oSIST prEN 16756:2014
(Main)Antimicrobial wound dressings - Requirements and test methods
Antimicrobial wound dressings - Requirements and test methods
This standard specifies requirements and test methods for the antimicrobial activity of antimicrobial wound dressings. It is designed for microbicidal and microbistatic dressings.
Test methods specifically for microbial binding are not included in the standard.
Antimikrobielle Wundauflagen - Anforderungen und Prüfverfahren
Diese Norm legt Anforderungen und Prüfverfahren für die antimikrobielle Wirkung antimikrobieller Wundauflagen fest. Sie ist vorgesehen für mikrobizide und mikrobistatische Auflagen.
Prüfverfahren speziell für mikrobielle Bindungen sind nicht in dieser Norm enthalten.
Pansements antimicrobiens - Exigences et méthodes d'essai
La présente norme spécifie les exigences et méthodes d’essai applicables à l’activité antimicrobienne de pansements antimicrobiens. Elle s’applique aux pansements microbicides et microbiostatiques.
Les méthodes d’essai spécialement applicables à la fixation microbienne ne sont pas incluses dans la norme.
Antimikrobni sanitetni material - Zahteve in preskusne metode
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 16756:2014
01-september-2014
Antimikrobni sanitetni material - Zahteve in preskusne metode
Antimicrobial wound dressings - Requirements and test methods
Antimikrobielle Wundauflagen - Anforderungen und Prüfverfahren
Pansements antimicrobiens - Exigences et méthodes d'essai
Ta slovenski standard je istoveten z: prEN 16756
ICS:
11.120.20 Sanitetni materiali, obveze in Wound dressings and
komprese compresses
oSIST prEN 16756:2014 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST prEN 16756:2014
---------------------- Page: 2 ----------------------
oSIST prEN 16756:2014
EUROPEAN STANDARD
DRAFT
prEN 16756
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2014
ICS 11.120.20
English Version
Antimicrobial wound dressings - Requirements and test methods
Pansements antimicrobiens - Exigences et méthodes Antimikrobielle Wundauflagen - Anforderungen und
d'essai Prüfverfahren
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee CEN/TC 205.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16756:2014 E
worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
Contents
Page
Foreword . iv
Introduction .v
1 Scope .6
2 Normative references .6
3 Terms and definitions .6
4 Classification .7
5 Requirements .7
5.1 Microbicidal dressings .7
5.2 Microbistatic dressings .7
5.3 Performance Table.7
6 Information to be supplied .7
Annex A (normative) Validation of dilution-neutralisation .9
A.1 Principle .9
A.2 Neutralizer selection .9
A.3 Preparation of microbial suspension .9
A.4 Test validation .9
A.5 Neutralizer toxicity . 10
A.6 Test organism viability . 11
A.7 Neutralisation Effectiveness . 11
A.8 Interpretation of data . 12
Annex B (informative) Neutralizers . 13
Annex C (informative) Rationale . 14
C.1 Title . 14
C.2 Microbial strains . 14
C.3 Cutting dressings . 15
C.4 Positive control . 15
C.5 Microbistatic requirements . 15
C.6 Performance Requirements . 15
C.7 Test method Information . 15
C.8 Media . 16
C.9 Incubation Temperatures . 16
C.10 Dressing Classification . 16
Annex D (informative) Apparatus . 17
Annex E (informative) Reagents and culture media . 18
E.1 General . 18
E.2 Water . 18
E.3 Tryptone Soya Agar . 18
E.4 Maximum recovery diluent . 18
E.5 Simulated Wound Fluid (SWF) . 18
E.6 Sabouraud agar (SAB) . 19
E.7 Neutralizer . 19
Annex F (informative) Reference strains. 20
F.1 Storage of strains . 20
F.2 Bacterial strains . 20
F.3 Yeasts and molds . 20
F.4 Other Strains . 20
Annex G (informative) Preparation of microbial suspensions . 21
G.1 Stock Cultures . 21
---------------------- Page: 4 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
G.2 Working Culture . 21
Annex H (normative) Test methods . 22
H.1 Introduction . 22
H.2 Equilibration of reagents . 22
H.3 Neutralizer . 22
H.4 Choice of experimental conditions . 22
H.5 Sample Size . 23
H.6 Direct contact test method . 23
H.7 Shaking method . 24
H.8 Two compartment method . 25
H.9 Recovery and enumeration of test organisms . 26
H. 10 Calculation and expression of results . 26
H.11 Test report . 29
Annex I (informative) Figures and illustrations . 30
Annex ZA (informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 93/92/EEC on medical devices . 32
Bibliography . 33
iii
---------------------- Page: 5 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
Foreword
This document (prEN 16756:2014) has been prepared by Technical Committee CEN/TC 205 “Non-active
medical devices”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive(s).
