SIST EN ISO 14730:2015
(Main)Ophthalmic optics - Contact lens care products - Antimicrobial preservative efficacy testing and guidance on determining discard date (ISO 14730:2014)
Ophthalmic optics - Contact lens care products - Antimicrobial preservative efficacy testing and guidance on determining discard date (ISO 14730:2014)
This International Standard specifies a procedure to be used in evaluating the antimicrobial preservative
activity of all preserved multidose contact lens care products, and provides guidance on methods for
determination of discard date as informative annexes.
This test is applicable to products for up to a 28-day discard date.
The test is not applicable to sterile products packaged in unit doses for single use or multidose containers
designed with physical barriers to microbial contamination (e.g. aerosol containers).
NOTE 1 Principles of the test can be used to extend discard dating beyond 28 d. See Annexes B, C, D and E.
NOTE 2 Use of multiple or mixed microbial challenges and/or inclusion of contact lenses or other organic
load can influence the apparent antimicrobial activity of a particular product. The evaluation of these variables
together with testing against a larger panel of microorganisms and testing of samples from partially used
containers can be of value in developing a contact lens care product, but are excluded from the scope of this
International Standard.
Augenoptik - Kontaktlinsenpflegemittel - Konservierungsmittelbelastungstest und Anleitung zur Feststellung der Aufbrauchfrist (ISO 14730:2014)
Diese Internationale Norm legt ein Verfahren fest, welches zur Bewertung der Konservierungs-leistung aller konservierten Mehrdosis-Kontaktlinsenpflegemittel einzusetzen ist, und sie stellt in informativen Anhängen einen Leitfaden zu Verfahren zur Bestimmung der Aufbrauchfrist zur Verfügung.
Der Test gilt für Produkte mit einer Aufbrauchfrist von bis zu 28 Tagen.
Der Test gilt nicht für sterile Produkte, die in Einzeldosen zum einmaligen Gebrauch verpackt sind oder Mehrdosisbehälter, die aufgrund ihrer Konstruktion über eine physische Barriere gegen mikrobielle Verunreinigungen verfügen.
ANMERKUNG 1 Grundprinzipien des Tests dürfen auch dazu angewendet werden, eine längere Aufbrauchfrist als 28 Tage festzulegen. Siehe hierzu Anhänge B, C, D und E.
ANMERKUNG 2 Ein mehrfaches Animpfen, ein Animpfen mit einer mikrobiologischen Mischprobe, die Einbeziehung von Kontaktlinsen oder der Zusatz einer organischen Verunreinigung kann möglicherweise die antimikrobielle Wirkung eines bestimmten Produktes beeinflussen. Die Bewertung dieser Faktoren sowie die Prüfung eines erweiterten Spektrums von Mikroorganismen und das Testen von Proben aus angebrochenen Behältern können zwar bei der Entwicklung eines Kontaktlinsenpflegemittels wichtig sein, werden aber bei dieser Internationalen Norm nicht berücksichtigt.
Optique ophtalmique - Produits d'entretien des lentilles de contact - Essais de l'efficacité de conservation antimicrobienne et lignes directrices pour la détermination de la durée d'utilisation après première ouverture (ISO 14730:2014)
L'ISO 14730:2014 spécifie la méthode suivie pour évaluer l'activité de conservation antimicrobienne de tous les produits d'entretien des lentilles de contact conservés en emballages multi-doses et fournit des lignes directrices sur les méthodes à utiliser pour la détermination de la durée d'utilisation après première ouverture, en annexes informatives.
Očesna optika - Kontaktne leče in izdelki za vzdrževanje kontaktnih leč - Preskušanje učinkovitosti antimikrobnih sredstev za konzerviranje in napotek za določanje datuma za zavrženje (ISO 14730:2014)
Ta mednarodni standard določa postopek, ki se uporablja za vrednotenje dejavnosti antimikrobnih sredstev za konzerviranje vseh konzerviranih izdelkov za vzdrževanje kontaktnih leč z več odmerki, ter zagotavlja napotek za metode za določanje datuma za zavrženje v obliki informativnih dodatkov. Ta preskus se uporablja za izdelke z datumom za zavrženje do vključno 28 dni. Preskus se ne uporablja za sterilne izdelke v posameznih odmerkih za enkratno uporabo ali vsebnikih z več odmerki s fizičnimi pregradami za mikrobno onesnaženje (npr. vsebniki za aerosole).
