EN ISO 10993-10:1995

SLOVENSKI STANDARD
01-januar-2000
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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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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)
IS0 10993-10:1995(E)
Contents
Page
..............................................................................................
1 Scope
.....................................................................
2 Normative references
.......................................................................................
3 Definitions
....................................
4 General principles, step-wise approach
............................................................................
5 Irritation tests
.... 2
5.1 Factors to be considered in design and selection of tests
...................................................................
5.2 Skin irritation test
................................................................
5.3 Ocular irritation test
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4 Intracutaneous (intradermal) reactivity test
. . . . . . . . . . . . . . . . . . . . . . . . . . . .*.
6 Sensitization tests
. . 10
6.1 Factors to be considered in design and selection of tests
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2 Maximization sensitization test
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3 Closed patch sensitization test
Annexes
.......................................
A Preparation of materials for testing
......... 17
B Method for extraction of materials for biological tests
..........................................................
C Animals and husbandry
........................................................
D Additional irritation tests
.........................................................
E Background information
............................................................................
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
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
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.

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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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.
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
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.

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
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
No erythema
2 to 4,9
Moderate
Very slight erythema (barely perceptible)
5 to 8
Severe
Well-defined erythema
Moderate erythema
5.2.7 Presentation of results
Severe erythema (beet-redness) to
eschar formation preventing grading of
The test report shall include
erythema
Oedema formation
a) a description of the test material(s) or device;
No oedema
b) the intended use/application of the test material(s)
Very slight oedema (barely perceptible)
or device;
Well-defined oedema (edges of area
well-defined by definite raising)
c) a detailed description of the method employed in
Moderate oedema (raised approxi-
preparing the test material or device;
mately 1 mm)
Severe oedema (raised more than d) the test animals;
1 mm and extending beyond exposure
area)
e) method of application to the test sites;
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
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.
0 IS0
IS0 10993-10:1995(E)
24h or maximum
Withdraw from the study any animal showing maxi- conjunctival inflammation
mum effects on the grading system in table3 - ab- khemosis grade 4 together with redness grade 3) -
sence of a light reflex (iridial response 2) or cornea1 without evidence of recovery within 48 h, and kill it
opacity (grade 4) without evidence of recovery within humanely.
Table 3 - Classification system for grading ocular lesions
Numerical
Reaction
grading
1. Cornea
Degree of opacity (most dense area used)
No opacity 0
Scattered or diffuse areas, details of iris clearly visible
I*
Easily discernible translucent areas, details of iris slightly obscured
2*
Opalescent areas, no details of iris visible, size of pupil barely discernible 3*
Opaque, iris invisible 4*
Area of cornea involved
One-quarter (or less), not zero
Greater than one-quarter, but less than half
Greater than half, but less than three-quarters
Greater than three-quarters, up to whole area
2. Iris
Normal 0
Folds above normal, congestion swelling, circumcorneal injection (any or all or combination of these), iris still
reacting to light (sluggish reaction is positive) I*
No reaction to light, haemorrhage gross destruction (any or all of these) 2*
3. Conjunctivae
Redness (refers to palpebral and bulbar conjunctiva excluding cornea and iris)
Vessels normal 0
Vessels definitely injected above normal
More diffuse, deeper crimson red, individual vessels not easily discernible
2*
Diffuse beefy red
3*
Chemosis
No swelling
Any swelling above normal (include nictitating membrane)
Obvious swelling with partial eversion of lids
2*
Swelling with lids about half-closed
3*
Swelling with lids about half-closed to completely closed
4*
Discharge
No discharge
Any amount different from normal (does not include small amounts observed in inner canthus of normal ani-
mals)
Discharge with moistening of the lids and hairs just adjacent to lids
Discharge with moistening of lids and hairs, and considerable area round the eye
* positive result
0 IS0
IS0 10993-10:1995(E)
5.3.7 Evaluation of results 5.4 lntracutaneous (intradermal) reactivity
test
Differences between the test and control eyes shall
be characterized and explained in the terms of the
5.4.1 Principle
classification system used in table 3.
5.3.7.1 Acute exposure
5.4.2 Exclusion from test
If the treated eye in more than one animal shows a
positive response (asterisked figures in table3) at any
Any material shown to be a skin, eye or mucosal tis-
of the observations, then the material is considered
sue irritant or those with a pH of < 2 or 2 1 I,5 shall
an eye irritant and further testing is not required.
not be tested.
If only one of three eyes treated shows a positive re-
5.4.3 Test material
action or the reactions are equivocal, treat further
animals.
The test materials shall be extracts and shall be pre-
pared according to the procedures specified in
When further animals have been treated, the test
annex B.
material is considered to be an eye irritant if more
than half of the eyes treated in the test group exhibit
5.4.4 Animals and husbandry
a positive reaction (asterisked figures in table3) at any
stage of the observation.
