EN ISO 10993-10:1995
(Main)Biological evaluation of medical devices - Part 10: Tests for irritation and sensitization (ISO 10993-10:1995)
Biological evaluation of medical devices - Part 10: Tests for irritation and sensitization (ISO 10993-10:1995)
This part of ISO 10993 describes test methods: a) to evaluate the potential of devices and their constituent materials to produce irritation; and b) to evaluate the potential of devices and their constituent materials to produce sensitization. These test methods are recommended for most categories of device and mode of body contact given in ISO 10993-1. Of the tests listed, those appropriate to the end use of the device are to be selected. Guidance is also given for the preparation of materials specifically in relation to the above tests. Note 1: Guidance on the conduct of supplementary tests which may be required specifically for use in the oral, rectal, penile and vaginal areas is given in Annex D.
Biologische Beurteilung von Medizinprodukten - Teil 10: Prüfungen auf Irritation und Sensibilisierung (ISO 10993-10:1995)
Dieser Teil der ISO 10993 beschreibt Prüfverfahren für: a) die Beurteilung des Irritationspotentials von Medizinprodukten und seinen Bestandteilen; und b) die Beurteilung des Sensibilisierungspotentials von Medizinprodukten und seinen Bestandteilen. Diese Prüfverfahren werden für die meisten Kategorien von Medizinprodukten und Arten ihres Körperkontakts, wie in der ISO 10993-1 angegeben, empfohlen. Von den aufgeführten Prüfungen sind die auszuwählen, die der Endbestimmung des Produkts entsprechen.
Evaluation biologique des dispositifs médicaux - Partie 10: Essais d'irritation et de sensibilisation (ISO 10993-10:1995)
La CEI 60743:2013 s'applique à la terminologie utilisée pour décrire les outils, les dispositifs, les équipements et les méthodes utilisés dans les travaux sous tension. Elle normalise les noms des outils, des dispositifs et des équipements et permet leur identification en donnant des définitions et des illustrations. Elle contient des exemples d'illustration. Cette troisième édition annule et remplace la deuxième édition publiée en 2001 ainsi que son Amendement 1:2008. Cette édition constitue une révision technique qui inclut les modifications techniques majeures suivantes par rapport à l'édition précédente: l'Article 2 a été simplifié et renvoie directement à la CEI 60050-651; certaines définitions ont été incorporées dans les articles spécifiques existants. La présente édition est un complément à la CEI 60050-651. Différentes publications sous la responsabilité du CE 78 contiennent des termes et leurs définitions. La CEI 60050-651 (VEI 651) fournit des définitions précises, brèves et correctes de concepts acceptés au niveau international dans le domaine des travaux sous tension, et indique les termes sous lesquels ces concepts sont connus. Electropedia donne accès aux termes et aux définitions de la CEI 60050-651 (http://www.electropedia.org/). Chaque norme de produit présente les définitions nécessaires à la compréhension de certains termes dans un contexte particulier. Le Glossaire de la CEI (http://std.iec.ch/glossary) donne accès en ligne à ces informations.
