BS EN ISO 14730:2014

BSI Standards Publication

Ophthalmic optics —
Contact lens care products —
Antimicrobial preservative
efficacy testing and guidance
on determining discard date
BS EN ISO 14730:2014 BRITISH STANDARD

National foreword
This British Standard is the UK implementation of EN ISO
14730:2014. It supersedes BS EN ISO 14730:2000 which is withdrawn.
The UK participation in its preparation was entrusted to Technical
Committee CH/172/9, Contact lenses and contact lens care products.
A list of organizations represented on this committee can be
obtained on request to its secretary.
This publication does not purport to include all the necessary
provisions of a contract. Users are responsible for its correct
application.
© The British Standards Institution 2014. Published by BSI Standards
Limited 2014
ISBN 978 0 580 84870 4
ICS 11.040.70
Compliance with a British Standard cannot confer immunity from
legal obligations.
This British Standard was published under the authority of the
Standards Policy and Strategy Committee on 31 October 2014.
Amendments issued since publication
Date Text affected
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.
BS EN ISO 14730:2014
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
 BS EN ISO 14730:2014
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

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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.

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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.

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 BS EN ISO 14730:2014
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.

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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 KH2PO4, 8  000  mg/l NaCl, and 2  160  mg/l Na2HPO4 ⋅ 7H2O or
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.

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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
cell suspensions with DPBST or other suitable diluent to a concentration of between 1,0 × 107 cfu/ml
and 1,0  ×  108  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
to provide a final count of between 1,0 × 105 cfu/ml and 1,0 × 106 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.

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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
1,0 × 104 cfu/ml to 1,0 × 105 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
for moulds, except when colonies are observed only for the 100 or 10−1 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.

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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
not less than 1,0  ×  105  cfu/ml to 1,0  ×  106  cfu/ml for the initial inoculum or 1,0  ×  104 cfu/ml to
1,0 × 105 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 concentration of each challenge organism
shall be reduced again by at least a mean value of 3,0 logs by 28 d.

5.6.3 Moulds and yeasts

The number of each challenge organism recovered per millilitre shall remain at, or below, the initial
concentrations (within an experimental error of ±0,5 logs) within 14 d. At 28 d, the concentration of each
challenge organism shall remain at, or below, the concentrations (within an experimental error of ±0,5
logs) of each challenge organism after the rechallenge.

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5.7 Test report

The test report shall include
a) the title of this International Standard,
b) the identification of the product, including name of the product, batch number, expiry date,
manufacturer, storage conditions and active substances(s), and its/their concentration(s) (as available),
c) the name(s) of the operator(s),
d) deviations from the protocol,
e) date and time of incubation,
f) storage time for inoculated product, and
g) results obtained, including numbers of organisms recovered at each time point.

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Annex A
(informative)

Example of a membrane filtration procedure II

A.1 Materials and reagents

A.1.1 Culture media and reagents

A.1.1.1 Diluting fluid, with or without neutralizers.

A.1.1.2 Tryptone Soya Agar (TSA).

A.1.1.3 Dulbecco’s Phosphate-Buffered Saline, without calcium chloride and magnesium chloride
(DPBS): 200 mg/l KCl, 200 mg/l KH2PO4, 8 000 mg/l NaCl, and 2 160 mg/l Na2HPO4⋅7H2O or suitable diluent.

A.1.1.4 Dulbecco’s Phosphate-Buffered Saline, plus 0,05 % polysorbate 80 (DPBST) or suitable diluent.

A.1.1.5 Validated neutralizing agents/media, as required, for example, Dey-Engley Neutralizing

Broth (DEB) and Letheen Broth.

A.1.2 Test equipment

Usual laboratory equipment (such as sterile pipettes, petri dishes, containers) together with the following.

A.1.2.1 Sterile membrane filter.

A.1.2.2 Suitable sterile apparatus, for holding the sterile membrane filter and filtrate.

A.1.2.3 Suitable equipment for creating a vacuum or pressure, to cause the liquid phase of the
inoculated test solution to pass through the membrane filter aseptically.

The membrane filter should have a nominal pore size of not greater than 0,45 µm, a diameter of at least
47 mm and should be free of chemicals which could be toxic to microbial cells.