OPOMBA 1: Načela preskusa je mogoče uporabiti za podaljšanje datuma za zavrženje nad 28 dni. Glej dodatke B, C, D in E.
OPOMBA 2: Uporaba več ali mešanih mikrobnih izzivov in/ali vključitev kontaktnih leč ali druge organske obremenitve lahko vplivajo na navidezno antimikrobno dejavnost posameznega izdelka. Vrednotenje teh spremenljivk skupaj s preskušanjem v prisotnosti širšega spektra mikroorganizmov in preskušanjem vzorcev iz delno uporabljenih posod je mogoče koristno pri razvoju izdelka za vzdrževanje kontaktnih leč, vendar je zunaj področja uporabe tega mednarodnega standarda.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 14730:2015
01-januar-2015
1DGRPHãþD
SIST EN ISO 14730:2001
SIST EN ISO 14730:2001/AC:2001
2þHVQDRSWLND.RQWDNWQHOHþHLQL]GHONL]DY]GUåHYDQMHNRQWDNWQLKOHþ
3UHVNXãDQMHXþLQNRYLWRVWLDQWLPLNUREQLKVUHGVWHY]DNRQ]HUYLUDQMHLQQDSRWHN]D
GRORþDQMHGDWXPD]D]DYUåHQMH ,62
Ophthalmic optics - Contact lens care products - Antimicrobial preservative efficacy
testing and guidance on determining discard date (ISO 14730:2014)
Augenoptik - Kontaktlinsenpflegemittel - Konservierungsmittelbelastungstest und
Anleitung zur Feststellung der Aufbrauchfrist (ISO 14730:2014)
Optique ophtalmique - Produits d'entretien des lentilles de contact - Essais de l'efficacité
de conservation antimicrobienne et lignes directrices pour la détermination de la durée
d'utilisation après première ouverture (ISO 14730:2014)
Ta slovenski standard je istoveten z: EN ISO 14730:2014
ICS:
11.040.70 Oftalmološka oprema Ophthalmic equipment
SIST EN ISO 14730:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
SIST EN ISO 14730:2015
---------------------- Page: 2 ----------------------
SIST EN ISO 14730:2015
EUROPEAN STANDARD
EN ISO 14730
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2014
ICS 11.040.70 Supersedes EN ISO 14730:2000
English Version
Ophthalmic optics - Contact lens care products - Antimicrobial
preservative efficacy testing and guidance on determining
discard date (ISO 14730:2014)
Optique ophtalmique - Produits d'entretien des lentilles de Augenoptik - Kontaktlinsenpflegemittel -
contact - Essais de l'efficacité de conservation Konservierungsmittelbelastungstest und Anleitung zur
antimicrobienne et lignes directrices pour la détermination Feststellung der Aufbrauchfrist (ISO 14730:2014)
de la durée d'utilisation après première ouverture (ISO
14730:2014)
This European Standard was approved by CEN on 23 July 2014.
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
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14730:2014 E
worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN ISO 14730:2015
EN ISO 14730:2014 (E)
Contents Page
Foreword .3
2
---------------------- Page: 4 ----------------------
SIST EN ISO 14730:2015
EN ISO 14730:2014 (E)
Foreword
This document (EN ISO 14730:2014) has been prepared by Technical Committee ISO/TC 172 “Optics and
photonics” in collaboration with Technical Committee CEN/TC 170 “Ophthalmic optics” 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 April 2015, and conflicting national standards shall be withdrawn at the
latest by April 2015.
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 14730:2000.
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 14730:2014 has been approved by CEN as EN ISO 14730:2014 without any modification.
