SIST EN ISO 9363-1:2000
(Main)Optics and optical instruments - Contact lenses - Determination of cytotoxicity of contact lens material - Part 1: Agar overlay test and growth inhibition test (ISO 9363-1:1994)
Optics and optical instruments - Contact lenses - Determination of cytotoxicity of contact lens material - Part 1: Agar overlay test and growth inhibition test (ISO 9363-1:1994)
Specification of a procedure for the determination of cytotoxity of contact lenses by contaminating different cells.
Optik und optische Instrumente - Kontaktlinsen - Bestimmung der Zytotoxizität von Kontaktlinsenmaterial - Teil 1: Agar-Überschichtungs-Prüfung und Wachstumsinhibitions-Prüfung (ISO 9363-1:1994)
Dieser Teil von ISO 9363 legt zwei in-vitro-Verfahren zur Bestimmung der Zytotoxizita(t von Kontaktlinsenmaterialien fest: die Agar-U(berschichtungs- Pru(fung und die Wachstumsinhibitions-Pru(fung. Der Hauptzweck dieser Pru(fungen ist der Nachweis von auslaugbaren zytotoxischen Substanzen in oder auf Kontaktlinsen.
Optique et instruments d'optique - Lentilles de contact - Détermination de la cytotoxicité des matériaux des lentilles de contact - Partie 1: Essai de recouvrement par de l'agar-agar et essai d'inhibition de croissance (ISO 9363-1:1994)
Optics and optical instruments - Contact lenses - Determination of cytotoxicity of contact lens material - Part 1: Agar overlay test and growth inhibition test (ISO 9363-1:1994)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 9363-1:2000
01-julij-2000
Optics and optical instruments - Contact lenses - Determination of cytotoxicity of
contact lens material - Part 1: Agar overlay test and growth inhibition test (ISO
9363-1:1994)
Optics and optical instruments - Contact lenses - Determination of cytotoxicity of contact
lens material - Part 1: Agar overlay test and growth inhibition test (ISO 9363-1:1994)
Optik und optische Instrumente - Kontaktlinsen - Bestimmung der Zytotoxizität von
Kontaktlinsenmaterial - Teil 1: Agar-Überschichtungs-Prüfung und Wachstumsinhibitions-
Prüfung (ISO 9363-1:1994)
Optique et instruments d'optique - Lentilles de contact - Détermination de la cytotoxicité
des matériaux des lentilles de contact - Partie 1: Essai de recouvrement par de l'agar-
agar et essai d'inhibition de croissance (ISO 9363-1:1994)
Ta slovenski standard je istoveten z: EN ISO 9363-1:1999
ICS:
11.040.70 Oftalmološka oprema Ophthalmic equipment
SIST EN ISO 9363-1:2000 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 9363-1:2000
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SIST EN ISO 9363-1:2000
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SIST EN ISO 9363-1:2000
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SIST EN ISO 9363-1:2000
INTERNATIONAL ISO
STANDARD 9363-1
First edition
1994-1 l-l 5
Optics and Optical instruments - Contact
lenses - Determination of cytotoxicity of
contact lens material -
Part 1:
Agar overlay test and growth inhibition test
Optique et instruments d’optique - LentilIes de contact - D&ermination
de Ia cytotoxicitk des matkiaux des lentilles de contact -
Partie 7: Essai de recouvrement par de I’agar-agar et essai d’inhibition de
croissance
Reference number
ISO 9363-1: 1994(E)
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SIST EN ISO 9363-1:2000
ISO 9363=1:1994(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. Esch 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.
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.
International Standard ISO 9363-1 was prepared by Technical Committee
ISO/TC 172, Optics and optica/ Instruments, Subcommittee SC 7,
Oph thalmic, endoscopic, me trological ins trumen ts and tes t me thods.
Annex A forms an integral pat of this part of ISO 9363. Annex B is for
information only.