A.2 Test method and results

A.2.1 Moisten the sterile membrane filter (A.1.2.1) in a sterile filter assembly (A.1.2.2) with sterile
DPBST (A.1.1.4), or suitable diluent.

A.2.2 Aseptically transfer a measured volume of the inoculated test solution into sterile DPBST (A.1.1.4)
or diluting fluid.

A.2.3 Transfer the diluted solution to the membrane and filter immediately with the aid of vacuum or
pressure. Dilute the sample applied to the filter with 50 ml to 100 ml of dilution fluid and thoroughly mix
to ensure uniform distribution of the sample over the entire area of the filter.
NOTE This will decrease the probability of multiple colony-forming units being placed on the filter at the
same location.

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A.2.4 Wash the membrane filter with several volumes of diluting fluid which may contain additional
neutralizing agents as needed. The actual volume should be determined empirically for each formulation
for each challenge organism.
NOTE Three volumes of diluting fluid (100  ml each) are usually sufficient to remove and/or dilute the
antimicrobial agent.

A.2.5 Incubate the membrane filter with appropriate media to allow growth of colony-forming units on
the surface of the filter.

This can be accomplished by aseptic removal of the membrane filter from the filter assembly unit and
placement of the membrane on the surface of a sterile agar plate which does not have obvious liquid on
the surface; or the membrane can be enclosed in an agar sandwich. Alternatively, a sterile membrane
filter unit can be used which requires addition of sterile media to the sealed filter and incubation of the
membrane in situ. Media should be used which are appropriate for the type of challenge organism and
the specific formulation under test. Time of the incubation should be established.

A.2.6 Determine the average number of colony-forming units on the countable membrane filters (3 cfu
to 100 cfu/47 mm filter for bacteria and yeast and 3 cfu to 10 cfu/47 mm filter for moulds). Calculate and
document the number of colony-forming units per millilitre of inoculated solution.

A.3 Controls
Confirm neutralizer efficacy by transferring an aliquot of the uninoculated test solution into 50  ml to
100 ml of sterile diluting fluid using the same ratio of volume of test solution to volume of diluting fluid.
Apply the entire volume to the membrane and filter using vacuum or pressure. Wash the filter with several
volumes of the diluting fluid using the same volume as used for the test procedure. Transfer 5 cfu to 100 cfu
challenge organisms (one species per filter) into 100  ml of diluting fluid and apply to the membrane.
Incubate the membrane filter in contact with media as described in the test procedure (see A.2.5).
Repeat the procedure using diluting fluid not exposed to the test solution. Compare counts with those
derived by the same method but using a suitable diluent (e.g. DPBST), instead of the test solution. Confirm
the inoculum on a suitable medium in triplicate (unless otherwise justified). Ensure that the recovery in
the neutralizer broth is at least 50 % of the inoculum.

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Annex B
(informative)

Discard date procedure I

B.1 Principle

B.1.1 The test consists of inoculating the test samples with a high level of organism (approximately
106 cfu/ml) on 0 d. The test samples are rechallenged with a low organism inoculum level (approximately
103 cfu/ml) in the test formulation.

B.1.2 Inoculation times are at initial, 2 weeks, 25 %, 50 %, 75 %, and 100 % of the proposed discard date.

B.1.3 Sampling times should include 1 week, 2 weeks, 3 weeks, 4 weeks, and 25 %, 50 %, 75 %, and
100 % of the proposed discard date, and 14 d after the proposed discard date.

B.1.4 Test samples should meet the test criteria for ISO Preservative Efficacy of Multidose Preserved
Contact Lens Care Products at 28 d.

B.1.5 Stasis should be shown after rechallenges.

B.2 Test methods

B.2.1 Materials and reagents

B.2.1.1 Test organisms

Test organisms should be as specified in 5.1.1.

B.2.1.2 Test media

Test media should be as specified in 5.1.2.

B.2.1.3 Laboratory equipment

Laboratory equipment should be as specified in 5.1.3.

B.2.1.4 Test samples

Challenge tests are conducted to support discard after opening date, using three lots of contact lens care
solutions representative of the product to be marketed.
Culture maintenance should be as specified in 5.2.

B.2.2 Preparation of microbial challenge (Inoculum)

Test organisms should be cultured and harvested as specified in 5.3.