3
---------------------- Page: 5 ----------------------
SIST EN ISO 14730:2015
---------------------- Page: 6 ----------------------
SIST EN ISO 14730:2015
INTERNATIONAL ISO
STANDARD 14730
Second edition
2014-10-01
Ophthalmic optics — Contact lens
care products — Antimicrobial
preservative efficacy testing and
guidance on determining discard date
Optique ophtalmique — Produits d’entretien des lentilles de contact
— Essais de l’efficacité de conservation antimicrobienne et lignes
directrices pour la détermination de la durée d’utilisation après
première ouverture
Reference number
ISO 14730:2014(E)
©
ISO 2014
---------------------- Page: 7 ----------------------
SIST EN ISO 14730:2015
ISO 14730:2014(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2014
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
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 14730:2015
ISO 14730:2014(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Test methods . 2
5.1 Materials and reagents . 2
5.2 Test sampling and culture maintenance . 2
5.3 Preparation of microbial challenge (Inoculum) . 3
5.4 Inoculum challenge test procedure . 3
5.5 Controls . 5
5.6 Performance criteria . 5
5.7 Test report . 6
Annex A (informative) Example of a membrane filtration procedure II . 7
Annex B (informative) Discard date procedure I . 9
Annex C (informative) Discard date procedure .12
Annex D (informative) Discard date procedure III .16
Annex E (informative) Discard date procedure IV.19
Annex F (informative) Test organisms from other culture collections .22
Bibliography .23
© ISO 2014 – All rights reserved iii
---------------------- Page: 9 ----------------------
SIST EN ISO 14730:2015
ISO 14730:2014(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 172, Optics and photonics, Subcommittee
SC 7, Ophthalmic optics and instruments in collaboration with the Technical Committee CEN/TC 170,
Ophthalmic optics.
This second edition cancels and replaces the first edition (ISO 14730:2000), of which it constitutes a
minor revision.
iv © ISO 2014 – All rights reserved
---------------------- Page: 10 ----------------------
SIST EN ISO 14730:2015
ISO 14730:2014(E)
Introduction
Contact lens care products (CLCP) are used with contact lenses. These products rinse, clean, disinfect,
store, wet, aid the comfort of, and condition contact lenses. Some products have one function, while
others are multifunctional.
Usually, products manufactured for use with hydrogel lenses may be used with rigid gas-permeable
(RGP) or poly (methyl methacrylate) (PMMA) lenses, but products specifically used for RGP or PMMA
contact lenses are not usually suitable for hydrogel lenses.
Most CLCPs are manufactured as solutions and are commonly packaged and sold in multidose containers.
Dry products are sold as tablets or granules and shall be dissolved in a suitable solvent immediately
prior to use.
If the contact lens care product solution does not have any antimicrobial activity itself, an antimicrobial
preservative can be added to the product to inhibit the growth of microorganisms that might be
introduced from repeated dispensing during use and subsequent storage. All antimicrobial agents
have the potential for toxicity to the user. For maximum protection to the user, the concentration of the
preservative should be such that it provides adequate preservative activity with minimum toxicity.
There are differences between ophthalmic preparations and contact lens care products and some
of these differences are significant in relation to preservative efficacy testing. Typically, ophthalmic
preparations are packaged in small-volume containers and are used for short periods on compromised
eyes. Contact lens care products are distributed in larger volume containers and are used with contact
lenses on a long term basis on healthy eyes. The potential risks for contact lens care products are the
solution/lens interaction causing ocular irritation and the risks of the solution contamination by the
repeated (daily) use of the product.
Thus, when contact lens care products are formulated, the risk of adverse patient reaction due to the
lens and/or solution interaction has to be weighed against the benefits of safety derived from the
maintenance of the antimicrobial activity of the solution.
This International Standard gives the test procedure and performance criteria for preservative efficacy.
It has been adapted from Pharmacopoeias which give a time limitation in their test procedure of 28 d.
The informative annexes give four examples of preservative efficacy test procedures developed by
contact lens care product manufacturers to show preservative efficacy for products whose discard
dates are over 28 d.
© ISO 2014 – All rights reserved v
---------------------- Page: 11 ----------------------
SIST EN ISO 14730:2015
---------------------- Page: 12 ----------------------
SIST EN ISO 14730:2015
INTERNATIONAL STANDARD ISO 14730:2014(E)
Ophthalmic optics — Contact lens care products —
Antimicrobial preservative efficacy testing and guidance
on determining discard date
1 Scope
This International Standard specifies a procedure to be used in evaluating the antimicrobial preservative
activity of all preserved multidose contact lens care products, and provides guidance on methods for
determination of discard date as informative annexes.