Healthy young adult albino rabbits of either sex from
a single strain weighing not less than 2 kg shall be
A severe reaction in only one animal is considered
used.
sufficient to label as an irritant.
The animals shall be acclimatized and cared for as
specified in annex
C .
5.3.7.2 Repeated exposure
A minimum of three animals shall be used initially to
evaluate the test material.
The test material is considered an eye irritant if more
than half of the animals in the test group exhibit a
If the response in the initial test is equivocal or not
positive reaction (asterisked figures in table3) at any
clear, additional testing shall be considered.
stage of the observation.
5.4.5 Test procedure
On the day before the test, closely clip the fur on the
5.3.8 Presentation of results
backs of the animals allowing a sufficient distance on
both sides of the spine for injection of the extracts.
The test report shall include
Inject intracutaneously 0,2 ml of the extract obtained
a) a description of the test material(s) or device;
with the polar solvent at five sites on one side of each
rabbit (see figure2). Use the smallest sized needle,
b) the intended use/application of the test material(s)
appropriate to the viscosity of the test material, for
or device;
the intradermal injections.
c) a detailed description of the method employed in
Similarly, inject 0,2 ml of the polar solvent control at
preparing the test material or device;
five posterior sites on the same side of each rabbit
(see figure 2).
d) the test animals;
Repeat the above procedures for the extract obtained
e) method of instillation; with the non-polar solvent and the non-polar solvent
control on the other side of each rabbit (see figure2).
how the ocular readings were performed and a
f 1
If other solvents are used, repeat the above steps for
record of the observations;
the extract obtained with the other solvents and the
solvent controls.
g) assessment of the results.
IS0 10993-10:1995(E)
Cranial
0,2 ml injections
of polar extract
I
6 # # 6
7 # # 7
0,2 ml injections
of polar solvent 4
8 # # 8
control
9 # # 9
\ 10 # #
Caudat
Figure 2 - Arrangement of injection sites
5.4.6 Observation of animals
Note the appearance of each injection site immedi-
Table 4 - Classification system for
ately after injection and at 24 h, 48 h and 72 h after
intracutaneous (intradermal) reactions
injection.
Numerical
Reaction grading
Grade the tissue reaction for erythema and oedema
according to the classification system given in table4
Erythema and eschar formation
for each injection site and at each time interval ob-
No erythema 0
served, and record the results.
Very slight erythema (barely perceptible) 1
NOTE 7 lntradermal injection of an oil frequently elicits
Well-defined erythema 2
an inflammatory response.
Moderate erythema 3
Injection of an appropriate vital dye such as Trypan
Severe erythema (beet-redness) to
blue or Evans blue, intravenously, may be undertaken
eschar formation preventing grading of
erythema 4
at the 72 h reading to assist in evaluation of the re-
sponse by delineating the area of irritation.
Oedema formation
No oedema 0
Non-invasive techniques may be used to assist in the
Very slight oedema (barely perceptible) 1
evaluation if they are available.
Well-defined oedema (edges of area
5.4.7 Evaluation of results well-defined by definite raising) 2
Moderate oedema (raised approxi-
Determine the Primary Irritation Index as follows.
mately 1 mm) 3
Severe oedema (raised more than
For each animal, add together the Primary Irritation
1 mm and extending beyond exposure
Scores for both erythema and oedema separately for
area) 4
each test extract at each time specified and divide by
the total number of observations. A similar assess-
Total possible score for irritation
ment is made of the sites injected with the reagent
control. Subtract that score for the reagent control
NOTE - Other adverse changes at the injection sites
from the score for the test material to obtain the Pri-
shall be recorded and reported.
mary Irritation Score to be used in determining the
Primary Irritation Index.
0 IS0
IS0 10993-10:1995(E)
Only use the 24 h, 48 h and 72 h observations for 6.1 Factors to be considered in design and
calculations.
selection of tests
Add the Primary Irritation Scores of each animal and
The biochemical and physical characteristics of the
divide the total by the number of animals. This value
test material may influence the choice of test. The
is the Primary Irritation Index (PII).
maximization test requires intradermal injections;
consequently if the test material cannot be injected
The Primary Irritation Index is characterized by num-
intradermally, the closed patch or alternative method
ber and description in table 5.
shall be used.
A solvent should be selected that optimizes exposure
Table 5 - Primary irritation response categories
by solubilization and penetration. The concentration
in rabbit
of test material should be the highest possible with-
Mean score (PII)l)
Response category
out affecting the ability to interpret the results. The
.
concentration of the test material at the skin surface
Negligible 0 to 0,4
is an important criterion for topical administration and
0,5 to I,9
Slight
not the total volume of test material. The latter will
Moderate 2 to 4,9
be determined by the capacity of the patch system.