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ISO 14730:2014(E)


B.2.3 Inoculum challenge test procedure

B.2.3.1 Prepare a composite sample of greater than 250 ml from individual test samples.

B.2.3.2 Prepare a separate 50 ml quantity of test formulation for each test organism. Place the sample
into a 250 ml flask or specimen container.

B.2.3.3 Prepare 50 ml containers of a suitable diluent for bacteria, yeast, and mould controls.

B.2.3.4 Inoculate formulation samples and controls with 0,5  ml aliquot of the 108  cfu/ml organism
suspension to achieve a final concentration of approximately 106 cfu/ml. Ensure complete dispersion of
the inoculum by adequate mixing.

B.2.3.5 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 contact lens care product is sensitive to light, it should be protected during the period of the test.

B.2.3.6 At 1 week, 2 weeks, 3 weeks, 4 weeks, and at 25 %, 50 %, 75 %, and 100 % of the proposed
discard date, and 14 d after the proposed discard date, take 1,0 ml aliquots of the inoculated product for
determination of viable counts.

B.2.3.7 Rechallenge the formulation samples at 2 weeks, 25 %, 50 %, 75 %, and 100 % of the proposed
discard date, using a 0,05  ml/50  ml ratio of a 106  cfu/ml organism suspension to provide a final
concentration of approximately 103 cfu/ml. Repeat the viable count determinations at each time point
prior to rechallenging.

B.2.3.8 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.

If an antimicrobial agent in the formulation cannot be adequately inactivated or neutralized, eliminate

it using a validated membrane filtration procedure (see Annex A).

B.2.3.9 Determine the viable count of organisms in appropriate dilutions by preparation of plates in
triplicate (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 may also contain antimicrobial inactivators or
neutralizers, if required.

B.2.3.10 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 should be determined. Minimum incubation times should be based
on recovery medium control testing (see B.2.4.2).

B.2.3.11 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/plate for bacteria and yeast, and 8 cfu to 80 cfu/plate for
moulds, except when colonies are observed only for the 100 or 10−1 dilution plates.

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ISO 14730:2014(E)


B.2.3.12 When plates for all dilutions of a sample at a single time point indicate no recovery of
microorganisms, it should be documented.

B.2.3.13 The concentration of survivors is calculated at each time point.

B.2.4 Controls

B.2.4.1 Inoculum controls

Fresh control samples are run at each inoculation date by inoculating the saline (for bacteria and yeast)
or saline/Tween®1) 80 for mould in the same manner as for the sample inoculation. The theoretical
numbers can be found by calculating the cumulative sum of the last inoculum, plus the last survivor
count in the formulation sample.

B.2.4.2 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 in 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 shall be used.
Qualify the neutralization of the product initially and periodically.

B.2.5 Performance criteria

The discard date is supported if the number of surviving bacteria are reduced by three logs by day 14
and the number of bacteria and fungi do not increase in numbers thereafter.2)

B.2.6 Test report

The test report should be as specified in 5.7.

1)    Tween is an example of a suitable product available commercially. This information is given for the convenience
of users of this International Standard and does not constitute an endorsement by ISO of this product.
2)    The number of organisms recovered from a test sample does not exceed the sum of the microbial counts from
the inoculation plus the last count of survivors within a ±0,5 log variance (i.e. multiplication of the organisms did not
occur).

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ISO 14730:2014(E)


Annex C
(informative)

Discard date procedure

C.1 Principle

C.1.1 The test consists of challenging the preparation with a specified inoculum of suitable
microorganisms, storing the inoculated preparation at a specified temperature, withdrawing samples
from the container at specified time intervals, and counting the organisms in the samples removed. The
capability of the product to prevent re-growth is confirmed by counting of viable organisms over longer
time periods.

C.1.2 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.

C.1.3 The preparation shall meet the requirements for an adequately preserved contact lens care
product initially, and following a simulated use for the intended discard date interval (performance
criteria are included in C.2.7).

C.1.4 Appropriate measures should be taken to inactivate or remove residual antimicrobial agents
during culturing and counting of survivors, and the effectiveness of these measures should be validated.
The action of this process during the test should be demonstrated by the construction of suitable controls.

C.2 Test methods

C.2.1 Materials and reagents

C.2.1.1 Test organisms

Test organisms should be as specified in 5.1.1.

C.2.1.2 Test media

Test media should be as specified in 5.1.2.

