SIST EN ISO 16954:2015

SLOVENSKI STANDARD
SIST EN ISO 16954:2015
01-september-2015
=RER]GUDYVWYR3UHVNXVQHPHWRGH]DUDYQDQMH]YRGRY]RER]GUDYVWYHQLKHQRWDK
SRQDþLQXELRILOPD,62
Dentistry - Test methods for dental unit waterline biofilm treatment (ISO 16954:2015)
Zahnheilkunde - Prüfverfahren zur Biofilmbehandlung der wasserführenden Leitungen
einer dentalen Behandlungseinheit (ISO 16954:2015)
Médecine bucco-dentaire - Méthodes d'essais pour le traitement du biofilm dans les
conduites d'eau de l'unit dentaire (ISO 16954:2015)
Ta slovenski standard je istoveten z: EN ISO 16954:2015
ICS:
11.060.20 =RERWHKQLþQDRSUHPD Dental equipment
SIST EN ISO 16954:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16954:2015

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SIST EN ISO 16954:2015

EUROPEAN STANDARD
EN ISO 16954

NORME EUROPÉENNE

EUROPÄISCHE NORM
July 2015
ICS 11.060.20
English Version
Dentistry - Test methods for dental unit waterline biofilm
treatment (ISO 16954:2015)
Médecine bucco-dentaire - Méthodes d'essais pour le Zahnheilkunde - Prüfverfahren zur Biofilmbehandlung der
traitement du biofilm dans les conduites d'eau de l'unit wasserführenden Leitungen einer dentalen
dentaire (ISO 16954:2015) Behandlungseinheit (ISO 16954:2015)
This European Standard was approved by CEN on 23 April 2015.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16954:2015 E
worldwide for CEN national Members.

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SIST EN ISO 16954:2015
EN ISO 16954:2015 (E)
Contents Page
European foreword .3

2

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SIST EN ISO 16954:2015
EN ISO 16954:2015 (E)
European foreword
This document (EN ISO 16954:2015) 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 2016, and conflicting national standards shall be withdrawn at
the latest by January 2016.
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.
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, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 16954:2015 has been approved by CEN as EN ISO 16954:2015 without any modification.

3

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SIST EN ISO 16954:2015

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SIST EN ISO 16954:2015
INTERNATIONAL ISO
STANDARD 16954
First edition
2015-07-01
Dentistry — Test methods for dental
unit waterline biofilm treatment
Médecine bucco-dentaire — Méthodes d’essais pour le traitement du
biofilm dans les conduites d’eau de l’unit dentaire
Reference number
ISO 16954:2015(E)
©
ISO 2015

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SIST EN ISO 16954:2015
ISO 16954:2015(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2015 – All rights reserved

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SIST EN ISO 16954:2015
ISO 16954:2015(E)

