EN ISO 10993-10:2010

Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization (ISO 10993-10:2010)

ISO 10993-10:2010 describes the procedure for the assessment of medical devices and their constituent materials with regard to their potential to produce irritation and skin sensitization.
ISO 10993-10:2010 includes:
pretest considerations for irritation, including in silico and in vitro methods for dermal exposure;
details of in vivo (irritation and sensitization) test procedures;
key factors for the interpretation of the results.
Instructions are given for the preparation of materials specifically in relation to the above tests and several special irritation tests are described for application of medical devices in areas other than skin.

Biologische Beurteilung von Medizinprodukten - Teil 10: Prüfung auf Irritation und Hautsensibilisierung (ISO 10993-10:2010)

Dieser Teil der ISO 10993 beschreibt das Verfahren für die Beurteilung von Medizinprodukten und ihren
Bestandteilen hinsichtlich ihres Potentials, eine Irritation und eine Hautsensibilisierung hervorzurufen.
Dieser Teil der ISO 10993 enthält:
a) vor der Prüfung durchzuführende Überlegungen hinsichtlich einer Irritation, einschließlich In-silico- und Invitro-
Verfahren zur dermalen Exposition;
b) Einzelheiten zur Durchführung von In-vivo-Prüfungen (Irritation und Sensibilisierung);
c) Schlüsselfaktoren für die Interpretation der Ergebnisse.
Anhang A enthält Anweisungen für die spezielle Vorbereitung von Materialien im Zusammenhang mit den
vorstehend angeführten Prüfungen. In Anhang B sind mehrere spezielle Prüfungen auf Irritation bei der
Anwendung von Medizinprodukten in anderen Bereichen als der Haut beschrieben.

Évaluation biologique des dispositifs médicaux - Partie 10: Essais d'irritation et de sensibilisation cutanée (ISO 10993-10:2010)

L'ISO 10993-10:2010 décrit le mode opératoire pour l'évaluation du potentiel des dispositifs médicaux et de leurs matériaux constitutifs à provoquer une irritation et une sensibilisation de la peau.
L'ISO 10993-10:2010 comprend
des considérations préalablement aux essais relatives à l'irritation, y compris des méthodes in silico et in vitro d'exposition dermique,
des informations détaillées relatives aux procédures d'essai in vivo (irritation et sensibilisation), et
des facteurs clés pour l'interprétation des résultats.
Des instructions sont fournies en vue de la préparation des matériaux, notamment pour les essais précités et plusieurs essais d'irritation spéciaux pour l'application de dispositifs médicaux dans des zones autres que la peau.

Biološko ovrednotenje medicinskih pripomočkov - 10. del: Preskusi draženja in preobčutljivosti kože (ISO 10993-10:2010)

Ta del ISO 10993 opisuje postopek za ocenjevanje medicinskih pripomočkov in materialov, iz katerih so narejeni, z vidika njihovega potenciala za povzročanje draženja in preobčutljivosti kože. Ta del ISO 10993 vključuje: a) obravnavo pred preskusom glede draženja, vključno z metodami izpostavljenost kože in silico ter in vitro; b) podrobnosti preskusnih postopkov in vivo (za draženje in preobčutljivost), in; c) ključne dejavnike pri razlagi rezultatov. Navodila za pripravo materialov, predvsem glede zgoraj navedenih preskusov, so navedena v Dodatku A. V Dodatku B je opisanih več posebnih preskusov draženja, kadar medicinski pripomočki uporabljajo drugje kot na koži.

