Iso 10993
Iso 10993
Iso 10993
of Medical Devices
Pugazhenthan Thangaraju and Shoban Babu Varthya
1 S
election and Qualification of Reference Materials
for Biological Tests (ISO 10993-8)
P. Thangaraju (*)
Department of Pharmacology, All India Institute of Medical Sciences (AIIMS),
Raipur, Chhattisgarh, India
S. B. Varthya
Department of Pharmacology, All India Institute of Medical Sciences (AIIMS),
Jodhpur, Rajasthan, India
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 163
P. S. Timiri Shanmugam et al. (eds.), Medical Device Guidelines and Regulations
Handbook, https://doi.org/10.1007/978-3-030-91855-2_11
164 P. Thangaraju and S. B. Varthya
3 P
hysicochemical, Morphological and Topographical
(PMT) Characterization of Materials (ISO 10993-19)
PMT characterization along with above framework is essential for judging a pro-
posed material to clinical established material or a prototype device to final device.
Few examples to understand the relation between PMT evaluation and clinical
effectiveness are as follows:
1. Porous materials as surfaces on orthopaedic implants can encourage tissue
ingrowth at the surface of the implant for better integration.
2. The use of material scaffolds and meshes aids in healing process.
3. The PMT characterization enables to understand the bacterial adherence to cath-
eters and their role in infections and blockages.
4. Alterations to the microtopography of surfaces, e.g. producing microgrooves or
other defined patterns, have been shown to influence the adhesion and direction
of movement of certain types of cells on that surface.
5. For certain medical devices, e.g. orthopaedic implants and vascular prostheses,
mechanical properties may influence biological responses such as tissue
remodelling.
4 F
ramework for Identification and Quantification
of Potential Degradation Products (ISO 10993-9)
Under this we will be looking at general principles for systemic evaluation of the
potential and observed biodegradation of medical devices and for the design and
performance of biodegradation studies. It depends on nature of material and its
anatomical location during use. Study design depends on chemical and physio-
chemical properties, surface morphology and biochemical properties. In case of
biodegradable material or material implanted for more than 30 days or toxic sub-
stance released from the material, biodegradable studies shall be conducted. But in
case if biodegradable products are similar to parent material or if safety data is
available, then there is no need to conduct further studies. When studies are planned
to study, then in vitro and in vivo studies are to be conducted.
Characterization of potential degradation products: Degradation products can be
particulate or soluble compound or ions; based on type of particulate matter, it must
be characterized (if particulate material, then size, shape, surface area and other
relevant characteristics recorded). Protocol of such study must aim at evaluating the
changes in bulk material and mechanism of change in its characteristics. Bulk mate-
rial undergoes changes during storage, or processing, or sterilization, during implan-
tation or after implantation, or leeching from the site of implantation. Substance
from implant can be released by chemical reaction, leeching, migration, depolymer-
ization or peeling of material.
166 P. Thangaraju and S. B. Varthya
A study must report nature of material and its intended use, assessment of degra-
dation and its rationale for same, description of degradation products related to test
material and methodologies including analytical test and statement of compliance to
good laboratory practice.
Test Period For devices whose intended use is longer than 30 d, test periods of
1 month, 3 months, 6 months and 12 months shall be used. For devices whose
intended use is less than 30 d, four alternative test periods shall be used, includ-
ing 30 d.
Description of the test material, batch or lot number, dimensions and number of
samples tested; test solution and conditions; detailed description and justification of
the test methods used, including (where appropriate) specificity, sensitivity, detec-
tion and quantification limits; method used to determine mass loss, including preci-
sion; mass/volume ratio of sample and shape of sample; sample pretreatment and
drying method; selected pH; test temperature; test periods; test results; (1) mass
balance; (2) molecular mass/distribution (cross-link density); (3) results of tests run
on the solution, debris and/or bulk polymer; (4) identified degradation products; (5)
rationale for final decision.
A potentiodynamic test and a potentiostatic test are used to identify and quantify
degradation from metals and alloys.
168 P. Thangaraju and S. B. Varthya
8 Compatibility Testing
8.2 S
elections of Tests for Interactions with Blood (ISO
10993-4)
In this section, we will discuss on devices coming into contact with blood. They
may be externally communicating devices like cannulae, IV catheters, cardiopul-
monary bypass circuit, capillary filters, guiding wires, etc. and implant devices like
AV shunts, IVC filters, ventricular assist devices, vascular stents, etc.
Characterization of Blood Interaction For characterization of devices, they need
to undergo certain test for interaction like haemolysis due to device or mechanical
force and thrombosis.
