February 1999
1st Working Draft Revision of ISO 10993-12:1996, Biological
evaluation of medical devices - Part 12: Sample Preparation and
Reference Materials (6 February 1999)
This International Standard specifies methods of sample
preparation and use of reference materials in biological
evaluation. Because of the many different biological assay
systems described in ISO 10993, the individual standards should
be consulted to ascertain the appropriateness of these
recommendations for a specific test system.
Sample preparation methods should consider both the biological
evaluation methods and the materials being evaluated. Each
biological test restricts selection of solid samples and
extraction solvents or conditions by its own methodology.
This part of ISO 10993 is based on existing national and
international specifications, regulations and standards wherever
possible. It is open to regular review whenever new research work
is presented to improve the state of scientific knowledge.
This part of ISO 10993 specifies requirements and gives guidance on procedures to be followed in the preparation of samples of medical devices for testing in biological systems in accordance with one or more parts of the ISO 10993 series. These include;
The following standards contain provisions
that, through reference in this text, constitute
provisions of this international Standard.
At the time of publication, the editions
indicated were valid. All standards are subject
to revision, and parties to agreements based
on this International Standard are encouraged
to investigate the possibility of applying
the most recent editions of the standards
indicated below. Members of international
Electrotechnical Commission (EC) and international
Organization for Standards (ISO) maintain
registers of currently valid international
Standards.
ISO/IEC Directives, Part 2: 1992, Methodology
for the development
of international Standards,
Annex B - Mention of reference materials
3.1 blank: Extraction vehicle not containing
the specimen used
for comparison with the extract liquid.
3.2 certified reference material (CRM): Reference
material,
accompanied by a certificate, one or more
of whose property
values are certified by a procedure which
establishes its
traceability to an accurate realization of
the unit in which the
property values are expressed, and for which
each certified value
is accompanied by an uncertainty at a stated
level of confidence
(see ISO Guide 30).
NOTE: standard reference material (SRM):
A trademark protected
certification supplied by the National Institutes
for Standards
and Technology, Gaithersburg. MD, USA.
3.3 extract liquid: Liquid that is tested
for biological response
after it has been used for extraction of
the device.
3.4 homogeneous: The condition of being of
uniform structure or
composition with respect to the biological
endpoint under study.
The RM is said to be homogeneous if the biological
response to a
specific test is found to lie within the
specified uncertainty
limits of the test, irrespective of the site
in the batch or lot
of material from where the test sample is
taken.
3.5 negative control: A negative control
is any well
characterized material or substance, which
when tested by the
procedure described, demonstrates the suitability
of the
procedure to yield a reproducible, appropriate
negative,
nonreactive or background response in the
test system.
3.6 positive control: A positive control
is any
well-characterized material or substance,
which when tested by
the procedure described, demonstrates the
suitability of the
procedure to yield a reproducible, appropriate
positive or
reactive response in the test system.
3.7 reference material (RM): A material or
substance, one or more of whose property
values are sufficiently homogeneous and well
established to be used for the calibration
of an apparatus, the assessment of a measurement
method, or for assigning values to materials
(see ISO Guide 30). For the purposes of the
standard, a reference material is any well-characterized
material or
substance, which when tested by the procedure
described,
demonstrates the suitability of the procedure
to yield a
reproducible, predictable response. The response
may be negative
or positive.
3.8 reference method: A thoroughly investigated
test method,
clearly and exactly describing the necessary
conditions and
procedures, for the evaluation of a specific
biological endpoint,
that has been shown to have accuracy and
precision commensurate
with its intended use and that can, therefore,
be used to
characterize a RM (see ISO Guide 30).
3.9 Stability of property values: The ability
of a material, when
stored under specified conditions, to maintain
the stated
biological response, within specified limits,
for a specific
period of time (see ISO Guide 30).
3.10 test material: Material, device, device
portion, or
component thereof subject to biological or
chemical testing.
3.11 test sample: Test material or extract
subject to biological
or chemical testing.