General Information

• Status: Withdrawn
• Publication Date: 31-Jul-2010
• Withdrawal Date: 20-Aug-2013
• ICS: 11.100 - Laboratory medicine
• ICS: 11.100.20 - Biological evaluation of medical devices
• Technical Committee: CEN/TC 206 - Biocompatibility of medical and dental materials and devices
• Drafting Committee: CEN/TC 206 - Biocompatibility of medical and dental materials and devices
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Start Date: 21-Aug-2013
• Completion Date: 21-Aug-2013
• Directive: 2007/47/EC - Directive 2007/47/EC of the European Parliament and of the council of 5 September 2007 amending Council Directive 90/385/EEC on the approximation of the laws of the Member States relating to active implantable medical devices, Council Directive 93/42/EEC concerning medical devices and Directive 98/8/EC concerning the placing of biocidal products on the market
• Directive: 90/385/EEC - Council Directive 90/385/EEC of 20 June 1990 on the approximation of the laws of the Member States relating to active implantable medical devices
• Directive: 93/42/EEC - Council Directive 93/42/EEC of 14 June 1993 concerning medical devices
• Mandate: M/BC/CEN/89/9 - Joint Standardization request to CEN/CENELEC concerning harmonized standards relating to horizontal aspects in the field of medical devices
• Ref Project: SIST EN ISO 10993-10:2010 - Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization (ISO 10993-10:2010)

Relations

• Replaces: EN ISO 10993-10:2009 - Biological evaluation of medical devices - Part 10: Tests for irritation and delayed-type hypersensitivity (ISO 10993-10:2002, including Amd 1:2006)
• Effective Date: 04-Jul-2009
• Replaced By: EN ISO 10993-10:2023 - Biological evaluation of medical devices - Part 10: Tests for skin sensitization (ISO 10993-10:2021)
• Effective Date: 19-Jan-2023
• Replaced By: EN ISO 10993-10:2013 - Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization (ISO 10993-10:2010)
• Effective Date: 21-Aug-2013

Standards Content (Sample)

SLOVENSKI STANDARD
01-oktober-2010
1DGRPHãþD
SIST EN ISO 10993-10:2009
%LRORãNRRYUHGQRWHQMHPHGLFLQVNLKSULSRPRþNRYGHO3UHVNXVLGUDåHQMDLQ
SUHREþXWOMLYRVWLNRåH,62
Biological evaluation of medical devices - Part 10: Tests for irritation and skin
sensitization (ISO 10993-10:2010)
Biologische Beurteilung von Medizinprodukten - Teil 10: Prüfung auf Irritation und
Hautsensibilisierung (ISO 10993-10:2010)
Évaluation biologique des dispositifs médicaux - Partie 10: Essais d'irritation et de
sensibilisation cutanée (ISO 10993-10:2010)
Ta slovenski standard je istoveten z: EN ISO 10993-10:2010
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.

EUROPEAN STANDARD
EN ISO 10993-10
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2010
ICS 11.100.20 Supersedes EN ISO 10993-10:2009
English Version
Biological evaluation of medical devices - Part 10: Tests for
irritation and skin sensitization (ISO 10993-10:2010)
Évaluation biologique des dispositifs médicaux - Partie 10: Biologische Beurteilung von Medizinprodukten - Teil 10:
Essais d'irritation et de sensibilisation cutanée (ISO 10993- Prüfung auf Irritation und Hautsensibilisierung (ISO 10993-
10:2010) 10:2010)
This European Standard was approved by CEN on 23 July 2010.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2010 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10993-10:2010: E
worldwide for CEN national Members.

Contents Page
Foreword .3
Annex ZA (informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC on Medical Devices .4
Annex ZB (informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 90/385/EEC on Active Implantable Medical Devices .5

Foreword
This document (EN ISO 10993-10:2010) has been prepared by Technical Committee ISO/TC 194 "Biological
evaluation of medical devices" in collaboration Technical Committee CEN/TC 206 “Biological evaluation of
medical devices” the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by February 2011, and conflicting national standards shall be withdrawn
at the latest by February 2011.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 10993-10:2009.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directives.
For relationship with EU Directives, see informative Annex ZA and ZB, which are integral parts of this
document.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 10993-10:2010 has been approved by CEN as a EN ISO 10993-10:2010 without any
modification.
Annex ZA
(informative)
Relationship between this European Standard and the Essential Requirements of EU
Directive 93/42/EEC on Medical Devices
This European Standard has been prepared under a mandate given to CEN by the European Commission
and the European Free Trade Association to provide a means of conforming to Essential Requirements of the
New Approach Directive 93/42/EEC on Medical Devices.