This test is applicable to products for up to a 28-day discard date.
The test is not applicable to sterile products packaged in unit doses for single use or multidose containers
designed with physical barriers to microbial contamination (e.g. aerosol containers).
NOTE 1 Principles of the test can be used to extend discard dating beyond 28 d. See Annexes B, C, D and E.
NOTE 2 Use of multiple or mixed microbial challenges and/or inclusion of contact lenses or other organic
load can influence the apparent antimicrobial activity of a particular product. The evaluation of these variables
together with testing against a larger panel of microorganisms and testing of samples from partially used
containers can be of value in developing a contact lens care product, but are excluded from the scope of this
International Standard.
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 14534, Ophthalmic optics — Contact lenses and contact lens care products — Fundamental requirements
ISO 18369-1, Ophthalmic optics — Contact lenses — Part 1: Vocabulary, classification system and
recommendations for labelling specifications
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 18369-1 apply.
4 Principle
4.1 The test consists of challenging the preparation with a specified inoculum of suitable microorganisms
at the commencement of the test and then rechallenging at day 14. The inoculated preparations are stored
at a specified temperature. Samples are withdrawn from the inoculated preparations at specified time
intervals and are cultured for determination of viable organisms. The capability of the product to prevent
re-growth is confirmed by counting of viable organisms over longer time periods.
4.2 The size of the microbial challenge chosen in this test is not intended to be representative of the
likely challenge in practice, but to provide countable numbers from which estimation of the rate and
extent of viability loss can be determined.
© ISO 2014 – All rights reserved 1
---------------------- Page: 13 ----------------------
SIST EN ISO 14730:2015
ISO 14730:2014(E)
4.3 The antimicrobial preservative properties of the product are adequate if, in the conditions of the
test, there is significant reduction of bacteria and no increase in yeasts and moulds in the inoculated
preparation after the times and at the temperatures specified. The performance criteria are given in 5.6.
4.4 Appropriate measures shall be taken to inactivate or remove residual antimicrobial agents during
culturing and counting of survivors. The effectiveness of these measures shall be validated.
5 Test methods
5.1 Materials and reagents
5.1.1 Test organisms
The strains listed in Table 1 shall be used.
NOTE Test organisms from other culture collections that can be used are listed in Annex F.
Table 1 — Test organisms
Pseudomonas aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538
Escherichia coli ATCC 8739
Candida albicans ATCC 10231
Aspergillus brasiliensis ATCC 16404
5.1.2 Culture media and reagents
5.1.2.1 Tryptone Soya Agar (TSA).
5.1.2.2 Sabouraud Dextrose Agar (SDA).
5.1.2.3 Dulbecco’s Phosphate-Buffered Saline, without calcium chloride and magnesium chloride
(DPBS).
Combine 200 mg/l KCl, 200 mg/l KH PO , 8 000 mg/l NaCl, and 2 160 mg/l Na HPO ⋅ 7H O or
2 4 2 4 2
suitable diluent.
5.1.2.4 Dulbecco’s Phosphate Buffered Saline, plus 0,05 % volumic mass polysorbate 80 (DPBST) or
suitable diluent.
5.1.2.5 Validated neutralizing agents/media as required, for example, Dey-Engley Neutralizing
Broth (DEB) and Letheen Broth.
5.1.3 Laboratory equipment
The following common laboratory equipment is required: sterile pipettes, swabs, tubes, petri dishes
(90 mm to 100 mm × 20 mm), etc. and suitable instruments for spectrophotometric determination of
cell density, for colony counting and for centrifugation.
5.2 Test sampling and culture maintenance
The product to be tested shall be representative of the product to be marketed. Aliquots should be taken
directly from the final product container immediately prior to testing.
2 © ISO 2014 – All rights reserved
---------------------- Page: 14 ----------------------
SIST EN ISO 14730:2015
ISO 14730:2014(E)
Three lots of product shall be tested. Each lot of product shall be tested with a separate inoculum
preparation for each challenge organism.
Maintain the test cultures as recommended by the curator of the appropriate culture collection.
Cultures should be no greater than five passes removed from the depository stock (ATCC, NCIB, NCTC,
NCPF or other recognized culture depository; see Annex F). Each pass is a subculture of the previous pass.
