vehicle should reflect the aim of the extraction. Nutrient broth is the preferred extraction
vehicle because of its ability to extract both polar and non-polar substances.
NOTE It is important to recognize that proteins from protein-rich or serum-containing nutrient broths are
known to bind, to some extent, extractables.
5.7.3 Extraction conditions
The extraction procedures shall be performed in accordance with ISO 10993-5.
The extraction shall be performed in sterile, chemically inert, closed containers by using aseptic
techniques with a volume of extraction vehicle based on the exposed surface area, in accordance with
ISO 10993-12.
With the exception of circumstances given below, the extraction shall be conducted under one of the
following conditions and shall be applied in accordance with the material characteristics and specific
conditions for use:
a) (24 ± 2) h at (37 ± 1) °C;
b) (72 ± 2) h at (37 ± 1) °C.
Other conditions which simulate the extraction that occurs during clinical use or provide an adequate
measure of the antibacterial properties of the material can be used but shall be justified and
documented.
Manipulation of the extract, such as by pH adjustment, filtering, centrifugation or other processing
methods prior to being applied to the bacteria should be avoided because it can influence the result. If
suchlike manipulation still is necessary, these details shall be recorded in the final report along with a
rationale for the additional steps.
5.7.4 Consecutive elution cycles
For providing an indication on long-term antibacterial activity, consecutive elution cycles are performed
[7]
by using the extraction vehicle (see 5.7.2) and extraction conditions (see 5.7.3) as described above .
The whole elution procedure shall be conducted by consecutively performing at least 10 single elution
steps. For each single elution step, the samples shall be stored in the dark at (37 ± 1) °C for either
(24 ± 2) h or (72 ± 2) h. After that period, the elution vehicle shall be renewed, and another elution
ISO 3990:2023(E)
step shall be performed. This procedure shall be repeated 10 times. For this purpose, extract samples
for a period of two weeks. Change the extraction vehicle four times a week in 24 h intervals and one
time at 72 h. This results in 10 changes. The extracts used for testing shall be 24 h extracts and no 72 h
extracts.
In the case antibacterial activity is observed for the extract after the 10th elution cycle, conduct a
further 10 elution cycles and test on an extract after 20 elution cycles in order to demonstrate a plateau
(i.e. a persisting effect) of the antibacterial activity. For this purpose, extract samples for a period of
four weeks. Change the extraction vehicle four times a week in 24 h intervals and one time at 72 h. This
results in 20 changes. The extracts used for testing should be 24 h extracts and no 72 h extracts.
According to ISO 10993-12, extracts should, if possible, be used immediately after preparation to
prevent sorption on to the extraction container or other changes in composition. If an extract is stored
for longer than 24 h refrigerated at 2 °C to 8 °C, then the stability and homogeneity of the extract under
the storage conditions shall be verified.
5.8 Preparation of materials for direct contact tests
5.8.1 Form of samples
The preferred sample of a solid material shall have a circular shape with a diameter of 10 mm and
a thickness of 1 mm to be used in 48-well plates. There should be at least one flat surface. If not,
adjustments shall be made to achieve flat surfaces.
In the case of materials that have another physical state than solid (gels, etc.), they can be tested in their
respective various shapes or sizes without modification in the antibacterial assays. These shapes shall
be similar among all samples. In these cases, the respected volumes of nutrient broth used for bacterial
culture need to be adjusted accordingly.
5.8.2 Principles of direct contact tests
Tests for direct contact shall be performed after a 24 h-storage in sterile water at (37 ± 1) °C following
mixing/curing as described by the manufacturer and after 10 consecutive elution cycles (see 5.7.4) to
[7]
provide an indication on long-term antibacterial activity .
If antibacterial activity is still observed for the material after the 10th elution cycle, a further test on
the material after 20 elution cycles should follow in order to demonstrate a plateau (i.e. a persisting
effect) of the antibacterial activity.
It shall be ensured that all surfaces of the samples are exposed to extraction. Samples sticking together
can result in unextracted surfaces and false positive findings. Use an appropriate rack (see Figure 1) to
mount samples for extraction. The volume of extraction vehicle used to extract samples shall be large,
i.e. at least 10 ml/cm sample surface.
Figure 1 — Example of a rack for mounting of samples
ISO 3990:2023(E)
Use one of the following extraction media:
a) nutrient broth used for bacterial culture in the respective set of experiments (see Annex A for
examples);
b) PBS.
For materials that are not pH-neutral, for example, cements, products containing alkaline glasses, or
that can be subject to corrosion, PBS should be used. In this instance, capacity to recharge shall be
considered as well (e.g. for glass ionomer-based materials).
6 Bacterial strains, nutrient broths and preparation of bacterial cultures
Established bacterial type or reference strains are preferred and, where used, shall be obtained
1)
from recognized repositories . Selection of bacterial strains shall be based on the relevance of these
organisms for the area of application of the respective material (e.g. Streptococcus spp. for materials
related to restorative dentistry or orthodontics). Due to restricted availability and potentially impaired
comparability of the obtained results, no clinical bacterial isolates should be used.
Annex A summarizes recommended bacterial type or reference strains with their corresponding
nutrient broths and solid growth media to be used for the set of experiments described in this document.
2)
All experiments shall be performed in accordance with appropriate microbiological practices .
If a stock culture of a bacterial strain is stored, storage shall be at −80 °C or below in the corresponding
nutrient broth but containing a cryoprotectant, e.g. glycerol. The nutrient broth shall meet the growth
requirements of the selected bacterial type strain.
Only bacterial strains free from any cross-contamination shall be used for the test. Before use, stock
cultures should be tested for the absence of cross-contamination (e.g. by MALDI-TOF analysis). Only
use pure cultures. The nutrient broth used for the experiment shall be sterile and free of any cross-
contamination.
Using the chosen bacterial type or reference strain and nutrient broth, prepare sufficient planktonic
bacterial cultures to complete the assay. Avoid sub-culturing bacterial cultures for more than five times
on agar plates or in liquid cultures because sub-culturing can change the properties of the chosen
bacterial type strain.
7 Test procedures
7.1 General
Assessment of antibacterial properties shall be made on the final finished product.
A minimum of three independent experiments with at least three replicates in each experiment shall be
used for samples and negative control materials and positive control materials.
1) For example, from the American Type Culture Collection (ATCC), the Deutsche Sammlung von Mikroorganismen
und Zellkulturen (DSMZ) or the National Collection of Type Cultures (NCTC).
2) For example, WHO Good Prac
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