C.2.1.3 Laboratory equipment

Laboratory equipment should be as specified in 5.1.3.

C.2.1.4 Test samples

The contact lens care product to be tested should be representative of the product to be marketed. Three
lots of product should be tested. Aliquots should be taken directly from the final product container
immediately prior to testing. A suitable number of containers should be utilized to ensure that enough
product will be available for challenge testing after simulated use (i.e. containers can be pooled in order
to provide sufficient product for testing).
Culture maintenance should be as specified in 5.2.

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ISO 14730:2014(E)


C.2.2 Preparation of microbial challenge (Inoculum)

Test organisms should be cultured and harvested as specified in 5.3.

C.2.3 Inoculum challenge test procedure

C.2.3.1 Prepare one tube containing a minimum of 10  ml of test solution per challenge organism.
Inoculate the sample tube of the product to be tested with a suspension of test organisms sufficient
to provide a final count of between 1,0 × 105 cfu/ml and 1,0 × 106 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.

C.2.3.2 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 contact lens care product is sensitive to light, it should be protected during the period of the test.

C.2.3.3 Take 1,0 ml aliquots of the inoculated product for determination of viable count at 7 d and 14 d.

C.2.3.4 After taking the 14 d sample, each sample is rechallenged as in C.2.3.1 by using an inoculum
level of 1,0 × 104 cfu/ml to 1,0 × 105 cfu/ml.

C.2.3.5 Take 1,0 ml aliquots of the inoculated product for determination of the viable count at 21 d and 28 d.

C.2.3.6 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.

If an antimicrobial agent in the formulation cannot be adequately inactivated or neutralized, eliminate

it using a validated membrane filtration procedure (see Annex A).

C.2.3.7 Determine the viable count of organisms in appropriate dilutions by preparation of plates in
triplicate (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 may also contain antimicrobial inactivators or
neutralizers, if required.

C.2.3.8 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 should be determined. Minimum incubation times should be
based on recovery medium control testing (see C.2.6.2).

C.2.3.9 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/plate for bacteria and yeast, and 8 cfu to 80 cfu/plate for
moulds, except when colonies are observed only for the 100 or 10−1 dilution plates.

C.2.3.10 When plates for all dilutions of a sample at a single time point indicate no recovery of
microorganisms, it should be documented.

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ISO 14730:2014(E)


C.2.3.11 The concentration of survivors is calculated at each time point. The concentration of viable
organisms following the 14 d rechallenge is the sum of the rechallenge inoculum concentration and the
14 d survivor concentration.

C.2.4 Simulated use

Using unchallenged product in original containers, subject the product to a simulated use by dispensing
an appropriate aliquot from the containers for a period of not less than the target discard date (i.e.
dispense 1 ml every 3 d for three months).
A suitable number of containers shall be utilized to ensure that enough product will be available for
challenge testing after simulated use (i.e. containers can be pooled in order to provide sufficient product
for testing).

C.2.5 Inoculum challenge following simulated use

At the end of the simulated use period, repeat the inoculum challenge test procedure as described in C.2.3.

C.2.6 Controls

C.2.6.1 Inoculum controls

The initial and rechallenge inoculum concentrations are calculated by dispersing an identical aliquot of
the inoculum into the same volume of a suitable diluent used in C.2.3.1 to achieve a final concentration of
not less 1,0 × 105 cfu/ml to 5,0 × 106 cfu/ml for the initial inoculum or 1,0 × 104 cfu/ml to 1,0 × 105 cfu/ml
for the rechallenge. The volume of inoculum shall not exceed 1 % of the sample volume. Ensure dispersion
of the inoculum by adequate mixing. Evaluate this control sample for colony-forming units per millilitre
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).

C.2.6.2 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 in 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 shall be used.
Qualify the neutralization of the product initially and periodically.

C.2.7 Performance criteria

C.2.7.1 General

Products shall meet the performance criteria for both the initial challenge and the final challenge
following simulated use.

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ISO 14730:2014(E)


C.2.7.2 Bacteria

The number of organisms recovered per millilitre should be reduced by a value of not less than 3,0 logs
at 14 d. After the rechallenge at 14 d, the concentration of bacteria should be reduced again by at least
a mean value of 3,0 logs by 28 d.