5 to 8
Severe
Damage to the skin cannot be avoided when Freund’s
1) The Primary Irritation Index (PII) is determined by complete adjuvant is injected intradermally.
adding the Primary Irritation Score for each animal and
dividing the total score by the number of animals. The times of exposure for all phases of each exper-
iment need to be sufficient for experimental success.
They should be varied only to the extent that they
5.4.8 Presentation of results
achieve this end. All phases (preliminary, induction
and challenge) are critical but the scoring intervals are
The test report shall include
critical only to the extent that they demonstrate the
delayed and persistent characteristic of hypersen-
a) a description of the test material(s) or device;
sitivity. Scoring of induction sites is not generally in-
formative and is not critical for interpretation.
b) the intended use/application of the test material(s)
or device;
The actual position of patch sites on the flank of the
guinea-pig is not a critical parameter, provided all the
c) a detailed description of the method employed in
challenge sites are conducted on naive skin.
preparing the test material or device;
Comparison of test animals at challenge with the ap-
d) the test animals;
propriate controls is essential for indication of a posi-
tive test result, though the severity of lesions will aid
e) method of injection;
in the interpretation.
f) how the site readings were performed and a rec-
Borderline reactions at challenge are best clarified by
ord of the observations;
rechallenge. Histopathology is not generally of diag-
nostic value.
g) assessment of the results.
A positive test does not necessarily exclude the test
material or device from use because the doses of the
test substance in the test procedure may be exag-
6 Sensitization tests
gerated compared to actual conditions of use. A
There are several methods for determining skin sen- positive test using any of the validated procedures
sitization in guinea-pigs. The two most commonly indicates the need for additional data, either in
used methods are the maximization (Magnusson & guinea-pigs or humans, that would allow risk assess-
ment of intended human exposure. The relative sen-
Kligman) and closed patch (Buehler) methods. It is
only necessary to evaluate by one of these methods. sitizing potencies of substances can be defined in
The maximization test (6.2) is regarded as the most terms of the minimum (lowest) induction concen-
sensitive and is the preferred method, particularly tration required to induce a given level of
with regard to the evaluation of extracts. A list of sensitization.lW This entails, as a minimum, verifi-
cation with appropriate concentrations and vehicles.
alternative methods is given in annex E.
0 IS0 IS0 10993-10:1995(E)
6.2.4 Test procedure
Repeating this assay with other techniques and util-
ization of open challenges (to avoid non-specific ef-
fects of tape) are of scientific and practical value.
Provocative tests followed by ad libitum use tests
with appropriate diagnostic patch testing can effec-
6.2.4.1 Preparation
tively determine safe levels of use.
For products to be used extensively on normal and
Clip the fur on all treatment sites, the day prior to
abnormal skin, no substantial risk is acceptable.
treatment.
However, many ingredients, in spite of sensitizing
potential, are fully acceptable in products at reason-
For intradermal injections, inject 0,l ml per site.
able concentrations because of their inherent benefit.
For all topical applications, saturate a patch of filter
paper of the appropriate dimensions with the test
material and apply the patch to the clipped skin sur-
6.2 Maximization sensitization test
face under an occlusive dressing wound around the
torso of the animal.
6.2.1 Principle
6.2.4.2 Preliminary tests
Assessment of the potential of the material under test
to produce skin sensitization in the guinea-pig.
NOTE 9 The preliminary tests are intended to determine
the concentrations of the test materials to be used in the
main test in 6.2.4.3.
6.2.2 Test material
Consideration shall be given to the pretreatment of
If the test material is either a solid or a liquid it shall
all animals by injection with Freund’s complete adju-
be prepared as specified in annex A.
vant.
If the test material is to be tested as an extract, it shall
be prepared as specified in annex B.
Inject a range of concentrations of the test material
or extract (in the selected solvent) intradermally into
at least two animals.
6.2.3 Animals and husbandry
Select for the intradermal induction phase in the main
test the highest concentration that does not cause
Healthy young adult albino guinea-pigs of either sex
extensive destruction of the skin and does not other-
from a single outbred strain, weighing 300 g to
wise adversely affect the animals.
500 g at the start of the test shall be used. If female
animals are used they shall be nulliparous and not
Topically apply a range of concentrations of the test
pregnant.
material or extract to the flanks of at least three addi-
tional animals. Remove the occlusive dressings and
The animals shall be acclimatized and cared for as
patches after 24 h, and assess the application sites
specified in annex C.
for erythema and oedema using the grading given in
table 6.
For testing powders or liquids, a minimum of ten ani-
mals shall be treated with the test material and a
Select
minimum of five animals acts as a control group. Ad-
ditional animals shall be used for the preliminary test.
a) if possible, for the topical induction phase in the
main test, the highest concentration that causes
For testing extracts, a minimum of ten animals shall
slight erythema but does not otherwise adversely
be treated with each extract and a minimum of five
affect the animals;
animals a
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