Biološko ovrednotenje medicinskih pripomočkov - 10. del: Preskusi draženja in občutljivosti (senzibilizacije) (ISO 10993-10:1995)
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 10993-10:2000
01-januar-2000
%LRORãNRRYUHGQRWHQMHPHGLFLQVNLKSULSRPRþNRYGHO 3UHVNXVLGUDåHQMDLQ
REþXWOMLYRVWL VHQ]LELOL]DFLMH ,62
Biological evaluation of medical devices - Part 10: Tests for irritation and sensitization
(ISO 10993-10:1995)
Biologische Beurteilung von Medizinprodukten - Teil 10: Prüfungen auf Irritation und
Sensibilisierung (ISO 10993-10:1995)
Evaluation biologique des dispositifs médicaux - Partie 10: Essais d'irritation et de
sensibilisation (ISO 10993-10:1995)
Ta slovenski standard je istoveten z: EN ISO 10993-10:1995
ICS:
11.100.20 %LRORãNRRYUHGQRWHQMH Biological evaluation of
PHGLFLQVNLKSULSRPRþNRY medical devices
SIST EN ISO 10993-10:2000 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 10993-10:2000
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SIST EN ISO 10993-10:2000
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SIST EN ISO 10993-10:2000
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SIST EN ISO 10993-10:2000
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SIST EN ISO 10993-10:2000
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SIST EN ISO 10993-10:2000
INTERNATIONAL IS0
STANDARD 10993-10
First edition
1995-03-I 5
Biological evaluation of medical devices -
Part 10:
Tests for irritation and sensitization
ivaluation biologique des dispositifs mkdicaux -
Partie IO: Essais d ‘irrita tion et de sensibilisa tion
Reference number
IS0 10993-I 0:1995(E)
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SIST EN ISO 10993-10:2000
IS0 10993-10:1995(E)
Contents
Page
1
..............................................................................................
1 Scope
1
.....................................................................
2 Normative references
1
.......................................................................................
3 Definitions
2
....................................
4 General principles, step-wise approach
2
............................................................................
5 Irritation tests
.... 2
5.1 Factors to be considered in design and selection of tests
3
...................................................................
5.2 Skin irritation test
5
................................................................
5.3 Ocular irritation test
8
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4 Intracutaneous (intradermal) reactivity test
10
. . . . . . . . . . . . . . . . . . . . . . . . . . . .*.
6 Sensitization tests
. . 10
6.1 Factors to be considered in design and selection of tests
11
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2 Maximization sensitization test
13
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3 Closed patch sensitization test
Annexes
16
.......................................
A Preparation of materials for testing
......... 17
B Method for extraction of materials for biological tests
19
..........................................................
C Animals and husbandry
20
........................................................
D Additional irritation tests
27
.........................................................
E Background information
29
............................................................................
F Bibliography
0 IS0 1995
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced
or utilized in any form or by any means, electronic or mechanical, including photocopying and
microfilm, without permission in writing from the publisher.
International Organization for Standardization
Case Postale 56 l CH-1211 Geneve 20 l Switzerland
Printed in Switzerland
ii
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SIST EN ISO 10993-10:2000
0 IS0 IS0 10993-10:1995(E)
Foreword
IS0 (the International Organization for Standardization) is a worldwide
federation of national standards bodies (IS0 member bodies). The work
of preparing International Standards is normally carried out through IS0
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. IS0
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 IS0 10993-10 was prepared by Technical Commit-
tee lSO/TC 194, Biological evaluation of medical devices.
IS0 10993 consists of the following parts, under the general title Biological
evaluation of medical devices:
- Part 1: Guidance on selection of tests
- Part 2: Animal welfare requirements
- Part 3: Tests for genotoxicity, carcinogenicity and reproductive
toxicity
- Part 4: Selection of tests for interactions with blood
- Part 5: Tests for cytotoxicity: in vitro methods
- Part 6: Tests for local effects after implantation
- Part 7: Ethylene oxide sterilization residuals
- Part 9: Degradation of materials related to biological testing
[Technical Report]
- Part IO: Tests for irritation and sensitization
- Part 1 I: Tests for systemic toxicity
- Part 12: Sample preparation and reference materials
- Part 13: Identification and quantification of degradation products
from polymers
. . .
III
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SIST EN ISO 10993-10:2000
0 IS0
IS0 10993-10:1995(E)
- Part 14: /den tifica tion and quantification of degradation products
from ceramics
- Part 15: /den tification and quantification of degradation products
from coated and uncoated metals and alloys
- Part 16: General guidance on toxicokinetic study design for degra-
dation products and leachables
- Part 17: Glutaraldehyde and formaldehyde residues in industrially
sterilized medical devices
Future parts will deal with other relevant aspects of biological testing.
