ISO 18189:2016

INTERNATIONAL ISO
STANDARD 18189
First edition
2016-06-01
Ophthalmic optics — Contact lenses
and contact lens care products —
Cytotoxicity testing of contact lenses
in combination with lens care solution
to evaluate lens/solution interactions
Optique ophtalmique — Lentilles de contact et produits d’entretien
des lentilles de contact — Essais de cytotoxicité des lentilles de
contact en association avec une solution d’entretien des lentilles de
contact pour évaluer les interactions solution/lentille
Reference number
©
ISO 2016
© ISO 2016, Published in Switzerland
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ii © ISO 2016 – All rights reserved

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Direct contact cytotoxicity test for lens/lens care solution combination .1
5.1 General . 1
5.2 Experimental procedure . 2
5.2.1 Basic procedure . 2
5.2.2 Material . 2
5.2.3 Preparation of test sample . 3
5.2.4 Methods . 3
6 Assessment of results . 5
7 Test report . 5
Annex A (normative) Measurement of zone of cell lysis for the direct contact cytotoxicity
test method for testing contact lens in combination with lens care solution .7
Bibliography . 9
Foreword
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The committee responsible for this document is ISO/TC 172, Optics and photonics, Subcommittee SC 7,
Ophthalmic optics and instruments.
iv © ISO 2016 – All rights reserved

INTERNATIONAL STANDARD ISO 18189:2016(E)
Ophthalmic optics — Contact lenses and contact lens
care products — Cytotoxicity testing of contact lenses
in combination with lens care solution to evaluate lens/
solution interactions
1 Scope
This International Standard describes an in vitro test method to assess the potential cytotoxic effects
that may arise due to interaction of contact lenses with contact lens care solutions.
NOTE The potential of a contact lens or a contact lens care solution to cause cytotoxicity by itself can be
evaluated in accordance with general guidance in ISO 10993-5.
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 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 and the following apply.
3.1
room temperature
temperature defined as 18 °C to 25 °C
4 Principle
The chemicals in a lens care solution can cause cytotoxic effects by direct contact with ocular tissues
or by indirect contact through contact lenses. Uptake of the care product preservative or other
solution ingredients by the lens and subsequent release of these chemicals in the ocular environment
can compromise ocular biocompatibility. The potential interactions between a lens care product and
various contact lens materials should be taken into account in designing the tests to fully evaluate the
cytotoxicity potential of a new contact lens or a lens care product.
5 Direct contact cytotoxicity test for lens/lens care solution combination
5.1 General
The following protocol describes the test method for evaluating potential cytotoxic effects of contact
lenses exposed to contact lens care solution. The cytotoxicity can result from contact lens/lens care
solution interactions.
With the exception of daily disposable contact lenses, the potential interaction of a new contact lens
with marketed representative multipurpose solutions to produce cytotoxicity shall be evaluated.
For evaluating a new contact lens care solution, the potential interaction of new contact lens care
solution with representative contact lenses to produce cytotoxicity shall be evaluated.
5.2 Experimental procedure
5.2.1 Basic procedure
The test contact lens is incubated in ~10 ml of contact lens care solution in a sterile compatible container
2+
for 24 h ± 2 h at room temperature. Similarly, a Dulbecco’s Phosphate Buffered Saline with Ca and
2+
Mg (DBPS)-treated control lens (“Lens Control”) is prepared by incubating the contact lens in ~10 ml
of DPBS in the same type of container for 24 h ± 2 h at room temperature.
For the purpose of this International Standard, a compatible container refers to a container in which
there is little to no uptake of the disinfecting agent and/or preservative. Rinsing of the container with
the contact lens care product may be used to reduce uptake by the container.
Following the 24 h ± 2 h soak period, the lenses may be cut in a pinwheel fashion (3 to 4 cuts
approximately 1/3 to 1/2 into the lens) and immediately used for cytotoxicity testing. If the lens is
not cut, it shall be placed on the cells in a concave manner. Each lens is placed in the centre on the cell
surface in a 60 mm diameter tissue culture plate containing subconfluent monolayer of L-929 cells in
1,6 ml Minimal Essential Medium (MEM) supplemented with 5 % fetal bovine serum (FBS).
Similarly, negative and positive controls are placed in the designated 60 mm diameter tissue culture
plates containing subconfluent monolayer of L-929 cells in 1,6 ml MEM supplemented with 5 % FBS.
The tissue culture plates are incubated at 37 °C ± 1 °C in 5 % ± 1 % CO for 24 h ± 2 h.
Following incubation, the lenses and the controls are removed from each plate and the cells are stained
with Trypan Blue to facilitate observation of dead or damaged cells. The cytotoxicity is assessed by
evaluating the cells macroscopically and microscopically (100×) for any abnormal cell morphology and
lysis around the test article and controls to determine the zone of lysis (if any).
5.2.2 Material
5.2.2.1 Cell line
L-929 cells [NCTC clone 929: CCL 1, American Type Culture Collection (ATCC), Manassas, VA, USA;
ECACC No. 88102702 or equivalent, European Collection of Cell Cultures, Salisbury, Wiltshire SP4 0JG,
UK]. Cell cultures shall be free of mycoplasma.
The passage number of the cells for testing should be 10 – 30.
5.2.2.2 Technical equipment
5.2.2.2.1 Incubator, 37 °C ± 1 °C, humidified, 5 % ± 1 % CO /air.
5.2.2.2.2 Laminar flow cabinet, standard: “biological hazard”.
5.2.2.2.3 Water bath, 37 °C.
5.2.2.2.4 Inverse phase contrast microscope.
5.2.2.2.5 Laboratory burner.
5.2.2.2.6 Centrifuge.
2 © ISO 2016 – All rights reserved

5.2.2.2.7 Laboratory balance.
5.2.2.2.8 Cell counter or hemocytometer.
5.2.2.2.9 Tissue culture flasks and 60 mm diameter tissue culture plat
...