In Vitro Tests Haematocrit, anticoagulant (type and amount), test sample prepara-
tion, test sample age, blood/blood component age, test sample storage, aeration and
pH, temperature, proper randomization, test sample surface area to blood volume
ratio and, for dynamic studies, fluid flow conditions, especially flow rate, wall shear
rate and pressure(s).
They help for simulation of test device and continuous monitoring for device, but its
usage was restricted by size of the animal, duration of implantation and cost.
Avoidance of costly animal models; high replication testing of test objects alongside
controls and reference materials using the same batch of blood and, at the same
time, use of human or animal blood where flow, temperature and anticoagulation are
standardized; worst-case scenario testing, where activation products accumulate
without clearance by kidneys or liver or other organs and activation-inhibiting func-
tions of endothelial cells are absent; and isolation from confounding factors associ-
ated with device implantation/tissue injury associated with in vivo usage.
Thrombosis This is analysed from the distal organ microscopy after autopsy. In
this we look for per cent of occlusion, surface area covered by thrombus and surface
area free from thrombus.
the level of coagulation activity. Partial thromboplastin time (PTT) is to test the
activation of intrinsic coagulation pathway. The activated partial thromboplastin
time (APTT) must be avoided because the activator used in this test may interfere
with material activation.
Methods to Test the Activity of Platelets Medical devices can cause platelet
depletion due to adhesion, aggregation and/or sequestration. To assess the activity
of platelets, platelet granule-release proteins beta-thromboglobulin (ß-TG) and
platelet factor 4 (PF4), thromboxane B2 (TxB2) and platelet morphological changes
are to be tested.
Three categories of test are used for in vitro cytotoxicity studies: extract test, direct
contact test and indirect contact test. These are primarily to study the biological
response of mammalian cells to medical devices and/or their extracts.
Endpoints under this test are (1) assessments of cell damage by morphological
means, (2) measurements of cell damage, (3) measurements of cell growth and (4)
measurements of specific aspects of cellular metabolism.
1. The extract test: In this test we simulate or exaggerate the condition intended
for use to assess the potential toxic effects in sample by fusion, melting or any
alteration of the chemical structure, unless this is expected during clinical appli-
cation. In case where two or more substances are mixed in sample, then extrac-
tion test shall be applied before washing the sample to remove residues.
Extraction vehicles in this test can be culture medium with serum, physiological
saline solution or other vehicles.
2. Neutral red uptake (NRU) cytotoxicity test: In this test BALB/c 3T3 cell lines
are used and seeded in 96-well cell plate for 24 h. At different concentrations test
sample will be applied for 24 h. The IC50 (i.e. the concentration producing 50%
reduction of NRU) is calculated from the concentration-response and expressed
as a dilution percentage of the extract.
ISO 10993: Biological Evaluation of Medical Devices 171
3. Colony formation cytotoxicity test: V79 cells are seeded into six-well plates
and maintained in culture for 24 h to start growing in a logarithmic phase. They
are then exposed to the test compound over a range of concentrations. They are
incubated for 6 days to make colonies large enough to count. Colonies are fixed
with methanol, stained with Giemsa solution and counted. If the extract exhibits
a cytotoxic effect on the cells, the IC50 (the concentration inhibiting plating
efficiency to 50%) is calculated and expressed as a percentage of the extract.
4. MTT cytotoxicity test (direct contact test): Test protocol is based on the mea-
surement of the viability of cells via metabolic activity. L929 cells are seeded
into 96-well plates and maintained in culture for 24 h (≈1 doubling period) to
form a semi-confluent monolayer (see Reference [5] for more information on
cell maintenance and culture procedures). They are then exposed to the test com-
pound over a range of concentrations. After 24 h exposure, the formazan forma-
tion is determined for each treatment concentration and compared to that
determined in control cultures. For each treatment the percentage inhibition of
growth is calculated.
5. XTT cytotoxicity test (indirect contact test): This is based on the measurement
of the viability of cells via mitochondrial dehydrogenases. L929 cells are seeded
into 96-well plates and maintained in culture for 24 h (≈1 doubling period) to
form a semi-confluent monolayer (see Reference [5] for more information on
cell maintenance and culture procedures). They are then exposed to the test com-
pound over a range of concentrations. After 24 h exposure, the formazan forma-
tion is determined for each treatment concentration and compared to that
determined in control cultures. For each treatment the percentage inhibition of
growth is calculated.
9 Toxicity Screening
9.2 E
valuation and Testing Within a Risk Management
Process (ISO 10993-1:2009)
To prevent the potential risk arising from the medical devices (from now we called
it as devices) to humans, their biological evaluation is essential. Biological evalua-
tion is done by in vitro and ex vivo tests using animal to identify potential adverse
response.