This part of IS0 10993 is a harmonization of numerous standards and
guidelines, including BS 5736, OECD Guidelines, U.S. Pharmacopeia and
the European Pharmacopoeia. It is intended to be the overall guidance
document for the selection and conduct of tests enabling evaluation of ir-
ritation and senitization responses relevant to material and device safety.
Annexes A, B and C form an integral part of this part of IS0 10993. An-
nexes D, E and F are for information only.
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SIST EN ISO 10993-10:2000
0 IS0 IS0 10993-10:1995(E)
Introduction
This part of IS0 10993 assesses possible contact hazards from device-
released chemicals that may produce skin and mucosal irritation, eye irri-
tation, and delayed contact sensitization.
Some materials that are included in these devices have been tested, and
their skin or mucosal irritation or sensitization potential has been docu-
mented. Other materials and their chemical components have not been
tested and may act differently when exposed to biological tissues. It is
incumbent upon the manufacturer to evaluate each device for its human
toxic potential prior to marketing.
Traditionally, small animal tests are performed prior to human testing to
help predict human response. More recently, in vitro tests have been
added as an alternative. Despite progress and considerable effort in this
direction, a review of findings suggests that currently no satisfactory in
vitro test has been devised to eliminate the requirement for in vivo testing.
Where appropriate, the preliminary use of in vitro methods is encouraged
as screening tests prior to animal testing. In order to reduce the number
of animals used, these standards use a step-wise approach with review
and analysis of test results at each stage.
It is incumbent upon the investigator to conduct these studies using good
scientific laboratory practices, complying with regulations related to animal
welfare. Since the number of animals is restricted, the data obtained may
be insufficient to warrant the application of statistics.
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SIST EN ISO 10993-10:2000
INTERNATIONAL STANDARD 0 IS0 IS0 10993-10:1995(E)
Biological evaluation of medical devices -
Part 10:
Tests for irritation and sensitization
IS0 10993-I : 1992, Biological evaluation of medical
1 Scope
devices - Part 7: Guidance on selection of tests.
This part of IS0 10993 describes test methods:
I so 10993-I 2:--l), Biological evaluation of medical
devices
- Part 12: Sample preparation and reference
a) to evaluate the potential of devices and their con-
materials.
stituent materials to produce irritation; and
to evaluate the potential of devices and their con-
b)
stituent materials to produce sensitization.
3 Definitions
These test methods are recommended for most cat-
For the purposes of this part of IS0 10993, the defi-
egories of device and mode of body contact given in
nitions given in IS0 10993-I and the following defi-
IS0 10993-I. Of the tests listed, those appropriate to
nitions apply.
the end use of the device are to be selected. Guid-
ance is also given for the preparation of materials
3.1 (allergic contact) sensitization; delayed con-
specifically in relation to the above tests.
tact hypersensitivity: Allergic response involving
immunological systems that have been activated by
NOTE 1 Guidance on the conduct of supplementary tests
pnor exposure.
which may be required specifically for use in the oral, rectal,
penile and vaginal areas is given in annex D.
3.2 irritation: Localized inflammatory response to
single, repeated or continuous application of the test
substance, without involvement of an immunological
mechanism.
2 Normative references
3.3 oedema: Swelling due to abnormal infiltration
of fluid into the tissues.
The following standards contain provisions which,
through reference in this text, constitute provisions
3.4 erythema: Reddening of the skin or mucous
of this part of IS0 10993. At the time of publication,
membrane.
the editions indicated were valid. All standards are
subject to revision, and parties to agreements based
3.5 eschar: Scab or discoloured slough of skin.
on this part of IS0 10993 are encouraged to investi-
gate the possibility of applying the most recent edi-
tions of the standards indicated below. Members of 3.6 corrosion: Production of irreversible tissue
IEC and IS0 maintain registers of currently valid damage at the site of contact with the skin following
International Standards. the application of a test substance.
1) To be published.
