ISO/DIS 10993-6

Biological evaluation of medical devices

Évaluation biologique des dispositifs médicaux

Standards Content (sample)

DRAFT INTERNATIONAL STANDARD ISO/DIS 10993-6
ISO/TC 194 Secretariat: DIN
Voting begins on: Voting terminates on:
2004-12-23 2005-05-23

INTERNATIONAL ORGANIZATION FOR STANDARDIZATION • МЕЖДУНАРОДНАЯ ОРГАНИЗАЦИЯ ПО СТАНДАРТИЗАЦИИ • ORGANISATION INTERNATIONALE DE NORMALISATION

Biological evaluation of medical devices —
Part 6:
Tests for local effects after implantation
Évaluation biologique des dispositifs médicaux —
Partie 6: Essais concernant les effets locaux après implantation
[Revision of first edition (ISO 10993-6:1994)]
ICS 11.100.20
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© International Organization for Standardization, 2004
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ISO/DIS 10993-6
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ISO/DIS 10993-6
Contents Page

Foreword ............................................................................................................................................................vi

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

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

3 Terms and definitions ...........................................................................................................................2

4 Common provisions for implantation test methods..........................................................................2

4.1 General...................................................................................................................................................2

4.2 Preparation of specimens for implantation. .......................................................................................2

5 Test methods, general aspects............................................................................................................3

5.1 Tissue and implantation site ................................................................................................................3

5.2 Animals...................................................................................................................................................3

5.3 Test periods...........................................................................................................................................3

5.4 Surgery and testing conditions ...........................................................................................................5

5.5 Evaluation...............................................................................................................................................5

5.5.1 General...................................................................................................................................................5

5.5.2 Macroscopic assessment.....................................................................................................................5

5.5.3 Implant retrieval.....................................................................................................................................5

5.5.4 Microscopic assessment......................................................................................................................6

5.5.5 Evaluation of responses.......................................................................................................................7

6 Test report..............................................................................................................................................7

Annex A (informative) General considerations regarding implantation periods and tissue

responses of degradable/resorbable materials .................................................................................8

Annex B (normative) Test methods for implantation in subcutaneous tissue.............................................9

B.1 Field of application................................................................................................................................9

B.2 Principle..................................................................................................................................................9

B.3 Test specimens......................................................................................................................................9

B.4 Test animals and implant sites ............................................................................................................9

B.5 Implantation procedure.........................................................................................................................9

B.5.1 Implantation along dorsal midline .....................................................................................................10

B.5.2 Implantation in neck............................................................................................................................10

B.6 Implantation period.............................................................................................................................10

B.7 Evaluation of biological response .....................................................................................................10

B.8 Format of test report ...........................................................................................................................10

Annex C (normative) Test method for implantation in muscle ....................................................................11

C.1 Field of application..............................................................................................................................11

C.2 Principle................................................................................................................................................11

C.3 Test specimens....................................................................................................................................11

C.4 Test animals and implant sites ..........................................................................................................11

C.5 Implantation procedure.......................................................................................................................11

C.6 Implantation period.............................................................................................................................12

C.7 Evaluation of biological response .....................................................................................................12

C.8 Format of test report ...........................................................................................................................12

Annex D (normative) Test method for implantation in bone ........................................................................13

D.1 Field of application..............................................................................................................................13

D.2 Principle................................................................................................................................................13

D.3 Test specimens....................................................................................................................................13

D.3.1 General.................................................................................................................................................13

D.3.2 Shape of implant specimens..............................................................................................................13

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D.3.3 Size of test specimens ........................................................................................................................13

D.4 Test animals and implant sites...........................................................................................................13

D.4.1 Test animals.........................................................................................................................................13

D.4.2 Implant sites.........................................................................................................................................14

D.5 Implantation procedure.......................................................................................................................14

D.6 Implantation period..............................................................................................................................14

D.7 Evaluation of biological response .....................................................................................................14

D.8 Format of test report ...........................................................................................................................14

Annex E (informative) Format for documentation and evaluation of local biological effects after

implantation..........................................................................................................................................15

Annex ZA (informative) Clauses of this International Standard addressing essential requirements

or other provisions of EU Directives 90/385/EEC for Active Implantable Medical Devices

and 93/42/EEC for Medical Devices ...................................................................................................17

Annex ZB (normative) Normative references to international publications with their relevant

European publications ........................................................................................................................18

Bibliography ......................................................................................................................................................19

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ISO/DIS 10993-6
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 10993-6 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices.