The primary role of this document is to serve as a framework to plan a biological
evaluation. A secondary role is to utilize scientific advances in our understanding of
172 P. Thangaraju and S. B. Varthya
Primary aim of ISO is protection of humans from any adverse events or potential
adverse events due to medical devices. In this respect, animal studies are conducted
to study the biological effects of medical devices. These tests are conducted in
humanly approach. Animal welfare primarily focuses on minimization of animal
use, minimizing or eliminating the pain and distress and replacement of animal test,
if alternative tests are available. Animal welfare primarily deals with vertebrate non-
human species.
In this part we will be discussing what essential prerequisites for testing medical
devices in animals are.
Essential Requirement for Minimizing or Eliminating Pain and Distress in
Animals Under this all experiments are conducted in accordance with legal provi-
sions of their jurisdiction and ethical provisions. Number of animals in each group
must be based on literature search, data sharing and replacement of animals wher-
ever possible and appropriate test strategy and study design. All these experiments
are conducted in good laboratory care and presence of competent personnel and
expert veterinarian service to alleviate their suffering.
Reuse Reuse of animals is warranted to reduce the cost of animal welfare and
minimize number of animals used, but in such condition reuse must be based on
scientific objective, because pain and distress in the course of experiment may inter-
fere with other test. Whenever animals are reused, it must be well documented.
In this part we will be discussing about test methods for assessment of local effects
by direct contact samples. This test applies for solid and non-absorbable; non-solid,
such as porous materials, liquids, gels, pastes and particulates; and degradable and/
or absorbable, which may be solid or non-solid.
Route of exposure: The test route of exposure shall be the most clinically relevant to
the use of the device, where possible. If an alternative route of exposure is neces-
sary, it shall be justified.
Dosing: Under this test sample is administered either single dose per day or multiple
doses in a day depending on the volume of administration, and test sample is
administered mostly at physiological temperature of animal.
Clinical observation:
Respiratory system: Dyspnoea (abdominal breathing, gasping), apnoea, cyanosis,
tachypnoea and/or discharge through nostrils
Motor activities: Decrease/increase of somnolence, loss of righting, catalepsy,
ataxia, unusual locomotion, prostration, tremors and fasciculation
CNS: Convulsion, reflexes (corneal, righting, myotactic, light, startle reflex).
Ophthalmological: Lacrimation, pupil size (miosis and mydriasis), extraocular
muscles (exophthalmos, ptosis), lens (opacity), iritis, conjunctivitis, chromodac-
ryorrhea, relaxation of nictitating membrane
Cardiovascular signs: Bradycardia, tachycardia, arrhythmia, vasodilation and
vasoconstriction
Gastrointestinal: Soft stool, diarrhoea, emesis, diuresis, rhinorrhoea, etc.
Dermatological signs: Oedema, erythema, etc.
Other systems if required:
Clinical pathology: To analyse the toxic effects of devices, blood test along with
other analytical tests is performed. Sample collection is done as per protocol.
Haematology (clotting potential (PT, APTT), haemoglobin concentration, haemato-
crit, platelet count, red blood cell count, white blood cell count, WBC
differential)
Clinical chemistry (LFT, RFT, lipid profile, blood glucose, serum electrolytes,
immunoglobulin, etc.)
Urine analysis for appearance, bilirubin, glucose, ketones, occult blood, protein,
sediment, specific gravity or osmolality, volume, etc.
Anatomic Pathology Under this gross morphological changes in the intact body
after euthanasia or death, discharge from all opening, and cranial, thoracic and
abdominal cavities. Gross morphology and histopathology of selected organs done
after harvesting adequate tissue.
Acute Systemic Toxicity This information gives about clinical effects on acute
exposure to device. Dosage regimen is established based on this study and mode of
toxic effects seen. Under this study, data regarding adverse clinical signs, body
weight change, gross pathological findings and death (if any) shall be recorded.
Repeated exposure systemic toxicity (subacute, subchronic and chronic sys-
temic toxicity):
1. Health hazards likely to arise from a prolonged exposure
176 P. Thangaraju and S. B. Varthya
Medical devices or their release chemicals may cause irritation of the skin and
mucous membrane or may cause sensitization leading to delayed type of hypersen-
sitivity reaction. To test such reaction, in vitro test, animal studies and human trials
are to be conducted. These tests are performed in case of devices intended as an
implant and externally communicating device.
Under this we follow stepwise approach:
Characterization of test material: In this part physicochemical characterization of
device shall be done. Details are already discussed.
Literature review: Through literature review essential for extracting information on
device or structurally similar components about physicochemical properties, irri-
tation and/or sensitization.