Experimental controls shall be used in biological evaluations to validate a test procedure and/or to evaluate the results from a new material. Depending on the biological test being used, negative and/or positive controls and blanks shall be used as appropriate. The same control may be applicable to different tests to allow cross-reference to established materials and test methods. Additional guidance on the selection of experimental controls is given in Annex B.
5.1 Reference or certified reference materials
shall be used in biological tests as control
materials to demonstrate the suitability
of the procedure to yield a reproducible,
predictable response, e.g. positive or negative.
Use of a reference material in this way will
ensure the comparability of the response
between laboratories. The property values
of any material used in this way shall be
characterized with each biological test procedure
for which the use of the material is desired.
A material characterized and then certified
for one reference test method or response,
e.g. sensitization, shall not be used as
a reference material for another, e.g. cytotoxicity,
without additional characterization.
5.2 Reference materials used as experimental
controls shall meet
the established quality assurance procedures
of the manufacturer
and test laboratory. They shall be identified
as to source,
manufacturer, grade, and type. Reference
materials used as
experimental controls shall be fabricated,
finished and their
surfaces cleaned and sterilized in a manner
appropriate to the
projected application in human subjects.
Where possible, the
above processes should be identical to the
processes employed in
the manufacture of the finished device.
5.2.1 Reference materials used as experimental
controls shall be
in the same physical class as the test sample,
i.e., polymer,
ceramic, metal, colloid, etc. Pure chemicals
may be used as
Reference Materials for mechanistically based
test procedures,
e.g. mutagenicity and immune sensitization.
5.3 A comparable, clinically accepted device
will satisfy the
requirement of 5.2.
6.1 Material screening versus medical device
biocompatibility
The uses of the reference materials described
in this document
are limited to biological screening testing
of the materials
intended for use in the manufacture of medical
devices. However,
while not intended for the purpose, they
are often used in the
performance assessment of the finished device.
The vertical
standard for the device, when available,
must address the
biological testing of the product in the
performance environment
of the device. Biological testing described
in the vertical
standard takes precedent over testing performed
to screen the
materials for suitability.
6.2 Misuse of CRMs
The attention of the users of this standard
is directed to the
discussion of "proper use" and
"misuse" of
CRMs in the introduction to ISO Guide 33.
This discussion points
out areas of both potential under and over
utilization of RMs and
CRMs. Users of this standard should also
note that the use of
calibration materials to evaluate the biological
response of
materials under investigation within a single
laboratory is
acceptable.
6.3 One or more property values
RMs or CRMs used to determine the biological
response in this way
must be evaluated with each biological test
procedure for which
the use of the material is desired. It is
not sufficient to
qualify a material for one type of reference
test method or
response, e.g. sensitization, and declare
it a reference material
for another, e.g. cytotoxicity, without additional
qualification
testing.
6.4 Long term availability of the material
During the development of this standard,
concern was expressed
about the future availability of reference
materials for
determination of biological response. It
is desirable for users
to obtain a commitment of not less than 5
years from suppliers of
the RMs or CRMs. A second, but less desirable,
option is the
publication by the source of the RM or CRM
of an "open
formulation" for the material, i.e.
publication of the
source materials and details of the processing
needed to insure
uniform batches of RM.
6.5 Certification of reference materials
for biological safety
testing
6.5.1 Individual laboratories certify reference
materials. The
individual laboratory determines the extent
of chemical, physical
and biological characterization. Commercially
available articles
may be used as reference materials (see Clause
5.3).
6.5.2 Certification of a reference material
is a procedure that
establishes the value of the biological response
of the material
under the test conditions specified, ensuring
a traceability of
the response, which leads to the issuance
of a certificate. The
biological response of the material should
be established through
interlaboratory tests.
7.1 It is preferable to test medical devices
in their final
product form whenever practical. When that
is not possible, the
second choice is representative portions
of the device (see
Clause 9). When these are not possible or
practical,
representative molded or extruded test specimens
of the
formulated material that have been preconditioned
by the same
processing as the final product shall be
tested.