C.2.7.3 Moulds and yeasts

The number of organisms recovered per millilitre should remain at, or below, the initial concentrations
(within an experimental error of ±0,5 logs) within 14 d. At 28 d, the concentration of moulds and yeast
should remain at, or below, the concentrations (within an experimental error of ±0,5 logs) of moulds and
yeasts after the rechallenge.

C.2.8 Test report

The test report shall be as specified in 5.7.

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BS EN ISO 14730:2014
ISO 14730:2014(E)


Annex D
(informative)

Discard date procedure III

D.1 Principle

D.1.1 The test consists of challenging the preparation with a specified inoculum of suitable
microorganisms, storing the inoculated preparation at a specified temperature, withdrawing samples
from the container at specified time intervals, and counting the organisms in the samples removed. The
capability of the product to prevent re-growth is confirmed by counting of viable organisms over longer
time periods.

D.1.2 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.

D.1.3 The preparation shall meet the requirements for an adequately preserved contact lens care
product throughout its intended discard date period (for performance criteria see D.2.5).

D.1.4 Appropriate measures should be taken to inactivate or remove residual antimicrobial agents
during culturing and counting of survivors, and the effectiveness of these measures should be validated.
The action of this process during the test should be demonstrated by the construction of suitable controls.

D.2 Test methods

D.2.1 Materials and reagents

D.2.1.1 Test organisms

Test organisms should be as specified in 5.1.1.

D.2.1.2 Test media

Test media should be as specified in 5.1.2.

D.2.1.3 Laboratory equipment

Laboratory equipment should be as specified in 5.1.3.

D.2.1.4 Test samples

The contact lens care product to be tested should be representative of the product to be marketed.
Three lots of product should be tested. Testing should be conducted in the actual product container. The
largest container size proposed for the product should be utilized.
Culture maintenance should be as specified in 5.2.

D.2.2 Preparation of microbial challenge (Inoculum)

Test organisms should be cultured and harvested as specified in 5.3.

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ISO 14730:2014(E)


D.2.3 Inoculum challenge test procedure

D.2.3.1 Inoculate the sample product to be tested with a suspension of test organisms sufficient
to provide a final count of between 1,0 × 105 cfu/ml and 1,0 × 106 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.

D.2.3.2 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 contact lens care product is sensitive to light, it should be protected during the period of the test.

D.2.3.3 Take 1,0 ml aliquots of the inoculated product for determination of viable count at 7 d, 14 d,
21 d, and 28 d, and continue sampling at 7 d intervals until the product contents are depleted.

D.2.3.4 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.

If an antimicrobial agent in the formulation cannot be adequately inactivated or neutralized, eliminate

it using a validated membrane filtration procedure (see Annex A).

D.2.3.5 Determine the viable count of organisms in appropriate dilutions by preparation of plates in
triplicate (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 may also contain antimicrobial inactivators or
neutralizers, if required.

D.2.3.6 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 should be determined. Minimum incubation times should be
based on recovery medium control testing (see D.2.4.2).

D.2.3.7 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/plate for bacteria and yeast, and 8 cfu to 80 cfu/plate for
moulds, except when colonies are observed only for the 100 or 10−1 dilution plates.

D.2.3.8 When plates for all dilutions of a sample at a single time point indicate no recovery of
microorganisms, it should be documented.

D.2.3.9 The concentration of survivors is calculated at each time point.

D.2.4 Controls

D.2.4.1 Inoculum controls

The initial inoculum concentration is calculated by dispersing an identical aliquot of the inoculum
into the same volume of a suitable diluent used in D.2.3.1 to achieve a final concentration not less than
1,0 × 104 cfu/ml to 5,0 × 105 cfu/ml. The volume of inoculum does not exceed 1 % of the sample volume.

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ISO 14730:2014(E)


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).

D.2.4.2 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 in 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 shall be used.
Qualify the neutralization of the product initially and periodically.

D.2.5 Performance criteria

D.2.5.1 Bacteria, moulds, and yeasts

The number of organisms recovered per millilitre should remain at, or below, the initial concentrations.

D.2.5.2 Determination of discard date

The product discard date should correspond to the interval prior to a time point which reveals an
increase in number for any organism.
EXAMPLE See Table D.1.