This second edition cancels and replaces the first edition (EN 30993-6:1994), 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
 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 the identification and quantification of potential degradation products

 Part 10: Tests for irritation and delayed-type hypersensitivity
 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

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 Part 16: Toxicokinetic study design for degradation products and leachables

 Part 17: Method for the establishment of allowable limits for leachable substances

 Part 18: Chemical characterization of materials
 Part 19: Physico-chemical, mechanical and morphological characterization

 Part 20: Principles and methods for immunotoxicology testing of medical devices

Future parts will deal with other relevant aspects of biological evaluation.
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DRAFT INTERNATIONAL STANDARD ISO/DIS 10993-6
Biological evaluation of medical devices — Part 6: Tests for
local effects after implantation
1 Scope

This part of the ISO 10993 specifies test methods for the assessment of the local effects after implantation of

biomaterials intended to be used in medical devices. The standard applies to solid biomaterials that are non-

degradable, degradable and/or resorbable, non-solid biomaterials, such as porous materials, and liquids,

pastes and particulates.

The test specimen is implanted into a site and tissue appropriate for the evaluation of the biological safety of

the material. This implantation test is not intended to determine the performance of the test specimen in terms

of mechanical or functional loading. This standard may also be applied to medical devices which are intended

to be used on surfaces breached or intact, in order to evaluate local tissue responses.

The local effects are evaluated by a comparison of the tissue response caused by a test specimen to that

caused by materials used in medical devices of which the clinical acceptability and biocompatibility

characteristics have been established. The objective of the test methods is to characterize the history of the

tissue response after implantation of a medical device/biomaterial including final integration or resorption /

degradation of the material. In particular for degradable/resorbable materials the degradation characteristics of

the material and the resulting tissue response should be determined.

This standard does not deal with systemic toxicity, carcinogenicity, teratogenicity or mutagenicity. However,

the long-term implantation studies intended for evaluation of local biological effects may provide insight into

some of these issues.

NOTE For the convenience of the user of this standard a bibliography is included.

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, Biological evaluation of medical devices – Part 1: Guidance on selection of tests

ISO 10993-2, Biological evaluation of medical devices – Part 2: Animal welfare requirements

ISO 10993-11, Biological evaluation of medical devices – Part 11: Tests for systemic toxicity

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

ISO 10993-16, Biological evaluation of medical devices – Part 16: Toxicokinetic study design for degradation

products and leachables
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ISO/DIS 10993-6
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 10993-1, ISO 10993-2, ISO 10993-

16 and the following apply.
3.1
degradation
chemical breakdown or decomposition of material
4 Common provisions for implantation test methods
4.1 General

The provisions of clause 4 shall apply to the test methods described in Annex B, C and D.

All animal studies shall be performed in a facility approved by a nationally recognized organisation and in

accordance with all appropriate regulations dealing with laboratory animal welfare. These studies shall be

performed under good laboratory practices or other recognized quality assurance systems, and comply with

the requirements in ISO 10993 -2.

It is important that the researcher plans the study in such detail that all relevant information can be extracted

from the use of each animal and each study (see ISO 10993-2, ISO 10993-11 and ISO 10993-16).

4.2 Preparation of specimens for implantation.

Test sample and reference material preparation shall be in compliance with 10993-12. The implant size and

shape shall be documented and justified. Test specimens for various implant sites are described in Annex B to

D. Physical characteristics (such as form, density, hardness, surface) can influence the character of the tissue

response to the test material and shall be recorded.

Each implant shall be manufactured, processed, cleaned of contaminants and sterilized by the method

intended for the final product and accordingly documented. After final preparation and sterilization, the implant

specimens shall be handled aseptically and in such a way as to ensure that they are not scratched, damaged

or contaminated in any way prior to or during implantation.

For materials used as scaffolds for tissue engineered medical products it may be appropriate not to use the

final preparation with cells, as the immune reaction of the laboratory animal to the cellular components of such

products may interfere with the resulting local tissue response.

For composite materials (e.g. bone cements, dental materials), the components may be mixed before use,

and set after varying time periods. Materials that are designed for use in devices with in situ polymerisation

shall be introduced in a manner such that in situ polymerisation occurs. For certain types of studies other

procedures may be used. The procedure used shall be documented.

Non-solid materials (including powders) may be contained in tubes for the purpose of testing for local effects

after implantation (see ISO 10993-12 for the selection of materials for tubes). Prepare the test material

according to the manufacturer’s instructions and insert the material into the tube until level with the top.

Exercise the utmost care to prevent contamination of the outer surface of the tube by the test material. Avoid

entrapment of air in the tube and ensure that the end surfaces of the inserted material in the tube and the tube

ends are smooth.

NOTE 1 Polyethylene (PE), polypropylene (PP), or polytetrafluoroethylene (PTFE) tubes are commonly used for this

purpose. PE tubes may be deformed by autoclaving. PTFE tubes are difficult to section in the microtome, and substitution

by PE or PP tubes of the same dimensions may be preferable when the tubes are to remain in the tissue blocks during

sectioning.
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Evaluation is done by comparison of the tissue reaction to that of a similar specimen/material of which the

clinical acceptability and biocompatibility characteristics have been established.

NOTE 2 For further guidance see ISO 10993-12.