7.2 The same test material selection procedure
applies when an
extract is required.
8.1 Test samples and reference materials
shall be handled to
prevent contamination. Any residues from
the manufacturing,
fabrication, cleaning, sterilization, etc.,
processes, if
present, should be considered to be integral
to the device,
device portion or component. Additional guidance
on the
preparation is given in Annex C.
8.1.1 Test samples from sterilized devices
and reference
materials shall be handled aseptically if
appropriate to the test
procedure.
8.1.2 Test samples from devices which are
normally supplied
non-sterile but are sterilized before use
shall be sterilized by
the method recommended by the manufacturer
and handled
aseptically if appropriate to the test procedure.
8.1.3 If test samples are cleaned prior to
sterilization, the
influence of the cleaning process and cleaning
agent shall be
considered.
8.2 If sterile test samples are required
for the test procedure,
the effect of the sterilization or resterilization
process on the
test sample and reference materials shall
be considered.
8.3 When test samples and reference materials
need to be cut into
pieces, the influence of previously unexposed
surfaces, e.g.
lumens or cut surfaces, shall be considered.
Techniques used for
cutting medical devices into representative
portions for testing
should be as clean as practical to prevent
contamination.
9.1 If a device cannot be tested as a whole,
each individual material in the final product
shall be represented proportionally in the
test sample.
9.1.1 The test sample of devices with surface
coatings shall
include both coating material and the substrate.
9.1.2 The test sample shall include a representative
portion of
the joint and/or seal if adhesives, radio
frequency (RF) seals,
or solvent seals are used.
9.1.3 Composite materials shall be tested
as finished materials.
9.1.4 Where extracts are used in the test
methods for evaluation
of materials that cure in-situ, e.g. cements,
adhesives and
monomers, initiation of the extraction shall
occur after the
specified minimum cure that may occur during
clinical use. For
test methods that use these materials directly,
e.g. direct
contact or agar overlay cytotoxicity, implantation,
some
genotoxicity tests, and direct contact hemolysis,
the material
shall be used as in clinical use, with in-situ
cure in the test
system. Modification of the delivery system
so that the
designated size or weight of the material
is delivered is
appropriate.
9.2 When different materials are present
in a single device, the
potential for synergies and interactions
shall be considered in
the choice of test sample.
9.3 The test sample may be chosen to maximize
the exposure of the
test system to the components where components
of a device are
known to have a potential for a biological
response.
If extracts of the device are required for
a test protocol, the
extraction media and conditions of extraction
used shall be
appropriate to the nature and use of the
final product.
Additional guidance on the extraction of
samples is given in
Annex D.
10.1 Containers for extraction.
10.1.1 The extraction shall be performed
in clean, chemically
inert closed containers with minimum headspace.
10.1.2 To ensure that the extraction vessels
do not adulterate
the extract of the test materials the extraction
vessels shall
be:
| Thickness
(mm) |
Extraction ratio
(surface area/volume) +-10% |
Example of
Materials |
| < 0.5 | 6cm2/ml | film, sheet,
tubing wall |
| 0.5 - 1.0 | 3cm2/ml | tubing wall, slab,
molded items |
| > 1.0 | 1.25cm2/ml | natural elastomer |
| Not applicable | 0.2g sample/ml
6cm2/ml |
powder, pellets
foam, non-absorbent |
---------------------------
1 The phase equilibrium of a material at
a temperature controls the relative amounts
of amorphous and crystalline phases present.
For the amorphous phase, the glass transition
temperature, Tg, dictates the polymer chain
mobility and the diffusion rate in the phase.
Usually, the diffusion rate is considerably
higher above the Tg compared with that below.
The diffusion rate is lowest in the crystalline
phase. The extraction conditions shall not
alter the phase equilibrium of the material.
Phase alteration may affect the amount and
type of extractables.