Once this standard is cited in the Official Journal of the European Union under that Directive and has been
implemented as a national standard in at least one Member State, compliance with the clauses of this
International Standard given in Table ZA.1 confers, within the limits of the scope of this European Standard, a
presumption of conformity with the corresponding Essential Requirements of that Directive and associated
EFTA regulations.
Table ZA.1 — Correspondence between this European Standard and Directive 93/42/EEC on medical
devices
Clause(s)/sub-clause(s) of this Essential Requirements (ERs) Qualifying remarks/Notes
European Standard of Directive 93/42/EEC
4, 5, 6, 7, 8, Annexes A, B and C 7.1, 7.2, 7.5 Within the limits of the Scope of this
standard.
6.5 and Annex C 6a Subclause 6.5 and Annex C contain
requirements that are relevant to clinical
investigations according to section 2 of
Annex X, as referred to in essential
requirement 6a of Directive 93/42/EEC.

General Note: Presumption of conformity depends on also complying with all relevant clauses/subclauses of
ISO 10993-1.
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling
within the scope of this standard.

Annex ZB
(informative)
Relationship between this European Standard and the Essential Requirements of
EU Directive 90/385/EEC on Active Implantable Medical Devices
This European Standard has been prepared under a mandate given to CEN by the European Commission
and the European Free Trade Association to provide a means of conforming to Essential Requirements of the
New Approach Directive 90/385/EEC on active Implantable Medical Devices.
Once this Standard is cited in the Official Journal of the European Union under that Directive and has been
implemented as a national standard in at least one Member State, compliance with the clauses of this
International Standard given in Table ZB.1 confers, within the limits of the scope of this standard, a
presumption of conformity with the corresponding Essential Requirements of that Directive and associated
EFTA regulations.
Table ZB.1 — Correspondence between this European Standard and Directive 90/385/EEC on Active
Implantable Medical Devices
Clause(s)/sub-clause(s) of this Essential Requirements (ERs) Qualifying remarks/Notes
European Standard of Directive 90/385/EEC
4, 5, 6, 7, 8, Annexes A, B and C 9 (first and second indents only) Within the limits of the Scope of
this standard.
6.5 and Annex C 5a Subclause 6.5 and Annex C
contain requirements that are
relevant to clinical investigations
according to section 2 of Annex
7, as referred to in essential
requirement 5a of Directive
90/385/EEC.
General Note: Presumption of conformity depends on also complying with all relevant clauses/subclauses of
ISO 10993-1.
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling
within the scope of this standard.

INTERNATIONAL ISO
STANDARD 10993-10
Third edition
2010-08-01
Biological evaluation of medical
devices —
Part 10:
Tests for irritation and skin sensitization
Évaluation biologique des dispositifs médicaux —
Partie 10: Essais d'irritation et de sensibilisation cutanée

Reference number
ISO 10993-10:2010(E)
©
ISO 2010
ISO 10993-10:2010(E)
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ii © ISO 2010 – All rights reserved

ISO 10993-10:2010(E)
Contents Page
Foreword .iv
Introduction.vi
1 Scope.1
2 Normative references.1
3 Terms and definitions .2
4 General principles — Step-wise approach .4
5 Pretest considerations.4
5.1 General .4
5.2 Types of material .5
5.3 Information on chemical composition .5
6 Irritation tests.6
6.1 In vitro irritation tests.6
6.2 In vivo irritation tests — Factors to be considered in design and selection of in vivo tests .6
6.3 Animal irritation test.7
6.4 Animal intracutaneous (intradermal) reactivity test .11
6.5 Human skin irritation test .14
7 Skin sensitization tests.15
7.1 Choice of test methods.15
7.2 Murine Local Lymph Node Assay (LLNA).15
7.3 Guinea pig assays for the detection of skin sensitization.18
7.4 Important factors affecting the outcome of the test .19
7.5 Guinea pig maximization test (GPMT).20
7.6 Closed-patch test (Buehler test) .23
8 Key factors in interpretation of test results.26
Annex A (normative) Preparation of materials for irritation/sensitization testing.27
Annex B (normative) Special irritation tests.29
Annex C (normative) Human skin irritation test .44
Annex D (informative) In vitro tests for skin irritation.48
Annex E (informative) Method for the preparation of extracts from polymeric test materials .54
Annex F (informative) Background information .57
Bibliography.61