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Treatment methods . 3
5 Test water and bacterial challenge suspensions . 3
5.1 Test water . 3
5.1.1 Reagents . 3
5.1.2 Preparation of hardness stock solution 1 . . 3
5.1.3 Preparation of hardness stock solution 2 . . 3
5.1.4 Preparation of test water prior to inoculation . 3
5.2 Bacterial challenge. 4
5.3 Inoculated test water . 4
6 Test apparatus . 4
6.1 Test apparatus design . 5
6.1.1 General. 5
6.1.2 Considerations specific to antimicrobial materials and materials which
prevent microbial adhesion. 6
6.2 Test apparatus operation . 6
6.2.1 Flow rates . 6
6.2.2 Flow patterns (on-off cycles) . 6
6.2.3 Test environment temperature and preconditioning period . 7
7 Test procedures . 7
7.1 Testing sequence . 7
7.2 Biofilm prevention or inhibition . 7
7.2.1 General. 7
7.2.2 Pretreatment of the test apparatuses . 8
7.2.3 Concurrent exposure to inoculated test water and the treatment method
for biofilm prevention or inhibition . 8
7.2.4 Microbiological sampling and testing . 8
7.2.5 Test duration . 8
7.2.6 Analysis of results . 9
7.3 Biofilm removal . 9
7.3.1 General. 9
7.3.2 Initial microbiological sampling and testing . 9
7.3.3 Application of the biofilm removal treatment method .10
7.3.4 Final microbiological sampling and testing .10
7.3.5 Analysis of results .10
8 Microbiological sampling and testing .11
8.1 Enumeration of bacteria levels in procedural water .11
8.1.1 Sampling.11
8.1.2 Total viable count test procedure .11
8.1.3 Alternative total viable count test procedure .11
8.2 Biofilm on waterline surfaces .12
8.2.1 Sampling.12
8.2.2 Biofilm assessment test procedure .12
9 Test Report .13
Bibliography .14
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SIST EN ISO 16954:2015
ISO 16954:2015(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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 106, Dentistry, Subcommittee SC 6, Dental equipment.
This first edition of ISO 16954:2015 cancels and replaces the first edition of ISO/TS 11080:2009, of which
it constitutes a technical revision.
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SIST EN ISO 16954:2015
INTERNATIONAL STANDARD ISO 16954:2015(E)
Dentistry — Test methods for dental unit waterline
biofilm treatment
1 Scope
This International Standard provides type test methods for evaluating the effectiveness of treatment
methods intended to prevent or inhibit the formation of biofilm or to remove biofilm present in dental
unit procedural water delivery systems under laboratory conditions.
This International Standard does not apply to devices intended to deliver sterile procedural water or sterile
solution. It also does not apply to lines, tubing, or hoses that deliver compressed air within the dental unit.
This International Standard does not establish specific upper limits for bacterial contamination
or describe test methods to be used in clinical situations. It also does not establish test methods for
evaluating any deleterious side effects potentially caused by treatment methods.
The test methods provided in this International Standard can be used to test other dental equipment
that delivers non-sterile water to the oral cavity.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. 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 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO 7494-1, Dentistry — Dental units — Part 1: General requirements and test methods
ISO 7494-2, Dentistry — Dental units — Part 2: Water and air supply
ISO 10523, Water quality — Determination of pH
ISO 19458, Water quality — Sampling for microbiological analysis
IEC 60601-1, Medical electrical equipment — Part 1: General requirements for basic safety and essential
performance
3 Terms and definitions
For the purposes of this document, the terms and definitions given in IEC 60601-1, ISO 1942, ISO 7494-1,
and ISO 7494-2 and the following apply.
3.1
biofilm
structured community of microorganisms inhabiting a self-developed extracellular biopolymeric matrix
attached to a surface
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SIST EN ISO 16954:2015
ISO 16954:2015(E)

3.2
dental unit
combination of interconnected dental equipment and dental instruments constituting a functional
assembly for use in the provision of dental treatment
[SOURCE: ISO 1942:2009, 2.86]
3.3
dental unit procedural water delivery system
system of components of a dental unit which convey water from a supply source to one or more outlets
used for dental treatment
3.4
procedural water
water supplied by the dental unit for use in the oral cavity
EXAMPLE Handpiece procedural water, multifunctional syringe water, scaler procedural water, or rinse cup
water.
[SOURCE: ISO 7494-2:2003, 3.1]
3.5
surrogate dental unit water system
test apparatus which accurately recreates the procedural water delivery system of a dental unit, including
design, construction, configuration, and operation of all water-bearing elements of the procedural water
delivery system, but not necessarily including other dental unit components which do not directly come
in contact with or control the flow of procedural water
3.6
test water
water having specified chemical and physical characteristics used for testing prior to the addition of the
specified bacterial challenge suspension
3.7
bacterial challenge suspension
consortium of specified bacteria suspended in a nutrient growth medium or buffered solution used to
inoculate test water
3.8
inoculated test water
prepared aqueous suspension used in testing, containing specified amounts of sterilized test water and
one or more bacterial challenge suspension(s)
3.9
test apparatus for the control group
apparatus used in testing in which no treatment method is applied and no antimicrobial material is
present in the waterline components
3.10
test apparatus for the test group
apparatus used in testing in which a treatment specified by the dental unit manufacturer is applied and
any antimicrobial materials specified by the manufacturer are present in the waterline components,
unless otherwise specified in the test requirements
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SIST EN ISO 16954:2015
ISO 16954:2015(E)

