1、BS ISO 18189:2016Ophthalmic optics Contactlenses and contact lens careproducts Cytotoxicitytesting of contact lenses incombination with lens caresolution to evaluate lens/solution interactionsBSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS ISO 18189:2016 BRITISH S
2、TANDARDNational forewordThis British Standard is the UK implementation of ISO 18189:2016.The UK participation in its preparation was entrusted to TechnicalCommittee CH/172/9, Contact lenses and contact lens care products.A list of organizations represented on this committee can beobtained on request
3、 to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 79530 5ICS 11.040.70Compliance with a British Stand
4、ard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 June 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 18189:2016 ISO 2016Ophthalmic optics Contact lenses
5、and contact lens care products Cytotoxicity testing of contact lenses in combination with lens care solution to evaluate lens/solution interactionsOptique ophtalmique Lentilles de contact et produits dentretien des lentilles de contact Essais de cytotoxicit des lentilles de contact en association av
6、ec une solution dentretien des lentilles de contact pour valuer les interactions solution/lentilleINTERNATIONAL STANDARDISO18189First edition2016-06-01Reference numberISO 18189:2016(E)BS ISO 18189:2016ISO 18189:2016(E)ii ISO 2016 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in
7、 SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can
8、be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 18189:2016ISO 18189:2016(E)Foreword iv1 S
9、cope . 12 Normative references 13 Terms and definitions . 14 Principle 15 Direct contact cytotoxicity test for lens/lens care solution combination . 15.1 General . 15.2 Experimental procedure . 25.2.1 Basic procedure 25.2.2 Material 25.2.3 Preparation of test sample 35.2.4 Methods . 36 Assessment of
10、 results 57 Test report . 5Annex 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 . 7Bibliography 9 ISO 2016 All rights reserved iiiContents PageBS ISO 18189:2016ISO 18189:2016(E)ForewordIS
11、O (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 co
12、mmittee 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 electro
13、technical 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 draf
14、ted 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 rig
15、hts. 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 constit
16、ute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/fo
17、reword.html.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 reservedBS ISO 18189:2016INTERNATIONAL STANDARD ISO 18189:2016(E)Ophthalmic optics Contact lenses and contact lens care products Cy
18、totoxicity testing of contact lenses in combination with lens care solution to evaluate lens/solution interactions1 ScopeThis 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
19、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 referencesThe following documents, in whole or in part, are normatively referenced in this document and are i
20、ndispensable 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
21、 for labelling specifications3 Terms and definitionsFor the purposes of this document, the terms and definitions given in ISO 18369-1 and the following apply.3.1room temperaturetemperature defined as 18 C to 25 C4 PrincipleThe chemicals in a lens care solution can cause cytotoxic effects by direct c
22、ontact 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
23、 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 combination5.1 GeneralThe following proto
24、col 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 co
25、ntact lens with marketed representative multipurpose solutions to produce cytotoxicity shall be evaluated. ISO 2016 All rights reserved 1BS ISO 18189:2016ISO 18189:2016(E)For evaluating a new contact lens care solution, the potential interaction of new contact lens care solution with representative
26、contact lenses to produce cytotoxicity shall be evaluated.5.2 Experimental procedure5.2.1 Basic procedureThe test contact lens is incubated in 10 ml of contact lens care solution in a sterile compatible container for 24 h 2 h at room temperature. Similarly, a Dulbeccos Phosphate Buffered Saline with
27、 Ca2+and Mg2+(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 t
28、o 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) an
29、d 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
30、 (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
31、 C in 5 % 1 % CO2for 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) fo
32、r any abnormal cell morphology and lysis around the test article and controls to determine the zone of lysis (if any).5.2.2 Material5.2.2.1 Cell lineL-929 cells NCTC clone 929: CCL 1, American Type Culture Collection (ATCC), Manassas, VA, USA; ECACC No. 88102702 or equivalent, European Collection of
33、 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 equipment5.2.2.2.1 Incubator, 37 C 1 C, humidified, 5 % 1 % CO2/air.5.2.2.2.2 Laminar flow cabinet, standard: “biological hazard”.