---------------------------
Documentation of sample preparation shall
include, but not be limited to:
B.1 The materials listed in the following
paragraphs may meet the
criteria for an appropriate experimental
control in selected
tests. It is the responsibility of the investigator
to make the
appropriates choices. See Table B 1.
Table B1 - Summary of reference materials
and controls
|
TEST |
POSITIVE CONTROL* |
NEGATIVE CONTROL* |
REFERENCE MATERIAL* |
|
IMPLANTATION |
PVC-org. Sn |
PE |
|
|
|
SPU-ZDEC |
Silicone |
|
|
|
Latex |
Alumina |
|
|
|
|
Stainless steel |
|
|
CYTOTOXICITY |
PVC-org. Sn |
PE |
|
|
|
SPU-ZDEC |
|
|
|
|
SPU-ZBEC |
|
|
|
|
Latex |
|
|
|
BLOOD COMPATIBILITY |
|
|
PVC 7506 PUR 2541 |
|
|
|
|
|
* Abbreviations on this Table refer to specific
materials available from sources designated
in Clauses B.1 and B.2.
B.2 Examples of solid reference materials
which have been used as
negative controls are, for example, high
density polyethylene(2,
low density polyethylene(3,4, silica-free
polydimethylsiloxane(4,5,
polyvinylchloride(6, polyetherurethane(7,
polypropylene(8,
medical grade latex, aluminum oxide ceramic
rods, stainless steel
and titanium alloys. This information is
given for the
convenience of the user of this part of ISO
10993 and does not
constitute an endorsement by ISO of the product.
B.3 Examples of materials which have been
used as positive
controls for solid samples are polyvinylchloride
containing
organo-tin additives(9, segmented polyurethane
films containing
zinc diethyl- or dibutyl-dithiocarbamate,
certain latex
formulations and solutions of zinc salts(10.
Substances which
have been used as positive controls for extract
samples are
dilutions of phenol and water. This information
is given for the
convenience of the user of this part of ISO
10993 and does not
constitute an endorsement by ISO of the product.
-----------------------
2 High density polyethylene (Negative Control
Plastic RS) can be
obtained from the US Pharmacopeia (Rockville,
MD USA).
3 PE 140 tubing is available from Rehau AG,
D-8673 Rehau,
Germany. PE film is available from Hoechst
AG, D6230 Frankfurt
80, Germany.
4 Biomaterials Program, Devices and Technology
Branch, National
Heart, Lung and Blood Institute, NIH, 312
Federal Building, 7550
Wisconsin Ave., Bethesda, MD 20892, USA.
5 SIK 8363 tubing is available from Rehau
AG, D-8673 Rehau,
Germany.
6 PVC 7506 and PVC 7536 tubing is available
from Rehau AG, D-8673
Rehau, Germany. PVC-DEHP and PVCTEHTM film
is available from
Hoechst AG, D-6230 Frankfurt 80, Germany.
7 PUR 2541 tubing is available from Rehau
AG, D-8673 Rehau,
Germany. PU film is available from Frontline
Filmblasning, S-60003
Norrkoping, Sweden.
8 PP 146 tubing is available from Rehau AG,
D-8673 Rehau,
Germany. PP film is available from Hoechst
AG, D6230 Frankfurt
80, Germany.
9 Positive control RS can be obtained from
the US Pharmacopeia,
Rockville, MD, 20852, USA.
10 AS 2696-1989 Medical Equipment - Single
use urethral catheters
(sterile) for general medical use.
-----------------------
This section provides general principles
on and practices of
material preparation and sample selection.
C.1 The material used in the biological assay
should be
representative of the composition, processes
and surface
characteristics of the final product. See
Clause 7.
C.1.1 In the case of plastic and rubber materials,
the
composition should include resin, polymer
and any additives.
Alternate components of a formulation should
be evaluated for
substitution. The formulation description
should specify the
history of the material, e.g., thermal, virgin
or regrind and the
maximum allowable regrind.