ISO 10993-10:2010(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. 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. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. 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.
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 rights.
ISO 10993-10 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices.
This third edition cancels and replaces the second edition (ISO 10993-10:2002), which has been technically
revised.
ISO 10993 consists of the following parts, under the general title Biological evaluation of medical devices:
⎯ Part 1: Evaluation and testing within a risk management process
⎯ 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 in vitro cytotoxicity
⎯ Part 6: Tests for local effects after implantation
⎯ Part 7: Ethylene oxide sterilization residuals
⎯ Part 9: Framework for identification and quantification of potential degradation products
⎯ Part 10: Tests for irritation and skin sensitization
⎯ Part 11: Tests for systemic toxicity
⎯ Part 12: Sample preparation and reference materials
⎯ Part 13: Identification and quantification of degradation products from polymeric medical devices
⎯ Part 14: Identification and quantification of degradation products from ceramics
⎯ Part 15: Identification and quantification of degradation products from metals and alloys
iv © ISO 2010 – All rights reserved

ISO 10993-10:2010(E)
⎯ Part 16: Toxicokinetic study design for degradation products and leachables
⎯ Part 17: Establishment of allowable limits for leachable substances
⎯ Part 18: Chemical characterization of materials
⎯ Part 19: Physico-chemical, morphological and topographical characterization of materials [Technical
Specification]
⎯ Part 20: Principles and methods for immunotoxicology testing of medical devices [Technical Specification]
ISO 10993-10:2010(E)
Introduction
This part of ISO 10993 assesses possible contact hazards from chemicals released from medical devices,
which may produce skin and mucosal irritation, eye irritation or skin sensitization.
Some materials that are included in medical devices have been tested, and their skin or mucosal irritation or
sensitization potential has been documented. Other materials and their chemical components have not been
tested and may induce adverse effects when in contact with human tissue. The manufacturer is thus obliged
to evaluate each device for potential adverse effects prior to marketing.
Traditionally, small animal tests are performed prior to testing on humans to help predict human response.
More recently, in vitro tests as well as human tests have been added as adjuncts or alternatives. 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 for screening purposes prior to animal testing. In order to
reduce the number of animals used, this part of ISO 10993 presents a step-wise approach, with review and
analysis of test results at each stage. An animal test is usually required prior to human testing.
It is intended that these studies be conducted using Good Laboratory Practice and comply with regulations
related to animal welfare. Statistical analysis of data is recommended and should be used whenever
appropriate.
This part of ISO 10993 is intended for use by professionals, appropriately qualified by training and experience,
who are able to interpret its requirements and judge the outcomes of the evaluation for each medical device,
taking into consideration all the factors relevant to the device, its intended use and the current knowledge of
the medical device provided by review of the scientific literature and previous clinical experience.
The tests included in this part of ISO 10993 are important tools for the development of safe products, provided
that these are executed and interpreted by trained personnel.
This part of ISO 10993 is based on numerous standards and guidelines, including OECD Guidelines,
U.S. Pharmacopoeia and the European Pharmacopoeia. It is intended to be the basic document for the
selection and conduct of tests enabling evaluation of irritation and dermal sensitization responses relevant to
safety of medical materials and devices.

vi © ISO 2010 – All rights reserved

INTERNATIONAL STANDARD ISO 10993-10:2010(E)

