FprEN ISO 10993-10

SLOVENSKI STANDARD
oSIST prEN ISO 10993-10:2020
01-april-2020
Biološko ovrednotenje medicinskih pripomočkov - 10. del: Preskusi
preobčutljivosti kože (ISO/DIS 10993-10:2020)

Biological evaluation of medical devices - Part 10: Tests for skin sensitization (ISO/DIS

Évaluation biologique des dispositifs médicaux -- Partie 10: Essais de sensibilisation

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

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be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 General principles — Step-wise approach ................................................................................................................................ 3

5 Pretest considerations ................................................................................................................................................................................... 4

5.1 General ........................................................................................................................................................................................................... 4

5.2 Types of material................................................................................................................................................................................... 4

5.2.1 Initial considerations ................................................................................................................................................... 4

5.2.2 Ceramics, metals and alloys ................................................................................................................................... 4

5.2.3 Polymers ................................................................................................................................................................................. 4

5.2.4 Biologically derived materials .............................................................................................................................. 4

5.3 Information on chemical composition ............................................................................................................................... 5

5.3.1 General...................................................................................................................................................................................... 5

5.3.2 Existing data sources ................................................................................................................................................... 5

6 Skin sensitization tests .................................................................................................................................................................................. 5

6.1 Choice of test methods ..................................................................................................................................................................... 5

6.2 Murine Local Lymph Node Assay (LLNA) ........................................................................................................................ 6

6.2.1 Principle .................................................................................................................................................................................. 6

6.2.2 Test sample preparation............................................................................................................................................ 6

6.2.3 Animals and husbandry ............................................................................................................................................. 7

6.2.4 Test procedure ................................................................................................................................................................... 7

6.2.5 Treatment groups ........................................................................................................................................................... 8

6.2.6 Determination of cellular proliferation and tissue preparation ............................................. 8

6.2.7 Results and interpretation ...................................................................................................................................... 9

6.2.8 Test report ............................................................................................................................................................................. 9

6.3 Guinea-pig assays for the detection of skin sensitization .................................................................................. 9

6.3.1 Principle .................................................................................................................................................................................. 9

6.3.2 Choice of test sample concentrations ............................................................................................................ 9

6.3.3 Induction..............................................................................................................................................................................10

6.3.4 Challenge .............................................................................................................................................................................10

6.4 Important factors affecting the outcome of the test ............................................................................................10

6.5 Guinea-pig maximization test (GPMT) ............................................................................................................................11

6.5.1 Principle ...............................................................................................................................................................................11

6.5.2 Test sample preparation.........................................................................................................................................11

6.5.3 Animals and husbandry ..........................................................................................................................................11

6.5.4 Test procedure ................................................................................................................................................................11

6.5.5 Observation of animals ........................................................................................................................................... .13

6.5.6 Evaluation of results ..................................................................................................................................................14

6.5.7 Test report ..........................................................................................................................................................................14

6.6 Closed-patch test (Buehler test) ...........................................................................................................................................14

6.6.1 Principle ...............................................................................................................................................................................14

6.6.2 Test sample preparation.........................................................................................................................................14

6.6.3 Animals and husbandry ..........................................................................................................................................14

6.6.4 Test procedure ................................................................................................................................................................15

6.6.5 Observation of animals ........................................................................................................................................... .16

6.6.6 Evaluation of results ..................................................................................................................................................16

6.6.7 Test report ..........................................................................................................................................................................16

7 Key factors in interpretation of test results ..........................................................................................................................17

Annex A (normative) Preparation of materials for sensitization testing....................................................................18

Annex B (informative) Method for the preparation of extracts from polymeric test materials ..........20

Annex C (informative) in vitro tests for skin sensitization .........................................................................................................23

Annex D (informative) Background information .................................................................................................................................36

Annex E (informative) Reference to the general safety and performance requirements ..........................39

Annex ZA (informative) Relationship between this European Standard and the essential

requirements of Directive 93/42/EEC [OJ L 169] aimed to be covered ....................................................41

Annex ZB (informative) Relationship between this European Standard and the essential

requirements of Directive 90/385/EEC [OJ L 189] aimed to be covered ................................................43

Annex ZC (informative) Relationship between this European standard and the General

covered ......... ................................................................................................................................................................................................................44

Bibliography .............................................................................................................................................................................................................................46

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

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

Any trade name used in this document is information given for the convenience of users and does not

For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following

This document was prepared by Technical Committee ISO/TC 194 Biological and clinical evaluation,.

