EN ISO 14730:2000

SLOVENSKI STANDARD
01-november-2001
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Ophthalmic optics - Contact lens care products - Antimicrobial preservative efficacy
testing and guidance on determining discard date (ISO 14730:2000)
Augenpolitik - Kontaktlinsenpflegemittel - Konservierungsmittlebelastungstest und
Anleitung zur Feststellung der Aufbrauchfrist (ISO/FDIS 14730:2000)
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 apres premiere ouverture (ISO/FDIS 14730:2000)
Ta slovenski standard je istoveten z: EN ISO 14730:2000
ICS:
11.040.70 Oftalmološka oprema Ophthalmic equipment
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 14730
NORME EUROPÉENNE
EUROPÄISCHE NORM
September 2000
ICS 11.040.70
English version
Ophthalmic optics - Contact lens care products - Antimicrobial
preservative efficacy testing and guidance on determining
discard date (ISO 14730:2000)
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 14730:2000)
de la durée d'utilisation après première ouverture (ISO
14730:2000)
This European Standard was approved by CEN on 15 September 2000.
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 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 Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2000 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14730:2000 E
worldwide for CEN national Members.

Page 2
Foreword
Corrected 2001-04-11
The text of the International Standard ISO 14730:2000 has been prepared by Technical
Committee ISO/TC 172 "Optics and optical instruments" 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 March 2001, and conflicting national
standards shall be withdrawn at the latest by March 2001.
Annexes A to F of this Standard are for information only.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,
Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United
Kingdom.
Endorsement notice
The text of the International Standard ISO 14730:2000 was approved by CEN as a European
Standard without any modification.
NOTE: Normative references to International Standards are listed in annex ZA (normative).

Page 3
Annex ZA (normative)
Normative references to international publications
with their relevant European publications
This European Standard incorporates by dated or undated reference, provisions from other
publications. These normative references are cited at the appropriate places in the text and the
publications are listed hereafter. For dated references, subsequent amendments to or revisions
of any of these publications apply to this European Standard only when incorporated in it by
amendment or revision. For undated references the latest edition of the publication referred to
applies (including amendments).
NOTE Where an International Publication has been modified by common modifications,
indicated by (mod.), the relevant EN/HD applies.
Publication Year Title EN Year
ISO 14534 1997 Ophthalmic optics - Contact lenses EN ISO 14534 1997
and contact lens care products -
Fundamental requirements
INTERNATIONAL ISO
STANDARD 14730
First edition
2000-09-15
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 14730:2000(E)
©
ISO 2000
ISO 14730:2000(E)
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ii © ISO 2000 – All rights reserved

ISO 14730:2000(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 .3
5.3 Preparation of microbial challenge (Inoculum) .3
5.4 Inoculum challenge test procedure .4
5.5 Controls .5
5.6 Performance criteria.5
5.7 Test report .6
Annex A (informative) Example of a membrane filtration procedure .7
Annex B (informative) Discard date procedure I.9
Annex C (informative) Discard date procedure II.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 14730:2000(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO 14730 was prepared by Technical Committee ISO/TC 172, Optics and optical
instruments, Subcommittee SC 7, Ophthalmic optics and instruments.
Annexes A to F of this International Standard are for information only.
iv © ISO 2000 – All rights reserved

ISO 14730:2000(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, whilst 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 must 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
may be added to the product to inhibit the growth of microorganisms that may 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 for use 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 days. 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 days.
INTERNATIONAL STANDARD ISO 14730:2000(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 to be used 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 may be used to extend discard dating beyond 28 days. 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 may 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 normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
1�
ISO 8320-2:— , Contact lenses and contact lens care products — Vocabulary — Part 2: Contact lens care
products.
ISO 14534:1997, Ophthalmic optics — Contact lenses and contact lens care products — Fundamental
requirements.
3 Terms and definitions
For the purposes of this International Standard, the terms and definitions given in ISO 8320-2 apply.
4Principle
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
1�
To be published.
ISO 14730:2000(E)
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.
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 may 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 niger 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 suitable diluent.
2 4 2 4 2
5.1.2.4 Dulbecco's Phosphate Buffered Saline plus 0,05 % volumic mass polysorbate 80 (DPBST) or
suitable diluent.
2 © ISO 2000 – All rights reserved