Biological evaluation of medical devices —
Part 10:
Tests for irritation and skin sensitization
1 Scope
This part of ISO 10993 describes the procedure for the assessment of medical devices and their constituent
materials with regard to their potential to produce irritation and skin sensitization.
This part of ISO 10993 includes:
a) pretest considerations for irritation, including in silico and in vitro methods for dermal exposure;
b) details of in vivo (irritation and sensitization) test procedures;
c) key factors for the interpretation of the results.
Instructions are given in Annex A for the preparation of materials specifically in relation to the above tests. In
Annex B several special irritation tests are described for application of medical devices in areas other than skin.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 10993-1:2009, Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk
management process
ISO 10993-2, Biological evaluation of medical devices — Part 2: Animal welfare requirements
ISO 10993-9, Biological evaluation of medical devices — Part 9: Framework for identification and
quantification of potential degradation products
ISO 10993-12, Biological evaluation of medical devices — Part 12: Sample preparation and reference materials
ISO 10993-13, Biological evaluation of medical devices — Part 13: Identification and quantification of
degradation products from polymeric medical devices
ISO 10993-14 Biological evaluation of medical devices — Part 14: Identification and quantification of
degradation products from ceramics
ISO 10993-15, Biological evaluation of medical devices — Part 15: Identification and quantification of
degradation products from metals and alloys
ISO 10993-18, Biological evaluation of medical devices — Part 18: Chemical characterization of materials
ISO 14155-1, Clinical investigation of medical devices for human subjects — Part 1: General requirements
ISO 14155-2, Clinical investigation of medical devices for human subjects — Part 2: Clinical investigation plans
ISO 10993-10:2010(E)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 10993-1 and the following apply.
3.1
allergen
sensitizer
substance or material that is capable of inducing a specific hypersensitivity reaction upon repeated contact
with that substance or material
3.2
blank
extraction vehicle not containing the test material, retained in a vessel identical to that which holds the test
material and subjected to identical conditions to which the test material is subjected during its extraction
NOTE The purpose of the blank control is to evaluate possible confounding effects due to the extraction vessel,
vehicle and extraction process.
3.3
challenge
elicitation
process following the induction phase, in which the immunological effects of subsequent exposures in an
individual to the inducing material are examined
3.4
dose
dosage
amount of test sample administered (e.g. mass, volume) expressed per unit of body weight or surface area
NOTE The terms are often used interchangeably (more commonly dosage).
3.5
erythema
reddening of the skin or mucous membrane
3.6
eschar
scab or discoloured slough of skin
3.7
extract
liquid or suspension that results from exposing a test or control material to a solvent under controlled
conditions
3.8
induction
process that leads to the de novo generation of an enhanced state of immunological activity in an individual, to
a specific material
3.9
irritant
agent that produces irritation
3.10
irritation
localized non-specific inflammatory response to single, repeated or continuous application of a substance/material
NOTE Skin irritation is a reversible reaction and is mainly characterized by local erythema (redness) of the skin.
2 © ISO 2010 – All rights reserved