1
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SIST EN ISO 10993-10:2000
Q IS0
IS0 10993=10:1995(E)
sensitizers; guidance is therefore provided only in the
3.7 ulceration: Open sore representing loss of
conduct of in viva tests in species other than humans.
superficial tissue.
It is not necessary to use positive controls in every in
38 . necrosis: Death of cells and/or tissues.
vivo test. A positive control should be run periodically
to validate the test system and demonstrate a positive
3.9 negative control: Substance that closely re-
response.
sembles the test substance in form and, when tested
in accordance with this part of IS0 10993, is neither
If assessment is not possible using the above stages,
an irritant nor a sensitizer.
consideration should be given to non-invasive testing
in humans.
3.10 positive control: Substance that, when tested
in accordance with this part of IS0 10993, gives a re-
producible irritation or sensitization response.
5 Irritation tests
3.11 solvent: Substance (chemical, vehicle, me-
dium, etc.) used to moisten, dilute, suspend, extract
51 . Factors to be considered in design and
or dissolve the test substance.
selection of tests
3.12 reagent control: Solvent used to moisten, di-
Factors affecting the results of irritation studies in-
lute, suspend, extract or dissolve the test substance,
clude
which is evaluated concurrently with the moistened,
diluted, suspended, extracted or dissolved test sub-
the patch test unit;
a)
stance.
b) the degree of occlusion;
application of the test substance;
d
4 General principles, step-wise approach
d) the application site;
This part of IS0 10993 advocates a step-wise ap-
proach which may include any or all of the following:
e) the duration of exposure; and
literature review;
a)
the techniques used in evaluating the test.
f 1
in vitro tests (if available and when validated);
b)
Additional background information is provided in an-
nex E.
in viva tests;
d
While increased flexibility will allow the investigator
non-invasive human tests/clinical trials.
d)
to enhance the sensitivity of the test to suit conditions
of use and population exposure, consistency in pro-
The first stage is a literature review and shall include
cedure contributes to comparability of test results
an evaluation of chemical and physical properties, and
with different materials and from different labora-
information on structually related chemicals and ma-
tories.
terials. If not already known, the pH and pKa of the
material (liquid, solution or extracts of materials) shall
Provisions have been included in the test procedures
be measured prior to any in vivo or in vitro testing.
for evaluation of devices and materials that will have
repeated and/or prolonged exposure. The investigator,
The second stage provides for in vitro assessments.
in consultation with the device manufacturer, should
These should always be considered in preference to
design the study to exaggerate the anticipated con-
in vivo tests and should replace these as new in vitro
tact (time and/or concentration) in the clinical situ-
methods become available and validated.
ation. While use of an exaggerated concentration or
At the third stage acute in viva studies are undertaken extract of the material is acceptable, this should be
to test for materials not already classified as severe borne in mind during interpretation of the results.
irritants or strong sensitizers by stages a) or b). Ma-
For products intended to be used extensively on
terials that do not demonstrate an acute potential may
normal and abnormal skin, no substantial risk is
be further evaluated following repeated exposure.
L
normally accepted; however, many products, in spite
of a potential to irritate, are fully acceptable because
At the present time there are no validated in vitro
of their inherent benefit.
tests (other than simple screens) to detect irritants or
2
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SIST EN ISO 10993-10:2000
0 IS0 IS0 10993-10:1995(E)
5.2.4 Test procedure
If the pH of the test material is less than or equal to
2 or equal to or greater than 11,5, the material may
5.2.4.1 Preparation of animals
be declared an irritant and no further testing is re-
quired. However, experimental evidence suggests
On the day before the test, closely clip the fur on the
that acidity and alkalinity of the test material are not
backs of the animals a sufficient distance on both
the only factors to be considered in relation to the
sides of the spine for application and observation of
capacity of a substance to produce severe injury. The
all test sites (approximately 10 cm x 15 cm). Use only
concentration of the test material, its period of con-
animals with healthy intact skin.
tact, and many other physical and chemical properties
are also important.