C.1.2 Materials that may be re-sterilized
by the same or
alternative methods should be tested after
treatment by the
multiple sterilizations. For example, a material
that is
sterilized by radiation and re-sterilized
by ethylene oxide
should be tested after (a) irradiation and
(b) irradiation plus
ethylene oxide. If a "worst case"
exposure can be
identified with appropriate justification,
testing may be
performed after exposure to this treatment.
C.1.3 Ideally, all biological tests on a
material should have the
surface of the material exposed (as opposed
to the bulk phase) to
the cellular/biological environment. An alternative
method to
cutting the surface is fabrication of miniatures
of the device
using the same process (extrusion, dipping,
etc.), temperatures,
time, atmosphere, release agents, annealing,
curing, cleaning,
sterilization, etc., processes used in the
manufacture of the
device.
C.1.4 Metals used in biological tests should
be from the same
stock material used to fabricate the device
and using the same
machining, grinding, polishing, cleaning,
passivation, surface
treatment and sterilization used in the manufacture
of the final
product.
C.1.5 Ceramic materials used in biological
tests should be
manufactured from the same powder stock using
the same casting,
investing, molding, sintering, surface finishing
and
sterilization processes used to manufacture
the device.
C.1.6 Bioprosthetic materials should be tested
after they have
been preserved under the manufacturers maximum
and minimum
allowable fixation times to allow for varying
penetration of the
fixative.
C.2 Extraction conditions that may cause
particle generation
should be considered in the design of tests
on the material.
C.3 The amount of material and surface area
thereof shall be
appropriate to the biological and physical
constraints of the
test system. In practice, the use of a standard
sample size for a
specific assay is recommended.
The purpose of an extract of a medical device
is to provide a
suitable test sample for determining the
biological reactivity of
any leachable substances in a biological
system and to
demonstrate the hazard potential for the
use of the device in
humans. If extracts of the device are prepared,
the medium and
conditions of extraction used should be appropriate
to the nature
and use of the final product as well as to
the predictability
(test rationale, sensitivity, etc) of the
test method. Extraction
conditions, therefore, should ideally reflect
not only actual
in-use conditions of the products but also
predictability of the
tests.
D.1 This standard assumes that the amount
of extractable
substance(s) is/are related to the period
of extraction, the
temperature, the ratio of surface-area-of-material
to
volume-of-extractant and the nature of the
solvent.
D.2 The period of extraction should be sufficient
to maximize the
amount of material extracted. In practice,
standard time and
temperature conditions are recommended in
lieu of specific
chemical analyses. An alternative practice
is repeated extraction
followed by concentration to obtain sufficient
extractable
substance(s).
D.3 The extraction temperature should maximize
the amount of
extractable substances as well as simulate
any extremely high
temperatures the device may incur during
clinical use. This
simulation should not initiate significant
degradation of the
material. The extraction temperature is dependent
upon the
physicochemical characteristics of the device
material(s). For
example, for polymers, the extraction temperature
chosen should
be below the glass transition temperature.
If the glass
transition temperature is below the use temperature,
the
extraction temperature shall be below the
melting temperature.
Recommended conditions are shown in Section
10.2.1.
The following examples are presented to illustrate
the
interpretation of this section:
D.4 The ratio of the surface area of the device to the volume of extractant or solvent should be sufficient to:
In practice, the use of a standard area and
solvent volume is recommended as described
in Section 10.2.2 in lieu of device specific
parameters. Some test methods require concentration
of extracts to increase the sensitivity of
the test.
NOTE: Concentration of extracts may result
in loss of volatile
materials such as ethylene oxide.
D.5 The solvent(s) selected as extractants
shall:
In practice, the use of standard polar and
non-polar solvents are recommended in Section
10.2.3 in lieu of device specific solvents.
NOTE: Standardization of the parameters given
in Clauses D4 and
D5 permit the use of data obtained from biological
tests of
medical devices for other types of applications,
e.g. for the
estimation of risk and to develop standardized
databases.