This fourth edition cancels and replaces the third edition (ISO 10993-10:2010), which has been

Any feedback or questions on this document should be directed to the user’s national standards body. A

This document assesses possible contact hazards from chemicals released from medical devices, which

Some materials that are included in medical devices have been tested, and their skin sensitization

potential has been documented. Especially for dental material sensitizing properties were reported see

Reference [50]. 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

Traditionally, small animal tests are performed prior to testing on humans to help predict human

response. Since 2015, several in chemico and in vitro assays have been validated and OECD test

guidelines released to assess the skin sentization potential of chemicals. These test methods

developed to address a specific key event may not be sufficient to conclude on the presence or absence

of skin sensitisation potential of chemicals and should be considered in the context of integrated

approaches such as IATA, combining them with other complementary information. It should be noted

that the in vitro and in chemico tests for skin sensitization have so far been validated only for neat

chemicals and not for medical device. In order to apply these assays for the testing of skin sensitization

potential of medical devices, further validation for this specific area is essential. . 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 document presents a step-wise approach, with review

and analysis of test results at each stage. It is intended that, for regulatory submission, sensitization

study/studies be conducted using GLP/ISO 17025 as applicable to the respective country. and comply

with regulations related to animal welfare. Statistical analysis of data is recommended and should be

This document 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

The tests included in this document are important tools for the development of safe products, provided

This document 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 dermal sensitization responses relevant to the

This document specifies the procedure for the assessment of medical devices and their constituent

NOTE Instructions for the preparation of materials specifically in relation to the above tests are given in

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 10993-1:2018, Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk

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

ISO 10993-12, Biological evaluation of medical devices — Part 12: Sample preparation and reference

ISO 10993-13, Biological evaluation of medical devices — Part 13: Identification and quantification of

ISO 10993-14, Biological evaluation of medical devices — Part 14: Identification and quantification of

ISO 10993-15, Biological evaluation of medical devices — Part 15: Identification and quantification of

ISO 10993-18, Biological evaluation of medical devices — Part 18: Chemical characterization of materials

ISO 14155, Clinical investigation of medical devices for human subjects — Good clinical practice

For the purposes of this document, the terms and definitions given in ISO 10993-1 and the following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

substance or material that is capable of inducing a specific hypersensitivity reaction upon repeated

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

Note 1 to entry: The purpose of the blank control is to evaluate possible confounding effects due to the extraction

process following the induction phase, in which the immunological effects of subsequent exposures in

process that leads to the de novo generation of an enhanced state of immunological activity in an

localized non-specific inflammatory response to single, repeated or continuous application of a

Note 1 to entry: Skin irritation is a reversible reaction and is mainly characterized by local erythema (redness),

well-characterized material or substance that, when evaluated by a specific test method, demonstrates

the suitability of the procedure to yield a reproducible, appropriately negative, non-reactive or minimal

Note 1 to entry: In practice, negative controls include blanks, vehicles/solvents and reference materials.

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

Note 1 to entry: 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

material, device, device portion or component thereof that is sampled for biological or chemical testing

material, device, device portion, component, extract or portion thereof that is subjected to biological or

liquid used to moisten, dilute, suspend, extract or dissolve the test substance/material

The available methods for testing sensitization were developed specifically to detect skin sensitization

potential. Other types of adverse effects 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

This document 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,

b) literature review, including an evaluation of chemical and physical properties, and information on

the sensitization potential of any product constituent as well as structurally-related chemicals and

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. There are currently a number of internationally validated and accepted

in vitro tests to detect the skin sentization potential of chemicals; However, none of these tests has

NOTE 1 Sensitization potential of medical device extracts 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

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).

e) Clinical studies, in accordance with ISO 14155 and ethics principles, shall not be performed before

It is important to emphasise that pretest considerations may result in the conclusion that testing for

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-

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 leaching or degradation characteristics of the finished products. These components

These materials are normally less complex than polymers and biologically derived materials in terms of

These materials are normally chemically more complex than those in 5.2.2 in terms of composition. A

number of reaction products/impurities/additives/residual catalyst can be present and the degree or

These materials are inherently complex in their composition. They often also contain process