0 ISO 1994
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced
or utilized in any form or by any means, electronie or mechanical, incruding photocopying and
microfilm, without Permission in writing from the publisher.
zation for Standardization
International Organi
CH-1211 Geneve 20 l Switzerland
Case postale 56 l
Printed in Switzerland
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SIST EN ISO 9363-1:2000
INTERNATIONAL STANDARD o ISO ISO 9363=1:1994(E)
Optics and Optical instruments - Contact lenses -
Determination of cytotoxicity of contact lens material -
Part 1:
Agar overlay test and growth inhibition test
The growth inhibition test is designed to ascertain the
1 Scope
presence of extractable cytotoxic substances. The
growth rate of mammalian cells is significantly
This part of ISO 9363 specifies two in vitro methods
decreased in the presence of toxic substances.
for determining the cytotoxicity of contact lens
Usually, the growth rate is determined by comparing
materials:
the cell number or the Protein content of cells at
- the agar overlay test; and
different time intervals. Since there is a linear
relationship between cell number and Protein
- the growth inhibition test.
concentration under conditions of the assay to be
described, the growth rate is determined by Protein
The primary purpose of these tests is to reveal the
measurement. Cell counting tan be used as an
presence of leachable cytotoxic substances in or on
alternative method of assessment.
contact lenses.
In the growth inhibition test, medium extracts of
NOTES
contact lenses are added to the culture medium of
cells and the Protein content of the cell culture after
1 Attention is drawn to ISO 10993-5.
72 h in the presence of the extract is compared with
the Protein content in cell cultures without the extract.
2 A minimum of one in vitro test is recommended for
preclinical evaluation of new types of contact lenses. Either
In Order to ensure high quality work, the cytotoxicity
one of the following two in v;tro tests may be used for the
testing of contact lenses should be carried out in
in vitro requirement.
experienced laboratories according to GLP guidelines.
The Overall assessment of the results should be
carried out by an expert in the field of toxicology who
is informed about the final product and the conditions
2 Principle of its use and has appropriate Chemical and biological
data concerning it.
The proposed tests are designed to ascertain the
absence of extractable cytotoxic substances.
The agar overlay test is designed to assess the
3 Agar overlay test
presence of leachable toxic substances in solid ma-
terials. The test Sample is placed in contact with the
surface of an agar layer which covers a monolayer of
3.1 Apparatus and solutions
cells treated with a vital stain. After 24 h of incubation,
the presence of leachable toxic substances is
3.1.1 Apparatus
manifested by the loss of dye from the cells within
the diffusion zone of the soluble substance(s) leaching
from the Sample and by lysis of the cells within the Standard tissue culture facilities, including sterilization
zone if the concentrations and toxicity of the diffusing equipment (autoclave and membrane filtration),
substance(s) are sufficiently high. laminar airflow hood, 37 “C carbon dioxide-air
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SIST EN ISO 9363-1:2000
0 ISO
ISO 9363=1:1994(E)
incubator, water bath, tissue culture glass and plastics 3.1.6 Trypsin solution
Ware.
A suitable concentration (0,l g/l to 025 g/l) of trypsin
in PBS or other agent to dissociate the cell monolayer
shall be used for preparation of cell suspensions.
3.1.2 Culture medium
The culture medium shall be sterile.
3.2 Test material
NOTE 3 To ensure this, the culture medium may be
purchased sterile and ready for use, may be prepared from
The test material shall be representative of the final
sterile ingredients using aseptic techniques, or, when one
product. At least two contact lenses of each Sample
or more ingredients are not available in sterile form, may be
are necessary.
sterilized after preparation by membrane filtration.
The complete culture medium shall be Dulbecco’s 3.3 Controls
Modified Eagle Medium containing 3,7 g/l sodium
hydrogen carbonate, 10 % by volume fetal calf Serum
3.3.1 Positive control material
(FCS), 100 IU/mI Penicillin and 100 pg/mI streptomycin
or any other culture medium which tan be
The positive control shall be any material which, when
demonstrated to give reproducible results five times.
tested by the procedure described in 3.52, produces
a cytotoxic response.
3.1.3 Agar medium
3.3.2 Negative control material
The agar medium shall be comprised of one part
double concentration of sterile complete culture
The negative control shall be only material which,
medium (all Supplements are double concentration as
when tested by the procedure described in 3.5.2, is
weil) plus one part of sterile 3 g/l agarose or
known not to produce a cytotoxic response.
equivalent in bidistilled water or equivalent.