In vitro tests: In silico methods gaining popularity to identify potentially important
reactions and sensitization.
In vivo animal tests: In this test we will be demonstrating the potential irritation and
sensitization using positive controls. To test the sensitization, we use local lymph
node assay in mice, the occluded patch test in guinea pigs or the guinea pig maxi-
mization test (GPMT).
Non-invasive human tests/clinical trials: If test material is found negative in animal
studies, these are taken for further evaluation.
9.7 P
rinciples and Methods for Immunotoxicology Testing
of Medical Devices (ISO 10993-20)
9.8 T
ests for Genotoxicity, Carcinogenicity and Reproductive
Toxicity (ISO 10993-3)
Carcinogenicity Tests To test this, in general a single study for chronic toxicity
and carcinogenicity studies are conducted in case of where potential risk is associ-
ated with device.
Criteria Materials for which the degradation time is greater than 30 days; materi-
als introduced in the body and/or its cavities with a cumulative contact of greater
than 30 days.
178 P. Thangaraju and S. B. Varthya
Test Methods Under this test human safety factor of 100 (100 times to the maxi-
mum dose exposed by human). Dose should be physiologically compatible. And all
tests are based on OECD guidelines.
9.9 T
oxicokinetic Study Design for Degradation Products
and Leachable (ISO 10993-16)
Metabolism and Excretion In this test metabolic cages are to be used to collect
urine and faeces. In case of collecting volatile gases like CO2, in such situation
special devices are to be used.
In this we will be studying devices which are in contact with patients. For medical
devices sterilized by ethylene oxide (EO), it is important to ensure the levels of
residual EO, ethylene chlorohydrin (ECH) and ethylene glycol (EG) and risks to
patients.
Based on duration medical devices are classified into different categories to
avoid more than permissible limit of exposure.
Classification of medical devices is delivered to patients and their permissible
limit for EO and ECH.
Tolerable contact limits for surface contacting devices and implants (mg/sqcm):
Primary aim is to prevent localized irritation. Tolerable contact limit for EO > 10 μg/
cm2 or negligible irritation. For ECH 5 mg/cm2 or negligible irritation. But in
special situations their exposure limit further changes (e.g. EO in intraocular
lenses shall not exceed 0.5 μg EO per lens per day, or 1.25 μg per lens).
Material Composition Materials that contain a source of free chloride ions exhibit
a wide degree of variation in the concentration of ECH formed; therefore a single
device composed of two dissimilar materials may require a representative sample of
both materials to ensure accurate analysis.
Packaging Packaging material based on their packing density and the density of
the shipping container varies the penetration and dissipation of both EO gas and the
other possible residues, which may in turn affect ECH residue levels.
ECH Methods
If devices are classified under more than one category, rigorous testing is performed.
Allowable limits: In general, maximum allowable limit in case of prolonged
exposure and permanent contact are mentioned in the table under heading of 10.1.
10.2 E
stablishment of Allowable Limits for Leachable
Substances (ISO 10993-17)
for this test is limulus amoebocyte lysate (LAL) assay. Sterilization is a mandatory
process before preclinical evaluation of nanomaterials.
Nano-objects have been used as haptens or hapten carriers, which indicates that
they are capable of exerting an adjuvant activity affecting the immune system. For
silver nanoparticles, the effects on the immune system were found to be the most
sensitive parameter of systemic toxicity after intravenous administration for 28 d.
Sensitization To assess local toxicity, various tests, such as the Buehler test (BT),
guinea pig maximization test (GPMT), local lymph node assay (LLNA), human
patch test (HPT) and a modified GPMT (GPMT with surface application), are used.
In vivo tests like the direct peptide reactivity assay (DPRA), the human cell line
activation test (h-CLAT), etc. are used.
Ocular Irritation Test The Bovine Corneal Opacity and Permeability (BCOP) test
method and the Isolated Chicken Eye (ICE) test method.
Oral Mucosa Irritation Test After applying test agents in the oral cavity, gross
and histological examination done and results are evaluated based on severity of
local inflammation from changes.
Penile Irritation Test For acute exposure, note the appearance of the penis in 1 h
after the initial application (e.g. immediately prior to the next application) and sub-
sequent treatments. Also observe at 1, 24 and 48 h of post-application.
For prolonged repeated exposure tests, note the appearance of the penis at first
hour after the initial application and immediately prior to the next application.
Grade the skin surface reactions for erythema accordingly.