5.3 Preparation of microbial challenge (Inoculum)
Culture each test organism on agar slopes under the conditions given in Table 2.
Table 2 — Media and incubation conditions for growth of challenge organisms
Temperature
Organism Medium Incubation time
°C
P. aeruginosa TSA 30 to 35 18 h to 24 h
S. aureus TSA 30 to 35 18 h to 24 h
E. coli TSA 30 to 35 18 h to 24 h
either 20 to 25 42 h to 48 h
C. albicans SDA
or 30 to 35 18 h to 24 h
A. brasiliensis SDA 20 to 25 7 d to 10 d
Use sterile DPBST or suitable diluent to harvest each culture; wash the surface growth, transfer it to a
suitable vessel and vortex. Filter the spore suspensions through sterile glass wool, cheesecloth or gauze
to remove hyphal fragments.
After harvesting, the cultured organisms can be washed using centrifugation. The bacterial suspensions
can be filtered (e.g. 3 µm to 5 µm pore size) to produce a single cell dispersion. Then, adjust all challenge
7
cell suspensions with DPBST or other suitable diluent to a concentration of between 1,0 × 10 cfu/ml
8
and 1,0 × 10 cfu/ml. Estimate the approximate cell concentration of each suspension by measuring
the turbidity of the suspension or a dilution of the suspension using a spectrophotometer. The actual
concentration of colony-forming units per millilitre shall be determined for each suspension, e.g. by the
plate-count method, at the time of the test.
If centrifugation is used, each centrifugation should be conducted at 20 °C to 25 °C for no longer than the
equivalent of 10 min at 4 000 g or less.
Use bacterial and yeast cell suspensions on the day of preparation.
NOTE 1 Longer centrifugation times might be required at lower speeds.
NOTE 2 Spore suspensions can be used up to seven days following preparation by storage under refrigeration
(2 °C to 8 °C).
5.4 Inoculum challenge test procedure
5.4.1 Prepare one or more tubes (for each lot tested) containing a minimum of 10 ml of test solution per
challenge organism.
NOTE Sample tubes are used rather than lens cases to allow effective technical execution of the test. Since
incompatibilities can exist between solution ingredients and tube materials, tubes of an appropriate material
which is compatible with the ingredients should be considered.
Inoculate the sample tube of the product to be tested with a suspension of test organisms sufficient
5 6
to provide a final count of between 1,0 × 10 cfu/ml and 1,0 × 10 cfu/ml. Ensure that the volume of
inoculum does not exceed 1 % of the sample volume. Ensure complete dispersion of the inoculum by
adequate mixing.
© ISO 2014 – All rights reserved 3
---------------------- Page: 15 ----------------------
SIST EN ISO 14730:2015
ISO 14730:2014(E)
5.4.2 Store the inoculated product at 20 °C to 25 °C. The temperature shall be monitored using a
calibrated device and the temperature documented.
If the product is sensitive to light, it should be protected during the period of the test.
5.4.3 Take 1,0 ml aliquots of the inoculated product for determination of viable count at 7 d and 14 d.
5.4.4 After taking the 14 d sample, each sample is rechallenged as in 5.4.1 by using an inoculum level of
4 5
1,0 × 10 cfu/ml to 1,0 × 10 cfu/ml.
5.4.5 Take 1,0 ml aliquots of the inoculated product for determination of the viable count at 21 d and 28 d.
5.4.6 Subject each of the 1,0 ml aliquots, removed at the specified time intervals, to a suitable series of
decimal dilutions in validated neutralizing media. Mix the suspension well by vortexing vigorously and
let stand to allow neutralization to be completed. Neutralization conditions shall be based on recovery-
medium control testing (see 5.5.2).
If an antimicrobial agent in the formulation cannot be adequately inactivated or neutralized, eliminate
it using a validated membrane filtration procedure (see Annex A).
5.4.7 Determine the viable count of organisms in appropriate dilutions by preparation of triplicate plates
(unless otherwise justified) of a suitable recovery medium (e.g. TSA for bacteria and SDA for mould and yeast).
If membrane filtration has been employed to remove or neutralize antimicrobial agents, culture the
membranes on these media as appropriate.