NOTE 2 Abrasion of the test site is not necessary, as
evidence indicates similar responses between abraded and
As dose levels in test procedures can be exaggerated,
non-abraded sites.
a positive test does not necessarily exclude the ma-
terial from use.
If repeated exposure is required, follow the pro-
cedures in 5.2.4.2, 5.2.4.3 or 5.2.4.4, repeated for a
maximum of 21 days.
5.2 Skin irritation test
5.2.4.2 Powder or liquid sample
5.2.1 Principle
Apply 0,5 g or 0,5 ml of the test material directly to
each test skin site as shown in figure 1. If the sub-
Assessment of the potential of the material under test
stance is a powder, it should be slightly moistened
to produce dermal irritation.
with water or other suitable solvent before appli-
cation.
5.2.2 Test material
Cover the application sites with a 25 mm x 25 mm
non-occlusive dressing (such as a gauze patch) and
If the test material is either a solid or a liquid, it shall
then wrap the application site with a semi-occlusive
be prepared as specified in annex A.
bandage for a minimum of 4 h. At the end of the
contact time, remove the dressings and mark the
If the test material is to be tested as an extract, it shall
positions of the sites. Remove residual test substance
be prepared as specified in annex B.
by appropriate means, such as washing with luke-
warm water or other suitable, non-irritating solvent,
and careful drying.
5.2.3 Animals and husbandry
5.2.4.3 Extracts and extractants
Healthy young adult albino rabbits of either sex from
a single strain weighing not less than 2 kg shall be
the appropriate extract(s) to the
APPlY
used.
25 mm x 25 mm four-ply gauze patches (0,5 ml per
patch), one patch on each side of the animal as shown
cared for as
The animals shall be acclimatized and
in figure 1. Apply a control patch of gauze moistened
specified in annex C.
with the extracting medium to the other side.
One animal shall initia Ily be used to evaluate the test
Cover the application sites with a semi-occlusive
material.
bandage for a minimum of 4 h. At the end of the
contact time, remove the dressings and mark the
A well-defined response in th e one animal obviates
positions of the sites. Remove residual test substance
the need for additional testing.
by appropriate means, such as washing with luke-
Unless a well-defined response is observed for solid warm water or other suitable, non-irritating solvent,
or liquid materials, a minimum of two further animals and careful drying.
shall be used. For extracts, a minimum of two further
animals per extract shall be used. 5.2.4.4 Solid sample
If the response in the test using the minimum of three Apply the samples of the test material directly to the
animals is equivocal or not clear, additional testing skin on each side of each rabbit as shown in figure 1.
shall be considered. Similarly, apply the control samples to each rabbit.
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SIST EN ISO 10993-10:2000
IS0 10993=10:1995(E)
Cranial
Y Clipped dorsal region
/- Test intact site
Test intact site ’
: LT
/- Control intact site1 )
Control intact site’)
Caudal
1 ) If sample preparation requires this type of control.
Figure 1 - Location of skin application sites
reversibility or irreversibility of the lesions: this need
When testing solids (which may be pulverized if con-
not exceed 14 days.
sidered necessary), the test substance shall be
moistened sufficiently with water or, where necess-
Describe and grade the skin reactions for erythema
ary, an alternative solvent, to ensure good contact
and oedema according to the classification system
with the skin. When solvents are used, the influence
given in table 1 for each application site at each time
of. the solvent on irritation of skin by the test sub-
interval and record the results for the test report.
stance shall be taken into account.
NOTE 3 Histological and non-invasive techniques may
Cover the application sites with 25 mm x 25 mm
assist in certain cases.
non-occlusive dressings (such as a gauze patch) and
then wrap the application sites with a semi-occlusive
bandage for a minimum of 4 h. At the end of the
contact time, remove the dressings and mark the
5.2.6 Evaluation of results
positions of the sites. Remove residual test substance
by appropriate means, such as washing with luke-
For acute exposure, determine the Primary Irritation
warm water or other suitable, non-irritating solvent,
Index (PII) as follows.
and careful drying.