4 Treatment methods
Depending upon the specific technical approach of a treatment method and its intended benefits, the
performance objectives of a dental unit procedural water delivery system treatment method can include
one or both of the following:
— prevention or inhibition of biofilm formation on surfaces within the dental unit procedural water
delivery system;
— removal of biofilm from surfaces within the dental unit procedural water delivery system.
This International Standard specifies separate test methods for each of the above performance
objectives. These requirements can be expanded upon, for example to include additional replicates
or test scenarios. Additions to the test method shall follow the general principles of this International
Standard and be fully described in the test report.
5 Test water and bacterial challenge suspensions
5.1 Test water
This subclause specifies the preparation of test water prior to inoculation.
5.1.1 Reagents
5.1.1.1 Water, in accordance with ISO 3696:1987, grade 3.
5.1.1.2 Calcium chloride (CaCl ), or an equivalent molar quantity of a calcium chloride hydrate.
2
5.1.1.3 Magnesium chloride (MgCl ), or an equivalent molar quantity of a magnesium chloride hydrate.
2
5.1.1.4 Sodium bicarbonate (NaHCO ).
3
5.1.1.5 Tryptic soy broth (TSB), 1/3-strength, 10,0 g tryptic soy medium per litre broth.
5.1.1.6 Sodium hydroxide (NaOH), 1 mol/l.
5.1.1.7 Hydrochloric acid (HCl), 1 mol/l.
5.1.2 Preparation of hardness stock solution 1
Dissolve 74,0 g of calcium chloride (5.1.1.2) and 31,7 g of magnesium chloride (5.1.1.3) in 1,00 l water
(5.1.1.1). Hardness stock solution 1 shall be sterilized by heat or filter-sterilized using a 0,2 μm microfilter
and used within 24 h or stored at (5 ± 3) °C for up to 6 months.
5.1.3 Preparation of hardness stock solution 2
Dissolve 56,0 g of sodium bicarbonate (5.1.1.4) in 1,00 l water (5.1.1.1). Hardness stock solution 2 shall be
filter-sterilized using 0,2 μm microfilter and used within 24 h or stored at (5 ± 3) °C for up to 6 months.
Hardness stock solution 2 is not to be heat sterilized.
5.1.4 Preparation of test water prior to inoculation
For each litre of test water to be prepared, add 1,00 ml of 1/3-strength TSB (5.1.1.5) and 1,80 ml of
hardness stock solution 1 (5.1.2) to 1,00 l water (5.1.1.1) and steam sterilize. After the sterilized solution
has cooled, for each litre of test water add 4,00 ml of hardness stock solution 2 which has been filter-
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ISO 16954:2015(E)