The physical characteristics such as shape and especially the surface condition of the control(s) shall be

similar to that of the implant test specimens as is practically possible. When the test material is contained in a

tube, the control shall be a rod of the same material as the tube and with the same diameter as the outer

diameter of the tube. In general, a positive control is not necessary.
5 Test methods, general aspects
5.1 Tissue and implantation site

The test specimen shall be implanted into the tissues most relevant to the intended clinical use of the material.

The justification for the choice of tissue shall be documented. Test methods for various implantation sites are

given in Annex B to D. If other implantation sites are chosen, the general scientific principles behind the test

methods described in Annex B to D shall still be adhered to.

For degradable/resorbable materials the implantation site shall be marked in a manner suitable for

identification of the site at the designated time periods. The use of a permanent skin marker and/or a template

marking the placement of the specimen is recommended. In certain circumstances an appropriate negative

control may be used as a marker for the location of the implant site. A sham surgical procedure might be used

to evaluate the impact of the procedure on the tissue involved.

NOTE Markers for identification of the implant site of resorbable test specimens may be non absorbable sutures or

skin paints.
5.2 Animals

Animal husbandry shall be in accordance with ISO 10993-2. In general, small laboratory animals such as mice,

rats, hamsters or rabbits are preferred. The use of larger animals may be justified based upon special

considerations of the particular biomaterial under study.

Select an animal species with due considerations of the size of the implant test specimens, the number of

implants per animal, the intended duration of the test in relation to the expected lifespan of the animals, as

well as potential species differences regarding biological response.

For short-term testing in subcutaneous tissue, muscle, and bone, animals such as mice, rats, guinea pigs or

rabbits are commonly used. Select one species among these.

For long-term testing in subcutaneous tissue, muscle and bone, animals such as rats, guinea pigs, rabbits,

dogs, sheep, goats, pigs and other animals with a relatively long life expectancy are suitable. Select one

species among these.

NOTE Especially for biodegradable materials it is recommended that a pilot study in rodents be undertaken to

determine the expected rate of degradation before embarking on studies in larger animals.

The specimens of test and control materials shall be implanted under the same conditions in the same

species of the same age, sex, and strain in corresponding anatomical sites. The number and size of implants

inserted in an animal depends on the size of the species and the anatomical location. Whenever possible, the

reference control and the test specimen should be in the same animal.
5.3 Test periods

The test period shall be determined by the likely clinical exposure time or be continued until or beyond a

steady state has been reached with respect to the biological response. For non-degradable/resorbable

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ISO/DIS 10993-6

materials the short-term responses are normally assessed from 1 week up to 4 weeks and long-term tests

exceeding 12 weeks. The local biological response to implanted materials depends both on the properties of

the materials and on the trauma of surgery. The tissue configuration found in the vicinity of an implant

changes with time elapsed after surgery. Up to two weeks, the reaction due to the surgical procedure itself

may be difficult to distinguish from the tissue reaction evoked by the implant. In muscle and connective tissue,

depending on the species, and the severity of the surgical trauma, a steady state is seen in the cell population

after 9 to 12 weeks. Implantation in bone tissue may need longer observation periods before a steady state is

reached.

For degradable/resorbable materials the test period shall be related to the estimated degradation time of the

test product. See Annex A for general considerations regarding degradable/absorbable materials. Before

starting with animal studies an estimation of the degradation time shall be made. This can be done in vitro by

real time or accelerated degradation studies or in certain circumstances by mathematical modelling. In general

it is to be expected that long-term experiments (exceeding 12 weeks) are needed for the evaluation of

degradable materials.

The milestones for evaluation of degradable biomaterials will depend on the degradation rate of the materials.

The following may be used for guidance:
 Early responses may be determined up to 2 weeks after implantation;
 Short-term: local reaction to be evaluated at 2-12 weeks after implantation;
 Intermediate: when it is anticipated that degradation is taking place;

 Long-term: when it is estimated that degradation is nearing completion, or a steady state has been

reached.

Even in the absence of complete degradation, absorption, or restoration to normal tissue structure and

function, the overall data collected may be sufficient to allow characterization of the local effects after

implantation.

NOTE 1 In vivo degradation may need a rather long period of time for up to more then one year. Additional animals

may be used to extend the observation period when the implant has not been degraded completely within the expected

investigational time period.

NOTE 2 Although this standard does not address the issues of systemic toxicity (ISO 10993-11), it is recommended

that the information required to meet this standard (ISO 10993-6) be obtained from any systemic toxicity studies using

implantation.

For long term implantation studies, generally used observation periods are given in Table 1.

Table 1 — Selection of test periods for implantation in subcutaneous tissue, muscle and bone

Species Implantation period in weeks
12 26 52 78 (104)
Rats X X X
Guinea pigs X X X
Rabbits X X X X X
Dogs X X X X X
Sheep X X X X X
Goats X X X X X
Pigs X X X X X
Depending on the intended use of the test material, not all implantation
periods may be necessary (see ISO 10993-12). An observation period of
104 weeks may be of interest in selected instances
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