For each animal, add together the Primary Irritation
Scores for the test material for both erythema and
oedema at each time specified and divide by the total
number of observations (six: two at each time speci-
5.2.5 Observation of animals
fied). When vehicle controls are used, calculate the
Primary Irritation Score for the vehicle controls and
For acute (single exposure) tests, record the appear-
subtract that score from the score for the test ma-
ance of each application site at 1 h, 24 h, 48 h and
terial to obtain the Primary Irritation Score.
72 h following removal of the patches. Extended ob-
servation may be necessary if there are persistent
Only use 24 h, 48 h and 72 h observations for calcu-
lesions, in order to evaluate the reversibility or
lations. Observations made prior to dosing or after
irreversibility of the lesions: this need not exceed
72 h, to monitor recovery, are not used in the deter-
14 days.
mination.
For repeated exposure, record the appearances of the
Add the scores for each animal and divide the total
application site at 1 h after removal of the patches and
by the number of animals. This value is the Primary
immediately prior to the next application. After the last
Irritation Index.
exposure, note the appearance of each application
site at 1 h, 24 h, 48 h and 72 h following removal of
For repeated exposure, determine the Cumulative Ir-
the patches. Extended observation may be necessary
ritation Index as follows.
if there are persistent lesions, in order to evaluate the
4
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SIST EN ISO 10993-10:2000
Table 2 - Irritation response categories in rabbit
- Classification system for skin reaction
Table 1
Mean score
Numerical Response category
Reaction
grading
0 to 0,4
Negligible
Erythema and eschar formation
Slight 0,5 to I,9
0
No erythema
2 to 4,9
Moderate
1
Very slight erythema (barely perceptible)
5 to 8
Severe
2
Well-defined erythema
3
Moderate erythema
5.2.7 Presentation of results
Severe erythema (beet-redness) to
eschar formation preventing grading of
The test report shall include
4
erythema
Oedema formation
a) a description of the test material(s) or device;
0
No oedema
b) the intended use/application of the test material(s)
1
Very slight oedema (barely perceptible)
or device;
Well-defined oedema (edges of area
2
well-defined by definite raising)
c) a detailed description of the method employed in
Moderate oedema (raised approxi-
preparing the test material or device;
3
mately 1 mm)
Severe oedema (raised more than d) the test animals;
1 mm and extending beyond exposure
4
area)
e) method of application to the test sites;
8
Total possible score for irritation
f) how the site readings were performed and a rec-
ord of the observations;
NOTE - Other adverse changes at the skin sites shall
g) assessment of the results.
be recorded and reported.
5.3 Ocular irritation test
For each animal, add together the Irritation Scores for
both erythema and oedema at each time specified.
Divide this total by the total number of observations
5.3.1 Principle
to obtain the Irritation Score per animal.
Assessment of the potential of the material under test
Add the Irritation Scores of each animal and divide by
to produce ocular irritation.
the total number of animals. This value is the Cumu-
lative Irritation Index.
5.3.2 Exclusion from test
The Cumulative Irritation Index is compared to the
Materials and/or final products which have demon-
categories of Cumulative Irritation Index defined in
strated definite corrosion or severe irritation in a
table2 and the appropriate Category is recorded for
dermal study shall not be further tested for eye irri-
the report.
tation. Strongly acidic or alkaline substances (pH < 2
or 3 11,5) shall not be tested owing to their predictive
NOTE 4 The categories of Cumulative Irritation Index are
based on the data relating the Primary Irritation Index (PII) corrosive properties. These products shall be con-
for chemicals in rabbits to the primary irritation response in
sidered eye irritants.
humans for a number of chemicals that have been tested
in both species.
5.3.3 Test material
For any response, determine the Maximum Irritation
If the test material is a liquid, instil 0,l ml undiluted
Response, the time of onset of the response and the
into the lower conjunctival sac of one eye.
time to maximum response.