For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this document.
---------------------- Page: 6 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
Introduction
This standard describes a range of test methods for establishing whether a wound dressing exerts
antimicrobial activity.
The laboratory tests attempt to simulate conditions of application, through the use of appropriate test fluids,
temperature, organisms, and contact times reflecting the parameters found in clinical situations. Conditions
which may influence the action of wound dressings having antimicrobial properties should be included.
The conditions are intended to cover general purposes and to allow comparison between laboratories and
product types.
v
---------------------- Page: 7 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
1 Scope
This standard specifies requirements and test methods for the antimicrobial activity of antimicrobial wound
dressings. It is designed for microbicidal and microbistatic dressings.
Test methods specifically for microbial binding are not included in the standard.
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.
EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the
determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal
(including bacteriophages) activity.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
antimicrobial dressing
wound dressing which can be shown to exert antimicrobial activity when tested to the appropriate tests in
Annex H of this standard
3.2
negative control dressing
wound dressing which is the same dressing as the dressing to be tested but without the antimicrobial
treatment
Note 1 to entry: If this is not available, then use a non-medicated dressing from the same product group preferably
from the same manufacturer. If no similar product is available, a sterile gauze swab of the same weight shall be used
3.3
plate count method
method in which the number of microorganisms present after incubation is calculated by counting the number
of colonies according to a ten-fold dilution method
3.4
neutralizer
chemical agents used to inactivate, neutralize, or quench the antimicrobial properties of antimicrobial agents
3.5
test dressing
wound dressing which is to be tested to assess its antimicrobial activity
3.6
saturation volume
volume of fluid (SWF) absorbed by the dressing as determined by the method in H.4.3
Note 1 to entry: This volume of fluid is added to the dressing to prevent the dressing absorbing all of the challenge
media (ISWF).
6
---------------------- Page: 8 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
4 Classification
Classification of antimicrobial dressings is not presently standardized. The following two classes are thought
to be useful in the context of this standard when selecting appropriate test methods as described in Table 1.
Microbicidal – capability of the dressing to produce at least a 3-log reduction in the number of viable cells
from the challenge organisms when tested under the conditions in Annex H.
Microbistatic – capability of the dressing to prevent further growth of the initial inoculum but which does not
achieve at least a 3 log reduction when tested under the conditions in Annex H.
If a manufacturer is claiming compliance with this standard, they must comply with the requirements in
Monographs 6 and 7.
NOTE The editing panel recommend that the usefulness of these definitions be reviewed during the development of
the standard.
5 Requirements
5.1 Microbicidal dressings
When tested using the relevant method in Annex H, antimicrobial dressings shall demonstrate at least a 3-log
reduction in activity as determined by that particular test method over 24 hours and against all three test
organisms.
5.2 Microbistatic dressings
When tested using the relevant method in Annex H, microbistatic dressings shall prevent further growth of the
initial inoculum over 24 hours and against all three test organisms.
5.3 Performance Table
Table 1 shows the performance requirements for antimicrobial dressing pending on the two classifications.
Table 1 — Performance requirements for antimicrobial dressings
Performance requirements
Microbicidal Microbistatic
A ≥ 3 0 ≤ A < 3
A = Antimicrobial activity (see Annex H.10)
6 Information to be supplied
The following information shall be supplied on request:
the Annex number of the test method used;
details of the test dressing, negative control dressing, and the mean values of C C , T , and T
0 t 0 t;
the antimicrobial activity A;
the full test report (see Tables 2 and 3 as examples).
7
---------------------- Page: 9 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
Table 2 — Example for a test report (blank)
Test dressing ______________________________________________________________
Control dressing ______________________________________________________________
Test method Annex ________________________________________________________
Exposure period ______________________________________________________________
Organism Dressing Time 0 Log Time XX Log Antimicrobial
10 10
counts per counts per Activity (A)
sample (Ms) sample (Ms)
Control
S. aureus
Test
Control
P. aeruginosa
Test
Control
C. albicans
Test
Result _______________________________________
NOTE The standard test time is (24 ± 1) h. Any additional exposure times should be provided in the same
format.