ISO 10993-10:2010(E)
3.11
necrosis
cell death as a direct result of irreversible changes caused by injury or disease
NOTE One should be aware that tissue repair will occur either resulting in complete functional restoration or resulting
in scar formation.
3.12
negative control
any well-characterized material or substance that, when tested by a specific procedure, demonstrates the
suitability of the procedure to yield a reproducible, appropriately negative, non-reactive or minimal response in
the test system
NOTE In practice, negative controls include blanks, vehicles/solvents and reference materials.
3.13
oedema
swelling due to abnormal infiltration of fluid into the tissues
3.14
positive control
any well-characterized material or substance that, when evaluated by a specific test method, demonstrates
the suitability of the test system to yield a reproducible, appropriately positive or reactive response in the test
system
3.15
skin corrosion
production of irreversible damage to the skin, manifested as visible necrosis through the epidermis and into
the dermis, following application of a test sample
EXAMPLE The action of a compound/chemical/test sample resulting in ulceration of skin (see 3.19).
3.16
skin sensitization
allergic contact dermatitis
immunologically mediated cutaneous reaction to a substance
NOTE In the human, the responses can be characterized by pruritis, erythema, oedema, papules, vesicles, bullae or
a combination of these. In other species the reactions can differ and only erythema and oedema can be seen.
3.17
test material
material, device, device portion or component thereof that is sampled for biological or chemical testing
3.18
test sample
material, device, device portion, component, extract or portion thereof that is subjected to biological or
chemical testing or evaluation
3.19
ulceration
open sore representing loss of superficial tissue
3.20
vehicle
liquid used to moisten, dilute, suspend, extract or dissolve the test substance/material
ISO 10993-10:2010(E)
4 General principles — Step-wise approach
The available methods for testing irritation and sensitization were developed specifically to detect skin and
mucous membrane irritation and skin sensitization potential. Other types of adverse effect are generally not
predicted by these tests. For medical devices that are used as implants or external communicating devices,
intradermal testing is more relevant in approaching the application and so for detection of irritation activity,
intracutaneous testing shall be used as described in 6.4.
This part of ISO 10993 requires a step-wise approach, which shall include one or more of the following:
a) characterization of test material, involving chemical characterization and analysis of the test sample
according to the general principles described in ISO 10993-9, ISO 10993-13, ISO 10993-14,
ISO 10993-15 and ISO 10993-18;
b) literature review, including an evaluation of chemical and physical properties, and information on the
irritation and sensitization potential of any product constituent as well as structurally-related chemicals
and materials;
c) in accordance with ISO 10993-2, in vitro tests in preference to in vivo tests shall be considered, and
replacement of the latter as new in vitro tests are scientifically validated and become reasonably and
practicably available. For the evaluation of skin irritation and corrosion, in vitro alternatives are available
for chemicals; there are currently no internationally validated and accepted in vitro tests to detect
sensitizers;
d) in vivo animal tests: in order to ensure reproducibility and sensitivity, a test of a positive-control substance
for irritation and skin sensitization shall be included in each assay by the testing laboratory in order to
validate the test system and demonstrate a positive response; for guinea pig sensitization assays,
however, when consistency has been demonstrated over a six month or more extended period, a positive
control does not need to be included in every assay, but may be run at regular intervals which shall not
exceed six months.
NOTE 1 Sensitization can at the moment only be determined by an in vivo assay. This can be accomplished by using
the local lymph node assay (LLNA) in mice, the occluded patch test in guinea pigs or the guinea pig maximization test
(GPMT). For single chemicals the LLNA is now the preferred assay for determining the sensitizing potential.
See References [69] [88] [90].
NOTE 2 In vivo animal tests are appropriate when test materials cannot be characterized and risk assessments cannot
be undertaken using information obtained by the means set out in a), b) and c).
NOTE 3 For sensitization assays in guinea pigs, ten animals are normally used for positive control once every six
months. Fewer guinea pigs can be used when an assay with a positive control substance is performed more frequently
than once every six months. At least five test animals with a positive substance and five control animals should be used.
e) Non-invasive human tests/clinical trials; if the material has been demonstrated not to be an irritant, a
sensitizer or toxic in animals, studies on skin irritation may then be considered in humans.
Clinical studies in accordance with ISO 14155-1, ISO 14155-2 and to ethics principles shall not be performed
before the results of the other evaluations in a) to d) are known.
5 Pretest considerations
5.1 General
It is important to emphasise that pretest considerations may result in the conclusion that testing for irritation
and/or sensitization is not necessary.
The requirements given in Clause 5 of ISO 10993-1:2009 and the following apply.
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ISO 10993-10:2010(E)
Non-sterile samples shall be investigated by topical investigation only, as the possibility of microbial
contamination of the test sample could confound the final assay interpretation. In cases where the sterility of a