If the test material is a solid or granular product, grind
The Primary or Cumulative Irritation Index is charac-
to a fine dust. When gently compacted, instil that
terized by number and description in table2.
amount which occupies a volume of 0,l ml and does
5
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SIST EN ISO 10993-10:2000
0 IS0
IS0 10993-10:1995(E)
ophth al
100 mg into the lower conjunc- The use of an mosco Ipe, hand slit-lamp, or
not weigh more than
other suitable de vice, i S recom mended.
tival sac of one eye.
lnstil the test material as specified in 5.3.3.
NOTE 5 Some products may not be amenable to testing
directly in the eye. Mechanical damage can result in making
Following instillation hold the eyelids together for ap-
the test useless.
proximately 1 s.
If th e test material is contained in a pump spray, expel
NOTE 6 The contralateral eye of each animal serves as
and instil 0,l ml as for liquids.
an untreated control.
If the test material is contained in an aerosol con-
If repeated exposure of the material is anticipated and
tainer, examine by either
the test material has not demonstrated a significant
response in the acute test, a repeat exposure study
a) spraying a single burst of 1 s duration at a dis-
may be conducted. The duration of the exposure
tance of 10 cm directed at the open eye; or
should bear resemblance to the length of use of the
b) expelling the aerosol into a cool container and test material/device in the clinical situation.
treating as for a liquid.
5.3.6 Observation of animals
If the test material is such that it can only be applied
as an extract, prepare extracts as described in
For animals receiving a single instillation of test ma-
annex B. lnstil a 0,l ml aliquot of the extract into the
terial, examine both eyes of each animal approxi-
lower conjunctival sac of one eye.
mately 1 h, 24 h, 48 h and 72 I after instillation.
Under conditions identical with those used above,
Extended observation may be necessary if there are
prepare reagent controls, using both the polar and the
persistent lesions in order to ( etermine the progress
non-polar solvent, in the absence of the test material.
of the lesions or their reversal; this need not exceed
21 days. Extended observation cannot be justified for
5.3.4 Animals and husbandry
animals with severe lesions.
m
H ealthy adult albin o rabbits of either s ex fro
Young
Grade and record any reactions observed in accord-
ing 2 kg to 3 kg shall be used.
a single strain weigh
ance with the scale for grading ocular lesions given in
table 3.
The animals shall be acclimatized and cared for as
specified in annex C .
For animals receiving multiple instillations of test ma-
terial, examine both eyes of each animal immediately
to evaluate the test
One an imal shall initially be
before and approximately 1 h after each instillation.
materia
If there is evidence of irritation after the last treat-
A well-defined response in the one animal obviates
ment, the observations may be extended. Extended
the need for additional testing.
observation may be necessary if there is persistent
Unless a well-defined response is observed for solid
cornea1 involvement or other ocular irritation in order
or liquid materials, a minimum of two further animals
to determine the progress of the lesions and their
shall be used. For extracts, a minimum of two further
reversibility.
animals per extract shall be used.
Grade and record any reactions observed in accord-
If the response in the test using the minimum of three
ance with the scale for grading ocular lesions given in
animals is equivocal or not clear, additional testing
table 3.
shall be considered.
Withdraw an animal immediately from the study and
humanely kill it, if at any time it shows
5.3.5 Test procedure
a) very severe ocular damage (e.g. sloughing and
No longer than 24 h before commencement of the
ulceration of conjunctival membrane, cornea1 per-
test, visually examine both eyes of each rabbit for
evidence of ocular abnormality. If either eye shows foration, blood or pus in the anterior chamber); or
any abnormality, the rabbit shall be replaced.
b) blood-stained or purulent discharge; or
When the eyes are examined, sodium fluorescein
c) significant cornea1 ulceration.
2 %I BP may be used to visualize any cornea1 damage.
6
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SIST EN ISO 10993-10:2000
0 IS0
IS0 10993-10:1995(E)
24h or maximum
Withdraw from the study any animal showing maxi- conjunc
...
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