34、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 reservedBS ISO 18189:2016ISO 18189:2016(E)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 diame
35、ter tissue culture plates.5.2.2.2.10 Pipetting aid.5.2.2.2.11 Pipettes.5.2.2.3 Chemicals, media and sera5.2.2.3.1 Eagle minimal essential medium (MEM).5.2.2.3.2 Fetal bovine serum (FBS).5.2.2.3.3 Trypsin/EDTA solution.5.2.2.3.4 Dulbeccos Phosphate Buffered Saline with Ca2+and Mg2+(DPBS).5.2.2.3.5 Pe
36、nicillin/streptomycin solution.5.2.2.3.6 Trypan Blue.5.2.3 Preparation of test sampleThe contact lenses should be handled aseptically with forceps. Each lens is individually soaked in 10 ml of appropriate contact lens solution in a sterile compatible container for 24 h 2 h with gentle stirring (cont
37、inual agitation on a shaker at 50 r/min) at room temperature. Aseptic procedure should be followed.Each lens may be cut in a pinwheel fashion (3 to 4 cuts approximately 1/3 to 1/2 into the lens) immediately following the 24 h soak period. The lens is held in a vertical fashion and the edge of the le
38、ns is gently tapped on sterile gauze to remove excess fluid and used for cytotoxicity testing immediately. If the lens is not cut, it shall be placed on the cells in a concave manner. The cytotoxicity test method is described in 5.2.4.3.5.2.4 Methods5.2.4.1 GeneralThe cells should be maintained and
39、cultured using the routine cell culture methods.5.2.4.2 Quality check of the assay: Positive, negative, and lens controls5.2.4.2.1 GeneralPositive control, negative control, and lens control shall be included in each test.5.2.4.2.2 Positive controlLatex glove is recommended as a positive control. A
40、1 cm 1 cm portion shall be placed on the cells in each designated positive control tissue culture plate for testing. Other validated positive control may be used. ISO 2016 All rights reserved 3BS ISO 18189:2016ISO 18189:2016(E)5.2.4.2.3 Negative controlHigh density polyethylene (HDPE) (0,5 mm thickn
41、ess) is recommended as a negative control. A 1 cm 1 cm portion shall be placed on the cells in each designated negative control tissue culture plate for testing. Other validated negative control may be used.5.2.4.2.4 Lens controlThe test contact lens soaked in DPBS shall be used as a lens control. T
42、he contact lenses should be handled aseptically with forceps. Each lens is individually soaked in 10 ml of DPBS solution in a sterile compatible container for 24 h 2 h with gentle stirring (continual agitation on a shaker at 50 r/min) at room temperature. Aseptic procedure should be followed.Each le
43、ns may be cut in a pinwheel fashion (3 to 4 cuts approximately 1/3 to 1/2 into the lens) immediately following the 24 h 2 h soak period. The lens is held in a vertical fashion and the edge of the lens is gently tapped on sterile gauze to remove excess fluid and used for cytotoxicity testing immediat
44、ely. If the lens is not cut, it shall be placed on the cells in a concave manner. The cytotoxicity test method is described in 5.2.4.3.5.2.4.2.5 Test acceptance criteriaFor a test to be considered valid, the following test acceptance criteria shall be met:a) The negative control shall have grades of
45、 1 in all four wells.b) The lens control shall have grades of 1 in all four wells.c) The positive control shall have grades of 3 in all four wells.For description of the reactivity grades, see Table 1.5.2.4.3 Test procedure5.2.4.3.1 Seed the L-929 cells at a density of 6 105cells per plate in the 60
46、 mm diameter tissue culture plates in 6 ml of MEM supplemented with 5 % FBS and incubate at 37 C 1 C in 5 % 1 % CO2for approximately 24 h to obtain subconfluent monolayers of cells prior to use. If antibiotics are used in the MEM medium, it should be documented in the worksheet.5.2.4.3.2 Verify the
47、subconfluency (80 %) and morphology of the cultures microscopically (100) before starting the test. Four cultures (i.e. four 60 mm plates with the cells) shall be used for each test and control article. Only a single test/control article section shall be placed in each plate.5.2.4.3.3 Discard the me
48、dium in each plate and replace with 1,6 ml of MEM.5.2.4.3.4 Place the test/control article in the centre on the cell surface in the designated plates.Place the lens which has been soaked in 10 ml of contact lens solution for 24 h 2 h and may have been cut in a pinwheel fashion as described in 5.2.3
49、in the centre on the cell surface in each of four 60 mm “Test article” tissue culture plates.Place a 1 cm 1 cm portion of latex (positive control) in the centre on the cell surface in each of four 60 mm “positive control” tissue culture plates.Place a 1 cm 1 cm HDPE (negative control) in the centre on the cell surface in each of four 60 mm “negative control” tissue culture plates.Place the lens which has been soaked in 10 ml of DPBS solution for 24 h 2 h and may have bee