ISO 14730:2000(E)
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.
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 Incubation
Organism Medium
°C time
P. aeruginosa TSA 30to 35 18hto24h
S. aureus TSA 30to 35 18hto24h
E. coli TSA 30to 35 18hto24h
either 20 to 25 42 h to 48 h
C. albicans SDA
or 30 to 35 18 h to 24 h
A. niger
SDA 20to 25 7dto 10d
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 may be washed using centrifugation. The bacterial suspensions may be
filtered (e.g. 3µm to 5µm pore size) to produce a single cell dispersion. Then adjust all challenge cell suspensions
7 8
with DPBST or other suitable diluent to a concentration of between 1 � 10 cfu/ml and 1 � 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.
ISO 14730:2000(E)
If centrifugation is used, each centrifugation should be conducted at 20 °Cto 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 may be required at lower speeds.
NOTE 2 Spore suspensions may be used up to seven days following preparation by storage under refrigeration (2 °Cto
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
5 6
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.
5.4.2 Store the inoculated product at 20 °Cto25 °C. The temperature must 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 may also contain antimicrobial inactivators or neutralizers, if
required.
5.4.8 Incubate bacterial recovery plates at 30 °Cto35 °C. Incubate yeast recovery plates at 20 °Cto 25 °Cor
30 °Cto35 °C. Incubate mould recovery plates at 20 °Cto25 °C. Incubation times for optimal recovery of bacteria,
4 © ISO 2000 – All rights reserved

ISO 14730:2000(E)
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,
0 –1
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.
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
5 6 4 5
1,0� 10 cfu/ml to 1,0� 10 cfu/ml for the initial inoculum or 1,0� 10 to 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 °Cto35 °C. Incubate yeast recovery plates at 20 °Cto 25 °Cor30 °Cto
35 °C. Incubate mould recovery plates at 20 °Cto25 °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.
ISO 14730:2000(E)
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.
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;
g) results obtained, including numbers of organisms recovered at each time point.
6 © ISO 2000 – All rights reserved

ISO 14730:2000(E)
Annex A
(informative)
Example of a membrane filtration procedure
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 KH PO , 8 000 mg/l NaCl, and 2 160 mg/l Na HPO �7H O or suitable diluent.
2 4 2 4 2
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 0,45µm, 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.
ISO 14730:2000(E)
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.
NOTE This may 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 may
be enclosed in an agar sandwich. Alternatively, a sterile membrane filter unit may 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.
8 © ISO 2000 – All rights reserved

ISO 14730:2000(E)
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 10 cfu/ml) on
0 d. The test samples are rechallenged with a low organism inoculum level (approximately 10 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, 2, 3, 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.
B.2.3 Inoculum challenge test procedure
B.2.3.1 Prepare a composite sample of greater than 250 ml from individual test samples.
ISO 14730:2000(E)
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 10 cfu/ml organism suspension
to achieve a final concentration of approximately 10 cfu/ml. Ensure complete dispersion of the inoculum by
adequate mixing.
B.2.3.5 Store the inoculated product at 20 °Cto25 °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 10 cfu/ml organism suspension to provide a final concentration of
approximately 10 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 °Cto35 °C. Incubate yeast recovery plates at 20 °Cto25 °C
or 30 °Cto35 °C. Incubate mould recovery plates at 20 °Cto 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
0 -1
when colonies are observed only for the 10 or 10 dilution plates.
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.
10 © ISO 2000 – All rights reserved

ISO 14730:2000(E)
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
2)
saline/Tween� 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 °Cto35 °C. Incubate yeast recovery plates at 20 °Cto 25 °Cor30 °Cto
35 °C. Incubate mould recovery plates at 20 °Cto25 °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 3 logs by day 14 and the number
3)
of bacteria and fungi do not increase in numbers thereafter.
B.2.6 Test report
The test report should be as specified in 5.7.
2) 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.
3) 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).
ISO 14730:2000(E)
Annex C
(informative)
Discard date procedure II
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 so 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 may be pooled in order to provide sufficient product for
testing).
Culture maintenance should be as specified in 5.2.
12 © ISO 2000 – All rights reserved

SIST EN ISO 14
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