Table 3 — Example for a test report (filled out)
Test dressing Silver foam_____________________________________________________
Control dressing Silver-free foam_________________________________________________
Test method Annex H.7_____________________________________________________
Exposure period 24 hours_______________________________________________________
Organism Dressing Time 0 Log Time 24 h Log Antimicrobial
10 10
counts per counts per Activity (A)
sample (Ms) sample (Ms)
Control 6.3 6.7 - 0.4
S. aureus
Test 6.3 2.1 4.2
Control 6.4 7.0 - 0.6
P. aeruginosa
Test 6.1 1.2 4.9
Control 6.1 6.2 - 0.1
C. albicans
Test 6.1 < 1.6 > 4.5
Result Microbicidal_____________________________
8
---------------------- Page: 10 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
Annex A
(normative)
Validation of dilution-neutralisation
A.1 Principle
The effectiveness of antimicrobial agents incorporated into wound dressings is measured by their ability to kill
microorganisms within a specified contact time. Consequently, accurate determination of antimicrobial activity
requires complete and immediate neutralisation of the antimicrobial agent. Inefficient or incomplete
neutralisation will permit killing or inactivation of microorganisms to continue beyond the experimental
exposure time, resulting in an overestimation of antimicrobial activity. A neutralizer is therefore required for
each antimicrobial dressing to inactivate or quench the microbicidal properties of the antimicrobial agent.
Neutralising agents may also be incorporated into agar to increase the neutralising capacity for the test
methods detailed in Annex H. Agar containing neutralising agents may be used for the enumeration of
microbial test suspensions providing its neutralisation effectiveness has been validated and it does not have
an inhibitory effect on organism viability.
A.2 Neutralizer selection
Refer to Annex B for examples of suitable neutralizers for certain antimicrobial agents. ASTM E1054-08 also
documents examples of appropriate neutralizers for antimicrobial agents.
The neutralizer selected should, where possible, minimize the neutralizer volume to allow the use of 1ml pour
plates to be used from the neat dilution, therefore maximising the sensitivity of the test.
A.3 Preparation of microbial suspension
NOTE The reference strains detailed in Annex F should be used in the neutralisation assay.
a) Prepare the microbial suspensions in accordance with Annex G.
b) Separately dilute each microbial suspension in MRD such that when a 100 µl volume is inoculated, the
final concentration within each neutralizer validation suspension contains 30 to 100 cfu/ml (see
Table A.1).
c) Using standard microbiological techniques perform an inoculum count on each suspension to confirm the
inoculum concentration.
A.4 Test validation
The neutralisation evaluation consists of three experimental tests (i.e. neutralizer toxicity, test organism
viability and neutralisation effectiveness test) for each test strain. At least three replicates must be performed
for each experimental condition. Table A.1 details the specific requirements of each experimental test which
are dependent on the chosen test method.
NOTE Each experimental test must simulate the test conditions as those detailed in Annex H (e.g. sample volumes,
dilution ratios and incubation conditions must replicate those in the chosen test method).
9
---------------------- Page: 11 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
Table A.1 — Requirements for the neutralisation validation
Test Method
Requirements Direct Contact Method Shaking Method Two Compartment
(see H.6) (see H.7 Method (see H.8)
Neutralizer toxicity 8.9 ml Neutralizer 4.4 ml Neutralizer 4.4 ml Neutralizer
+ 100 µl inoculum + 100 µl inoculum + 100 µl inoculum
+ 1 ml MRD + 0.5 ml SWF + 0.5 ml SWF
Test organism viability 8.9 ml MRD 4.4ml MRD 4.4 ml MRD
+ 100 µl inoculum + 100µl inoculum + 100 µl inoculum
+ 1 ml MRD + 0.5ml SWF + 0.5 ml SWF
Neutralisation Dressing sample as per Dressing sample as per Dressing sample as per
effectiveness H.5 H.5 H.5
+ 5 x 0.1 ml SWF + 5 ml SWF + the + 2.5 ml SWF + the
as per H.6.3 determined SV as per determined SV as per
H.4.3 H.4.3
Incubate (32 ± 2) °C for Incubate (32 ± 2) °C for Incubate (32 ± 2) °C for
desired contact time desired contact time desired contact time
8.9 ml Neutralizer 4.4 ml Neutralizer 4.4 ml Neutralizer
+ 100 µl inoculum + 100 µl inoculum + 100 µl inoculum
+ Dressing sample + 0.5 ml SWF sample + 0.5 ml SWF sample
A.5 Neutralizer toxicity
This is performed to demonstrate that the chosen neutralizer does not have any inhibitory effect on the
challenge organisms.
a) Transfer the required volume of suitable neutralizer (see E.7) to a test tube.
b) Inoculate a 100 µl volume of microbial suspension prepared in A.3.
c) Add the appropriate volume of MRD or SWF to the neutralizer/challenge organism suspension such that
the resulting suspension contains 30 to 100 cfu/ml of the challenge organism. Mix contents.
d) Allow the suspension to stand for an appropriate time (see note below).
e) After the exposure time, transfer 1ml volumes of the suspension to duplicate agar pour plates; agar
spread plates may be used by transferring a 0.1 ml volume onto duplicate plates, taking into account the
extra 10-fold dilution. If neutralizers are incorporated into agar for the test method, then use the same
medium for plating the suspension.
f) Repeat this procedure an additional two times, for a total of three replicates.
g) Incubate the agar plates under the same conditions as those used in the chosen test method.
h) Following incubation, count the number of colonies on each agar plate and determine the cfu/ml as
detailed in A.8.