Table D.1
Count at day
Organism
0 7 14 21 28 35
E.coli 105 101 102 102 103 103
P. aeruginosa 105 103 103 102 103 104
S. aureus 105 <10 <10 <10 <10 <10
C. albicans 105 105 104 102 <10 <10
A. brasiliensis 105 104 103 104 104 105
NOTE 1 Interval prior to time point which reveals an increase in growth:
E. coli: 7 d
P. aeruginosa: 21 d
A. brasiliensis: 14 d
NOTE 2 The discard date in the above hypothetical product is 7 d after opening.

D.2.6 Test report

The test report should be as specified in 5.7.

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 BS EN ISO 14730:2014
ISO 14730:2014(E)


Annex E
(informative)

Discard date procedure IV

E.1 Principle

E.1.1 The test consists of inoculating product bottles with a low level of organisms. The test samples are
repeatedly rechallenged with a low level of organisms inoculated into the product bottle according to schedule.

E.1.2 Inoculation times are at initial, 24  h, 1  week, 2  weeks, 4  weeks, 6  weeks, 8  weeks, 12  weeks,
16 weeks, 18 weeks, 24 weeks and further at 6-week intervals and/or until twice the desired or labelled
discard date is reached.

E.1.3 Sampling times begin at 24 h and immediately before each rechallenge and at the end point of the test.

E.1.4 To pass the test, all sample point counts shall be less than the total of the most recent challenge
plus the previous survivor count.

E.1.5 Discard-date testing is done as a supplement to preservative efficacy of multidose preserved

contact lens care products testing performed to verify the labelled shelf life. Discard testing requirements
shall be met throughout the labelled shelf life.

E.2 Test methods

E.2.1 Materials and reagents

E.2.1.1 Test organisms

Test organisms should be as specified in 5.1.1.

E.2.1.2 Test media

Test media should be as specified in 5.1.2.

E.2.1.3 Laboratory equipment

Laboratory equipment should be as specified in 5.1.3.

E.2.1.4 Test samples

Challenge tests are conducted directly into final product containers. A minimum of two sample containers for
each test organism, from each of three individual product lots representative of the product to be marketed,
are selected for testing at each interval in the stability-testing schedule selected for discard dating.
Culture maintenance should be as specified in 5.2.

E.2.2 Preparation of microbial challenge (Inoculum)

Test organisms should be cultured and harvested as specified in 5.3.

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ISO 14730:2014(E)


E.2.3 Inoculum challenge test procedure

E.2.3.1 On day 0, inoculate a series of the final product containers being tested with a single strain
of test organisms suspended to a level to produce a 1,0 × 103 cfu/ml to 2,0 × 103 cfu/ml count in the
volume of product being tested. The volume of inoculum should not exceed 0,5 % of the sample volume at
each challenge period, to reduce the risk of diluting the test product sample over repeated rechallenges.
Ensure dispersion of inoculum by adequate mixing.

E.2.3.2 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 contact lens care product is sensitive to light, it should be protected during the period of the test.

E.2.3.3 At 24 h, take 1,0 ml aliquots of the inoculated product for determination of viable counts.

E.2.3.4 Rechallenge with fresh cultures of the same inoculum organism, level and quantity as in E.2.3.1,
store at 20 °C to 25 °C until day 7 and repeat the viable count determination, rechallenge, and storage
steps. Duplicate this process at 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 18 weeks, 24 weeks and as
needed at 6-week intervals, and/or until two times the desired or labelled discard date has been reached.

E.2.3.5 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.

If an antimicrobial agent in the formulation cannot be adequately inactivated or neutralized, eliminate

it using a validated membrane filtration procedure (see Annex A).

E.2.3.6 Determine the viable count of organisms in appropriate dilutions by preparation of plates in
triplicate (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 may also contain antimicrobial inactivators or
neutralizers, if required.

E.2.3.7 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 should be determined. Minimum incubation times should be
based on recovery medium control testing (see E.2.4.2).

E.2.3.8 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/plate for bacteria and yeast, and 8 cfu to 80 cfu/plate for
moulds, except when colonies are observed only for the 100 or 10−1 dilution plates.

E.2.3.9 When plates for all dilutions of a sample at a single time point indicate no recovery of
microorganisms, it should be documented.

E.2.3.10 The concentration of survivors is calculated at each time point.