sterilized using a 0,2 μm microfilter. Adjust the pH to 7,0 to 8,0, measured according to ISO 10523, by
adding sodium hydroxide (5.1.1.6) or hydrochloric acid (5.1.1.7). The test water shall be used within
24 h or stored at (5 ± 3) °C for up to one week.
NOTE 1 The hardness of the prepared test water is approximately 1,8 mmol of calcium ions per litre (equivalent
to approximately 180 mg/l as CaCO ). This corresponds to the upper limit of the generally accepted range for
3
[17]
hard water.
NOTE 2 The test water has a concentration of approximately 10 mg TSB per litre, which yields a total organic
carbon (TOC) level of approximately 4 mg/l, although the exact TOC level might vary somewhat. This approximate
[1]
TOC level is consistent with the recommended upper limit of 4 mg/l for TOC in chlorinated drinking water and
is included in the test water to reduce the time for biofilm formation.
5.2 Bacterial challenge
Bacterial challenge suspensions used to inoculate the test water shall be prepared with the following
bacteria from the American Type Culture Collection (ATCC) or an international authorized ATCC distributor:
a) Pseudomonas aeruginosa (ATCC #700888);
b) Klebsiella pneumoniae (ATCC #13882).
Alternate strains of P. aeruginosa and K. pneumoniae (i.e. different ATCC numbers for the same bacterium
species) can be substituted if the specified strains are not available, provided that the isolation source
indicated by ATCC is water or a water system.
The bacterium species shall be separately reconstituted in sterilized dilute TSB, having a concentration
of 0,3 g TSB per litre. The reconstituted cultures shall be used within eight transfer passages. Cultivation
of the bacteria for inoculating test water shall be performed one day before preparing the inoculated
test water in accordance with 5.3.
Compliance with appropriate laboratory safety practices is critical when working with these bacteria,
including the handling of waste that can be contaminated with these bacteria.
5.3 Inoculated test water
On days of test apparatus operation inoculated test water shall be prepared within two hours before
the commencement of the daily flow program specified in 6.2.2 by inoculating sterilized test water (5.1)
1
with both of the reconstituted bacterial cultures (5.2), to achieve a concentration of 5 × 10 CFU/ml to
2 2 3
5 × 10 CFU/ml of each of the bacteria and a total bacterial concentration of 10 CFU/ml to 10 CFU/ml
in the inoculated test water. The temperature of the sterilized test water at the time of inoculation shall
be (23 ± 3) °C.
To ensure accurate concentrations of each bacterium species in the inoculated test water, it can be useful
to centrifuge and re-suspend each of the reconstituted bacterial cultures in sterile phosphate buffer
and determine the approximate bacterial concentration using turbidimetric measurements. Using these
results, the volume of each of the single-species bacterial suspensions to be added to the test water can
be calculated. Alternatively, other methods for achieving the specified inoculation range can be used.
6 Test apparatus
The test apparatuses shall consist of a specified number of either dental units or surrogate dental unit
water systems which closely replicate the dental unit procedural water system.
In order to achieve reproducible results, all components of the test apparatuses which are in contact
with the procedural water shall be new each time the test procedure for biofilm prevention or inhibition
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ISO 16954:2015(E)

(7.2) or the sequence of test procedures for biofilm prevention or inhibition (7.2) and for biofilm removal
(7.2) is performed.
NOTE As specified in 7.3.1, the test procedure for biofilm removal (7.3) is performed after first developing
biofilm in the test apparatuses per the test procedure for biofilm prevention or inhibition (7.2).
6.1 Test apparatus design
6.1.1 General
If the test apparatuses consist of dental units, the dental units shall represent the most challenging
model or configuration (when more than one model or configuration is available from the manufacturer).
Length of waterlines, number of branch waterlines and likelihood of stagnation are among the factors
that shall be considered in determining the most challenging model or configuration.
If the test apparatuses consist of surrogate dental unit water systems, the surrogate dental unit water
systems must be able to simulate the basic clinical performance parameters of a functioning dental
unit, including as described in 6.2. The surrogate dental unit water systems must represent the most
challenging model or configuration of the dental unit which the surrogate dental unit water systems are
intended to represent (when more than one model or configuration is available from the manufacturer).
Surrogate dental unit water systems shall accurately recreate the procedural water delivery system,
including design, construction, configuration and operation of the water-bearing elements of the
procedural water delivery system. Other components which do not directly come in contact with or
control the flow of procedural water need not be included, such as structural and decorative components.
The components of surrogate dental unit water systems shall be subject to the same environmental
conditions (i.e. light exposure and temperature) as components in the dental units which they represent.
Any air gap system or other backflow prevention device required to comply with the backflow prevention
requirements of ISO 7494-2 for isolating the procedural water from the incoming water shall be included
in the test apparatuses.
Critical elements to be recreated by the surrogate dental unit water systems includes the following:
— configuration of waterlines (placement of components, arrangement of branch lines, dead legs, etc.);
— tubing diameter(s);
— tubing length(s);
— tubing material(s);
— other componen
...