Rectal Irritation Test It is indicated in case the material contacts with the rectal
tissue during clinical use. In case a test material showed to be skin/eye irritant, those
with a pH < 2.0 or > 11.5, then it is characterized as rectal irritant; in such scenario
it is not essential to perform rectal irritation tests. A short catheter or cannula is
inserted in the rectum and test solution delivered and observed for changes. This test
should be repeated for 5 days. Observe for appearance of the perineum for signs of
discharge, erythema and irritation. Results are evaluated by macro- and microscopic
appearance of rectal tissue by pathologist.
186 P. Thangaraju and S. B. Varthya
Vaginal irritation test: It is indicated in case the material contacts with the vaginal
tissue during clinical use. In case a test material showed to be skin/eye irritant, those
with a pH < 2.0 or > 11.5, then it is characterized as rectal irritant; in such scenario
it is not essential to perform rectal irritation tests. Healthy young adult female albino
rabbits (n = 3) from a single strain weighing not less than 2 kg shall be used. A short
catheter or cannula is inserted in the vagina and test solution delivered and observed
for changes. This test should be repeated for 5 days. Observe for appearance of the
perineum for signs of discharge, erythema and irritation. Results are evaluated by
macro- and microscopic appearance of rectal tissue by pathologist.
Human Skin Irritation Test At least 30 volunteers shall complete the test, with no
less than one-third of either sex. Apply the test material to intact skin at a suitable
site, e.g. the upper outer arm, by means of an occlusive chamber containing a gauze
pad. The application site shall be the same in all volunteers and shall be recorded.
Generally, the patch shall measure at least 1.8 cm, preferably 2.5 cm in diameter.
The patch shall be held in contact with the skin by means of a suitable non-irritating
dressing, including non-irritating tape, for the duration of the exposure period.
Duration of exposure starts from 15 or 30 min to up to 4 h.
Clinical evaluation is done using observable changes on skin like erythema, dry-
ness and oedema.
In Vitro Tests for Skin Irritation Human skin models can be obtained commer-
cially (e.g. EpiDerm, EPISKIN, Vitrolife-Skin, TESTSKIN, LabCyte EPI-MODEL)
or be developed or constructed in the testing laboratory. The preferred ones are
EpiDerm and EPISKIN tests.
References
ISO 10993-1:2018 Biological evaluation of medical devices Part 1: Evaluation and testing within
a risk management process
ISO 10993-2:2006 Biological evaluation of medical devices Part 2: Animal welfare requirements
ISO 10993-3:2014 Biological evaluation of medical devices Part 3: Tests for genotoxicity, carci-
nogenicity and reproductive toxicity
ISO 10993-4:2017 Biological evaluation of medical devices Part 4: Selection of tests for interac-
tions with blood
ISO 10993-5:2009 Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity.
ISO 10993-6:2016 Biological evaluation of medical devices Part 6: Tests for local effects after
implantation
ISO 10993-7:2008 Biological evaluation of medical devices Part 7: Ethylene oxide sterilization
residuals
ISO 10993-8:2001 Biological evaluation of medical devices Part 8: Selection of reference materi-
als (withdrawn)
ISO 10993: Biological Evaluation of Medical Devices 187
ISO 10993-9:2010 Biological evaluation of medical devices Part 9: Framework for identification
and quantification of potential degradation products
ISO 10993-10:2013 Biological evaluation of medical devices Part 10: Tests for irritation and skin
sensitization
ISO 10993-11:2018 Biological evaluation of medical devices Part 11: Tests for systemic toxicity
ISO 10993-12:2012 Biological evaluation of medical devices Part 12: Sample preparation and
reference materials (available in English only)
ISO 10993-13:2010 Biological evaluation of medical devices Part 13: Identification and quantifi-
cation of degradation products from polymeric medical devices
ISO 10993-14:2009 Biological evaluation of medical devices Part 14: Identification and quantifi-
cation of degradation products from ceramics
ISO 10993-15:2009 Biological evaluation of medical devices Part 15: Identification and quantifi-
cation of degradation products from metals and alloys
ISO 10993-16:2018 Biological evaluation of medical devices Part 16: Toxicokinetic study design
for degradation products and leachables
ISO 10993-17:2009 Biological evaluation of medical devices Part 17: Establishment of allowable
limits for leachable substances
ISO 10993-18:2020 Biological evaluation of medical devices Part 18: Chemical characterization
of medical device materials within a risk management process
ISO/TS 10993-19:2006 Biological evaluation of medical devices Part 19: Physico-chemical, mor-
phological and topographical characterization of materials
ISO/TS 10993-20:2006 Biological evaluation of medical devices Part 20: Principles and methods
for immunotoxicology testing of medical devices
ISO/TR 10993-22:2017 Biological evaluation of medical devices Part 22: Guidance on
nanomaterials