NOTE Allow the resulting suspension to stand for the longest exposure period representative of that used in chosen
test method. Unless prior knowledge and experience in the use of the chosen neutralizer is known, a validation exercise
may be performed to determine optimal standing time for effective neutralisation and absence of toxicity.
10
---------------------- Page: 12 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
A.6 Test organism viability
This is performed to determine the population of the challenge organism used in the neutralisation assay.
Perform the test as detailed in A.5 but replace the neutralizer with required volume of MRD as detailed in
Table A.1.
A.7 Neutralisation Effectiveness
This is performed to show the neutralizer is effective at inactivating or quenching the microbicidal properties of
an antimicrobial agent.
a) Aseptically cut a sample of dressing to the size specified in Table A.1 and transfer to a sterile petri-dish.
b) For the direct contact method only, hydrate the dressing sample with an identical volume of saturation
fluid (SWF) to that determined in the test method (see H.4.3).
c) Depending on the chosen test method, manipulate the dressing as detailed in Table A.1:
Direct contact method: add 5 x 0.1 ml volumes of SWF across the dressing sample as described in
H.6
Shaking method: Transfer dressing sample to 5 ml of SWF (+ saturation volume) contained within
an Erlenmeyer flask
Two compartment method: Transfer dressing sample to 2.5 ml of SWF (+ saturation volume)
contained within a 6 well plate.
d) Incubate the dressing sample for the desired contact time, under the same conditions as those used in
the chosen test method (see Annex H).
e) Following the desired contact time, remove the dressing sample from the incubator.
f) Transfer an appropriate volume of neutralizer (see Table A.1) to a test tube. For the Direct Contact
Method, transfer the appropriate volume of neutralizer to a sterile stomacher bag.
g) Inoculate a 100 µl volume of microbial suspension prepared in A.3.
h) Depending on the chosen test method, perform the following step:
Direct contact method: Transfer the dressing sample to the neutralizer/challenge organism
suspension. Process the dressing in a stomacher blender as detailed in H.6;
Shaking method/Two compartment method: Remove a 0.5 ml volume of SWF from the
appropriate test vessel and transfer to the neutralizer/challenge organism suspension such that the
resulting suspension contains 30 to 100 cfu/ml of the challenge organism. Mix contents.
i) Allow the resulting suspension to stand for an appropriate time (see note below).
After the exposure time, transfer 1ml volumes of the suspension to duplicate agar pour plates; agar
spread plates may be used by transferring a 0.1 ml volume onto duplicate plates, taking into account the
extra 10-fold dilution. If neutralizers are incorporated into agar for test method, then use the same
medium for plating the suspension.
j) Repeat this procedure an additional two times, for a total of three replicates.
Incubate the agar plates under conditions appropriate for each test organism.
11
---------------------- Page: 13 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
Following incubation, count the number of colonies on each agar plate and determine the cfu/ml as detailed in
A.8.
NOTE Allow the resulting suspension to stand for the longest exposure period representative of that used in chosen
test method. Unless prior knowledge and experience in the use of the chosen neutralizer is known, a validation exercise
may be performed.
A.8 Interpretation of data
a) Apply the following formula to calculate the number N of surviving challenge organisms resulting from
each replicate of each test:
P1+P2
N= (CFU /ml)
2
where
P1 = counts on plate 1
P2 = counts on plate 2
b) Compare the mean cfu/ml from A.7 (Neutralisation effectiveness) and A.5 (Neutralizer toxicity) to A.6
(Organism viability).
c) Neutralisation is considered adequate if A.7 recovery population is ≥ 70 % of A.6 organism viability
population (see Note below).
d) The chosen neutralising medium is considered non-toxic if A.5 recovery population is within 70 % of A.6
organism viability population (see Note below).
NOTE As per USP 36, p.227 “Validation of microbial recovery from pharmacopoeial articles”.
12
---------------------- Page: 14 ----------------------
oSIST prEN 16756:2014
prEN 16756:2014 (E)
Annex B
(informative)
Neutralizers
Table B.1 — Examples of active agents and possible neutralizers
Antimicro
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.