3.4 Cells
Bring melted agarose to approximately 50 “C and
medium to 37 “C in a water bath or both to 42 “C and
One of the following cell lines shall be used:
mix aseptically. Use at 42 “C.
a) American Type Culture Collection CCL 1, NCTC
Clone 929 (connective tissue, mouse), clone of
3.1.4 Phosphate buffered Saline (PBS), Calcium- and Strain L (referred to hereafter as L 929 cells).
magnesium-free
b) Any other cell line, provided that when tested in
accordance with this part of ISO 9363 a repro-
The Phosphate buffered Saline shall consist of 8,0 g of
ducible toxic titre is obtained for the positive
sodium chloride, 0,2 g of potassium chloride, 2,9 g of
control material and no cytotoxicity is observed for
disodium hydrogen orthophosphate dodecahydrate
the negative control material and fresh cell culture
(Na2HP04. 12H,O), 0,2 g of potassium dihydrogen
medium.
orthophosphate ( KHZPOJ) dissolved in bidistilled water
or equivalent (cell culture grade) to give 1 000 ml of
NOTES
Solution. Adjust to pH 7,2, sterilize by an appropriate
method. Warm up to 37 “C before use.
4 The passage number should be recorded.
5 Stock cultures should be tested for the absence of
mycoplasma before use. Test for mycoplasma tan be
3.1.5 Vital stain
performed in accordance with W. C. Russe11 et al. or any
other established method. Only cells free from myco-
The vital stain shall be neutral red or an equivalent
Plasma should be used for the test.
vital stain. Neutral red vital stain shall be prepared as
follows:
3.5 Test procedure and evaluation
Stock Solution: 1,O g/l neutral red in bidistilled water.
3.5.1 Sample preparation
Adjust to pH 7,2, sterilize by filtration. Protect from
strong light.
The contact lenses should be applied directly. The
Vital stain: 1 :lO stock Solution in sterile PBS, prepare
agar overlay test does not require sterile samples,
freshly and protect from strong light.
although sterility is desirable.
2
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SIST EN ISO 9363-1:2000
0 ISO ISO 9363=1:1994(E)
Neutral red is a redox dye which particularly con-
3.5.2 Procedure
centrates in the lysosomes of living viable cells.
Decolorization of cells is a first sign of cell darnage
Use L 929 cells from an exponentially growing
and precedes detachment from the Substrate and cell
monolayer culture.
lysis. Depending on the water solubility and concen-
Prepare a working stock of cells. Remove the tration of toxic substances of low molecular mass in
medium, wash twice with PBS, add approximately the specimen, those cells under and around the
3 ml trypsin Solution per 75 cm* tissue culture flask Sample are decolorized (Zone-index). Frequently, a
and incubate until cells become detached (approxi- decolorization of the cells directly under the Sample is
mately 3 min at 37 “C with 0,25 % trypsin in PBS). observed but without cell lysis. However, a material is
judged as “cytotoxic” only if lysis is observed simul-
Stop the enzyme reaction by adding 10 ml of
taneously. If a material releases highly diffusible
complete culture medium, centrifuge for 10 min at
cytotoxic compounds in a low concentration, decolor-
100 g and bring to 2,5 x IO5 cells/ml medium. The
ization of the cells without lysis is possible. Therefore,
viability of the cells shall be more than 75 % as
a Zone-index of 2 or higher without cell lysis is also
determined by trypan blue staining or any other
considered a significant reaction. In any case, the
appropriate method.
reaction-index shall be given and the result has to be
interpreted.
Plate 10 ml (4,5 ml) of the adjusted cell Suspension
into 90 mm (60 mm) diameter disposable Petri dishes
NOTE 6 Hydrophylic contact lens materials tan passively
and incubate for 24 h at 37 “C in humidified air
absorb dye, giving a discoloration which would not be due
containing 5 % by volume carbon dioxide.
to cytotoxic effects.
Aspirate the medium after in
...
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