test sample cannot be guaranteed, but the sample is still considered to be non-contaminated, intradermal
administration may be justified.
5.2 Types of material
5.2.1 Initial considerations
It shall be taken into consideration that, during manufacture and assembly of medical devices, additional
chemical components may be used as processing aids, e.g. lubricants or mould-release agents. In addition to
the chemical components of the starting material and manufacturing process aids, adhesive/solvent residues
from assembly and also sterilant residues or reaction products resulting from the sterilization process may be
present in a finished product. Whether these components pose a health hazard/risk depends on the leakage
or degradation characteristics of the finished products. These components shall be taken into account for their
potential irritation/sensitization activity.
5.2.2 Ceramics, metals and alloys
These materials are normally less complex than polymers and biologically derived materials in terms of the
number of chemical constituents.
5.2.3 Polymers
These materials are normally chemically more complex than those in 5.2.2 in terms of composition. A number
of reaction products/impurities/additives may be present and the completeness of polymerization may vary.
5.2.4 Biologically derived materials
These materials are inherently complex in their composition. They often also contain process residues,
e.g. cross-linkers and anti-microbial agents. Biological materials can be inconsistent from sample to sample.
The methods in this part of ISO 10993 have not been designed for testing of biologically derived materials and
can therefore be less adequate. For example, the tests in this part of ISO 10993 do not consider cross-
species sensitization.
5.3 Information on chemical composition
5.3.1 General
Full qualitative data on the chemical constituents of the material shall be established. Where relevant to
biological safety, quantitative data shall also be obtained. If quantitative data are not obtained, the rationale
shall be documented and justified.
5.3.2 Existing data sources
Qualitative and quantitative information on the composition shall be obtained where possible from the supplier
of the starting material.
For polymers this often requires access to proprietary information; provision should be made for the transfer
and use of such confidential information.
Qualitative information about any additional processing additives (for example, mould-release agents) shall
also be obtained from appropriate members of the manufacturing chain, including converters and component
manufacturers.
ISO 10993-10:2010(E)
In the absence of any data on composition, a literature study to establish the likely nature of the starting
material and any additives is recommended, so as to assist in the selection of the most appropriate methods
of analysis for the material concerned.
The chemical composition of finalized products shall be determined in accordance with ISO 10993-18.
NOTE The composition of ceramics, metals and alloys can be specified in accordance with ISO or American Society
of Testing Materials (ASTM) standards and/or can be specified by the user. However, in order to obtain full qualitative and
quantitative details on composition, it can be necessary to request these from the supplier or manufacturer of the starting
material and also from component manufacturers to ensure that processing aids are also identified. Material master files
held by regulatory authorities are another source of data, where they are accessible.
6 Irritation tests
6.1 In vitro irritation tests
In vitro methods, the rat skin Transcutaneous Electrical Resistance (TER) test and the Human skin model test,
have been internationally validated and accepted as alternative tests to assess the skin corrosivity of
[9] [10]
chemicals (OECD Guidelines 430 and 431 ). National and international organizations continue working to
develop and validate in vitro tests for skin irritancy in parallel with the search for alternative methods; others
have been developing methods to quantify the responses of animals and humans in order to better define
endpoints using non-invasive techniques (see F.1).
NOTE In 2007 the ECVAM Scientific Advisory Committee (ESAC) evaluated the validation process of an in vitro
human skin model for the determination of skin irritation of chemicals. See Reference [101]. The use of in vitro human skin
models for assessing the potential of chemicals to induce skin irritation is described in Annex D.
The in vitro test for skin irritation has so far been validated only for neat chemicals and not for medical device
extracts. In order to apply these assays for the testing of irritation potential of medical devices, further
validation for this specific area is essential.
6.2 In vivo irritation tests — Factors to be considered in design and selection of in vivo
tests
Irritation testing of medical devices can be performed with the finished product and/or extracts thereof.
Factors affecting the results of irritation studies include the following:
a) the nature of the device used in a patch test;
b) the dose of the test material;
c) the method of application of the test material;
d) the degree of occlusion;
e) the application site;
f) the duration and number of exposures;
g) the techniques used in evaluating the test.
Additional background information is provided in Annex F.
Whilst flexibility with respect to the precise protocol followed allows the investigator to enhance the sensitivity
of the test to suit conditions of use and population exposure, consistency in procedure contributes to
comparability of test results with different materials and from different laboratories.
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ISO 10993-10:2010(E)
Provisions have been included in the test procedures for evaluation of devices and materials that will have