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 BS EN ISO 14730:2014
ISO 14730:2014(E)


E.2.4 Controls

E.2.4.1 Inoculum controls

At each time interval duplicate matched bottles containing a suitable diluent (e.g. DPBST) are inoculated
at the same time as the test samples for the determination of inoculum levels. The volume in the control
container should approximate the remaining volume in the test container. 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).

E.2.4.2 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 in 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 shall be used.
Qualify the neutralization of the product initially and periodically.

E.2.5 Performance criteria

E.2.5.1 The test is carried out to two times the desired or labelled discard time.

E.2.5.2 The numbers of organisms recovered should not be more than the sum of the most recent
inoculum plus the previous survivor count within a ±0,5 log variance, (i.e. multiplication of organisms
has not occurred).

E.2.5.3 The products should be capable of meeting these criteria throughout the labelled shelf life and
for the additional time of the discard-after-opening date at the end of the shelf life.

E.2.6 Test report

The test report should be as specified in 5.7.

© ISO 2014 – All rights reserved  21

BS EN ISO 14730:2014
ISO 14730:2014(E)


Annex F
(informative)

Test organisms from other culture collections

Table F.1 — Test organisms from other culture collections

Pseudomonas aeruginosa MUAVCR 278 CCM 1961 CIP 82.118 DSM 1128 IAM 10374
IFO 13275 NCIMB 8626 NRRL B-800
Staphylococcus aureus CIP 4.83 DSM 799 IFO 13276 NCIB 9518 NCTC 10788
Escherichia coli CIP 53.126 DSM 1576 NCDO 904 NCIB 8545
Candida albicans CBS 6431 CCY 29–3-106 CIP 48.72 DSM 1386 IFO 1594
NCPF 3179 NCYC 1363 VTT C-85161
Aspergillus brasiliensis CBS 733.88 DSM 1988 IMI 149007 IFO 9455 NCPF 2275
NOTE Cultures from various collections should be equivalent to ATCC strains.

Table F.2 — Culture collections and institutions

ATCC American Type Culture Collection, Rockville, MD, USA
MUAVCR Microbiologický ústav Akademie ved. Ceské republiky, Prague, Czech Republic
CBS Centraalbureau voor Schimmelcultures, Baarn, The Netherlands
CCM Czech Collection of Microorganisms, Ceská sbirka mikroorganizmu, Prírodovedecká
fakulta Masarykovy univerzity, Brno, Czech Republic
CCY Culture Collection of Yeasts, Chemický ústav SAV, Bratislava, Slovakia
CIP Collection de bactéries de l’Institut Pasteur, Paris, France
DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Ger-
many
IAM Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan
IFO Institute for Fermentation, Osaka, Japan
IMI International Mycological Institute, Kew, Surrey, UK
NCDO National Collection of Dairy Organisms, Shinfield, Reading, Berkshire, UK
NCIB National Collection of Industrial Bacteria, Aberdeen, Scotland, UK
NCIMB National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland, UK
NCPF National Collection of Pathogenic Fungi, Mycological Reference Laboratory, Central Pub-
lic Health Laboratory, London, UK
NCTC National Collection of Type Cultures, Central Public Health Laboratory, London, UK
NCYC National Collection of Yeast Cultures, Nutfield, Surrey, UK
NRRL Northern Regional Research Center, US. Department of Agriculture, Peoria, IL, USA
VTT Technical Research Centre of Finland, VTT Collection of Industrial Microorganisms,
Espoo, Finland

22  © ISO 2014 – All rights reserved

 BS EN ISO 14730:2014
ISO 14730:2014(E)


Bibliography

[1] Ph. Eur. 1994. Efficacy of antimicrobial preservation. In: European Pharmacopoeia 1994, 2nd ed.,
Part 11, 18th section. Maisonneuve S.A., Sainte-Ruffine, France, p.V111.14.1-V111.14.4
[2] USP 23. 1994. Antimicrobial preservatives  — Effectiveness. In United States Pharmacopoeia,
23rd rev. U.S. Pharmacopoeial Convention, Inc., Rockville, MD, p. 1681
[3] Urban/Hecker/Schiller. Analytical R & D Sandoz AG, Basel. Zbl. Bakt. Hyg. I. Abt. A. 1981,
172 pp. 478–484

© ISO 2014 – All rights reserved  23

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