If the pour-plate method is utilized, keep the agar for pour plates below 50 °C prior to pouring.
NOTE The agar media used for determination of viable counts can also contain antimicrobial inactivators or
neutralizers, if required.
5.4.8 Incubate bacterial recovery plates at 30 °C to 35 °C. Incubate yeast recovery plates at 20 °C to
25 °C or 30 °C to 35 °C. Incubate mould recovery plates at 20 °C to 25 °C. Incubation times for optimal
recovery of bacteria, yeast, and moulds shall be determined. Minimum incubation times shall be based on
recovery medium control testing (see 5.5.2). Record the number of cfu observed on countable plates.
Plates should be observed periodically during incubation to prevent the occurrence of uncountable
plates due to overgrowth.
5.4.9 Determine the average number of colony-forming units on countable plates. Calculate the
microbial reduction at the specified time points.
NOTE Countable plates refer to 30 cfu to 300 cfu per plate for bacteria and yeast, and 8 cfu to 80 cfu per plate
0 −1
for moulds, except when colonies are observed only for the 10 or 10 dilution plates.
5.4.10 The absence of microorganisms shall be documented, e.g. by recording a “0” or “NR” (no recovery),
when plates for all dilutions of a sample at a single time point have zero colonies.
5.4.11 The concentration of survivors is calculated at each point of time. The concentration of viable
organisms following the 14 d rechallenge is the sum of the rechallenge inoculum concentration and the
14 d survivor concentration.
4 © ISO 2014 – All rights reserved
---------------------- Page: 16 ----------------------
SIST EN ISO 14730:2015
ISO 14730:2014(E)
5.5 Controls
5.5.1 Inoculum controls
The initial and rechallenge inoculum concentrations are calculated by dispersing an identical aliquot of
the inoculum into the same volume as used in 5.4.1 of a suitable diluent to achieve a final concentration
5 6 4
not less than 1,0 × 10 cfu/ml to 1,0 × 10 cfu/ml for the initial inoculum or 1,0 × 10 cfu/ml to
5
1,0 × 10 cfu/ml for the rechallenge. The volume of inoculum does not exceed 1 % of the sample volume.
Ensure dispersion of the inoculum by adequate mixing. Evaluate this control sample for cfu/ml at the
beginning of the test in order to demonstrate the suitability of the medium used for growth of the test
organism and provide an estimate of the initial inoculum concentration. Plate the appropriate aliquot
from each tube onto the recovery agar plates in triplicate (unless otherwise justified).
5.5.2 Recovery medium control
Vortex a 1/10 dilution of the preserved product in the validated neutralizing broth (1 ml into 9 ml). Let
it stand to allow neutralization to be completed. Prepare a second control tube with 10 ml of a suitable
diluent (e.g. DPBST). Inoculate the tubes with sufficient inoculum to result in 10 cfu to 100 cfu of challenge
organism per plate. Incubate for an appropriate period of time at ambient temperature. Plate the
appropriate aliquot from each tube onto the recovery agar plates in triplicate (unless otherwise justified).
Incubate bacterial recovery plates at 30 °C to 35 °C. Incubate yeast recovery plates at 20 °C to 25 °C or
30 °C to 35 °C. Incubate mould recovery plates at 20 °C to 25 °C. Determine minimum incubation times
for optimal recovery of bacteria, yeast, and moulds.
Check that the recovery from the neutralizer broth is at least 50 % of the recovery in the second control
tube. Perform this control for each challenge organism.
If a dilution of greater than 1/10 is required for neutralization, then membrane filtration should be used.
Validate the neutralization of the product with each challenge organism initially and as appropriate.
5.6 Performance criteria
5.6.1 General
Products shall be capable of meeting these criteria throughout their labelled shelf life and at the discard date.
Meeting the criteria of 5.6.2 and 5.6.3 shall justify a 28 d period of use after opening (discard date).
NOTE Refer to Annexes B, C, D and E for suggested methods if a discard date longer than 28 d is desired.
5.6.2 Bacteria
The number of each challenge organism recovered per millilitre shall be reduced by a mean value of not
less than 3,0 logs at 14 d. After the rechallenge at 14 d, the
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.