repeated and/or prolonged exposure. The study shall be designed to exaggerate the anticipated contact (time
and/or concentration) in the clinical situation. This shall be borne in mind during interpretation of the result.
If the pH of the test sample is u 2,0 or W 11,5, the material shall be considered an irritant and no further
testing is required. However, experimental evidence suggests that acidity and alkalinity of the test material are
not the only factors to be considered in relation to the capacity of a material to produce severe injury. The
concentration of the test material, its period of contact, and many other physical and chemical properties are
also important.
In exceptional cases where further risk characterization/assessment is needed, it might be necessary to test
materials which are either an irritant or have a pH outside the range mentioned above. These cases shall be
justified and documented.
6.3 Animal irritation test
6.3.1 Principle
An assessment is made of the potential of the material under test to produce dermal irritation in a relevant
animal model.
The rabbit is the preferred test animal.
6.3.2 Test material
If the test material is a solid or a liquid, it shall be prepared as specified in Annex A.
The sensitivity of the assay shall be demonstrated. This can be done by including a positive control in the
assay. However, the use of a positive control to confirm sensitivity is only warranted when the testing
laboratory has not within the previous six months produced positive results using the test method.
NOTE A suitable positive control is sodium lauryl sulphate (SLS).
6.3.3 Animals and husbandry
Three healthy young adult albino rabbits of either sex from a single strain, weighing not less than 2 kg, shall
be used. If irritation is anticipated, consideration shall be given to testing in one animal first. Unless a well-
defined positive response [score greater than 2 for either erythema or oedema (see Table 1)] is observed, a
minimum of two additional animals shall be used. If the response in the test using the minimum of three
animals is equivocal, further testing shall be considered.
The animals shall be acclimatized and cared for as specified in ISO 10993-2.
ISO 10993-10:2010(E)
Table 1 — Scoring system for skin reaction
Reaction Irritation score
Erythema and eschar formation
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet-redness) to eschar formation preventing grading of erythema 4
Oedema formation
No oedema 0
Very slight oedema (barely perceptible) 1
Well-defined oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 mm) 3
Severe oedema (raised more than 1 mm and extending beyond exposure area) 4
Maximal possible score for irritation 8
Other adverse changes at the skin sites shall be recorded and reported.
6.3.4 Test procedure
6.3.4.1 Preparation of animals
The condition of the skin is a critical factor. Use only animals with healthy intact skin. Fur is generally clipped
within 24 h to 4 h of testing on the backs of the animals, a sufficient distance on both sides of the spine for
application and observation of all test sites (approximately 10 cm × 15 cm). Fur may be re-clipped to facilitate
observation and/or to accommodate repeated exposures. Depilatories may be used by trained technicians, if
the process has been validated at the testing facility. If repeated exposure is required, follow the procedures in
6.3.4.2.1, 6.3.4.2.2 or 6.3.4.2.3, repeated for a maximum of 21 d.
6.3.4.2 Application of test sample
6.3.4.2.1 Application of powder or liquid sample
Apply 0,5 g or 0,5 ml of the test material directly to each test skin site as shown in Figure 1. For solid and
hydrophobic materials, there is no need for moistening. If the material is a powder, it should be slightly
moistened with water or other suitable vehicle before application (see Annex A).
Cover the application sites with a 2,5 cm × 2,5 cm non-occlusive dressing (such as an absorbent gauze patch)
and then wrap the application site with a bandage (semi-occlusive or occlusive) for a minimum of 4 h. At the
end of the contact time, remove the dressings and mark the positions of the sites with permanent ink. Remove
residual test material by appropriate means, such as washing with lukewarm water or other suitable non-
irritating solvent, and careful drying.
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ISO 10993-10:2010(E)
Key
1 cranial end
2 test site
3 control site
4 clipped dorsal region
5 caudal end
Figure 1 — Location of skin application sites
6.3.4.2.2 Application of extracts and extract vehicle
Apply the appropriate extract(s) to the 2,5 cm × 2,5 cm absorbent gauze patches. Use a volume of extract
sufficient to saturate the gauze, generally 0,5 ml per patch. Apply one patch on each side of the animal as
shown in Figure 1. Apply a control patch of gauze moistened with the extract vehicle as shown in Figure 1.
Cover the application sites with a bandage (semi-occlusive or occlusive) for a minimum of 4 h. At the end of
the contact time, remove the dressings and mark the positions of the sites with permanent ink. Remove
residual test material by appropriate means, such as washing with lukewarm water or other suitable non-
irritating solvent and careful drying.
6.3.4.2.3 Application of solid sample
Apply the samples of the test material directly to the skin on each side of each rabbit as shown in Figure 1.
Similarly, apply the control samples to each rabbit. When testing solids (which may be pulverized if considered
necessary), the test material shall be moistened sufficiently with water or, where necessary, an alternative
solvent, to ensure good contact with the skin (see Annex A). When solvents are used, the influence of the
solvent on irritation of skin caused by the test material shall be taken into account.
Cover the application sites with 2,5 cm × 2,5 cm n
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