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Biocompatibility Test Methods

The following page describes some of the specific procedures recommended for PBL Learning
biocompatibility testing. This listing does not imply that all procedures are
necessary for any given device, nor does it indicate that these are the only available
Center
tests.
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1. Cytotoxicity (Tissue Culture)
Pharmaceutical
2. Sensitization Assays
and Biotech 
3. Irritation Tests Development
4. Acute Systemic Toxicity
Pharmaceutical
5. Subchronic Toxicity and Biotech
Manufacturing 
6. Genotoxicity
Quality Control
7. Implantation Tests
Medical
8. Hemocompatibility 
Device Testing
9. Carcinogenesis Bioassay
10. Reproductive And Developmental Toxicity GLP and
Device
11. Pharmacokinetics Studies
12. Preclinical Safety Testing
Assessing
13. Histopathology Services 
Biocompatibility

Cytotoxicity (Tissue Culture) Introduction to

Biocompatibility
Cell culture assays are used to assess the biocompatibility of a material or extract Testing
through the use of isolated cells in vitro. These techniques are useful in evaluating
the toxicity or irritancy potential of materials and chemicals. They provide an Biocompatibility
Planning Tool
excellent way to screen materials prior to in vivo tests.
(BioPT) 
Qualitative Cytotoxicity Tests Biocompatibility
There are three qualitative cytotoxicity tests commonly used for medical devices. Test Methods
The Direct Contact procedure is recommended for low density materials, such as
Biocompatibility
contact lens polymers. In this method, a piece of test material is placed directly onto Chemical
cells growing on culture medium. The cells are then incubated. During incubation, Characterization
leachable chemicals in the test material can di use into the culture medium and
contact the cell layer. Reactivity of the test sample is indicated by malformation, USP Class
degeneration and lysis of cells around the test material. Plastics
Tests
The Agar Di usion assay is appropriate for high density materials, such as
elastomeric closures. In this method, a thin layer of nutrient-supplemented agar is Sterilization

placed over the cultured cells. The test material (or an extract of the test material Validations
dried on filter paper) is placed on top of the agar layer, and the cells are incubated. A
Medical
zone of malformed, degenerative or lysed cells under and around the test material Device
indicates cytotoxicity. Sterility
Assurance
The MEM Elution assay uses di erent extracting media and extraction conditions to Tests
test devices according to actual use conditions or to exaggerate those conditions. 
Extracts can be titrated to yield a semi-quantitative measurement of cytotoxicity. Reusable
A er preparation, the extracts are transferred onto a layer of cells and incubated.  Device 
Validations
Following incubation, the cells are examined microscopically for malformation,
degeneration and lysis of the cells. (See All About Extracts section for more
Analytical
information on the selection of extracting media and conditions.) At least one type Methods 
of cytotoxicity test should be performed on each component of any device.

Quantitative Cytotoxicity – MTT Assay

Recent regulatory additions (ANSI/AAMI/ISO 10993-5:2009) on biocompatibility for

devices state that the qualitative cytotoxicity tests (direct contact, mem elution,
agar di usion) are appropriate for screening purposes, but that quantitative
evaluation is preferable.

Annex C of ISO 10993-5:2009 refers to the MTT cytotoxicity assay, which can
accurately quantify as few as 950 cells. The MTT is a colorimetric method that
measures the reduction of yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide by mitochondial succinate dehydrogenase. Because the
cellular reduction is only catalyzed by living cells, it is possible to quantify the
percentage of living cells in a solution.

The MTT can be used to evaluate the cytotoxicity of:

Extractable materials of medical devices

Toxic compounds

Toxins and environmental pollutants

Potential anti-cancer drugs 

 Antibodies to examine growth inhibiting potential

The major advantages of the MTT are its quantitative ability, that it can be done on
either extracts or by direct contact, and that the results are not subject to analyst
interpretation. Additionally, the MTT can be performed on 96-well microplates in a
standard reader (such as a Bio-Tek ELx808) allowing for fast screening of multiple
samples.

However, it should be noted that while the MTT is recommended, the MTT assay
does not discriminate a specific cellular death mechanism – such as apopotosis vs.
induced cell death. Additionally, it may underestimate cellular damage and only
detect death at the last stages of the cellular dying process.

Sensitization Assays
Sensitization studies help to determine whether a material contains chemicals that
cause adverse local or systemic e ects a er repeated or prolonged exposure. These
allergic or hypersensitivity reactions involve immunologic mechanisms. Studies to
determine sensitization potential may be performed using either specific chemicals
from the test material, the test material itself, or most o en, extracts of the test
material. The Materials Biocompatibility Matrix recommends sensitization testing
for all classes of medical devices.

The Guinea Pig Maximization Test (Magnusson-Kligman Method) is recommended

for devices that will have externally communicating or internal contact with the
body or body fluids. In this study the test material is mixed with complete Freund’s
adjuvant (CFA) to enhance the skin sensitization response.

The Closed Patch Test involves multiple topical doses and is recommended for

devices that will contact unbroken skin only.

The Murine Local Lymph Node Assay (LLNA) determinates the quantitative increase

in lymphocytes in response to a sensitizer. If a molecule acts as a skin sensitizer, it
will induce the epidermal Langherhans cells to transport the allergen to the draining
lymph nodes, which in turn causes T-lymphocytes to proliferate and
di erentiate. From an animal welfare perspective, this test is preferable to the Guinea
Pig Maximization Test or the Closed Patch Test.

Irritation Tests
These tests estimate the local irritation potential of devices, materials or extracts,
using sites such as skin or mucous membranes, usually in an animal model. The
route of exposure (skin, eye, mucosa) and duration of contact should be analogous
to the anticipated clinical use of the device, but it is o en prudent to exaggerate
exposure conditions somewhat to establish a margin of safety for patients.

In the Intracutaneous Test, extracts of the test material and blanks are injected
intradermally. The injection sites are scored for erythema and edema (redness and
swelling). This procedure is recommended for devices that will have externally
communicating or internal contact with the body or body fluids. It reliably detects
the potential for local irritation due to chemicals that may be extracted from a
biomaterial.

The Primary Skin Irritation test should be considered for topical devices that have
external contact with intact or breached skin. In this procedure, the test material or
an extract is applied directly to intact and abraded sites on the skin of a rabbit. A er
a 24-hour exposure, the material is removed and the sites are scored for erythema
and edema.

Mucous Membrane Irritation Tests are recommended for devices that will have
externally communicating contact with intact natural channels or tissues. These
studies o en use extracts rather than the material itself. Some common procedures
include vaginal, cheek pouch and eye irritation studies. (See All About
Extracts section for more information on extracts.)

Acute Systemic Toxicity

By using extracts of the device or device material, the Acute Systemic Toxicity test
detects leachables that produce systemic (as opposed to local) toxic e ects. The
extracts of the test material and negative control blanks are injected into mice
(intravenously or intraperitoneally, depending on the extracting media). The mice
are observed for toxic signs just a er injection and at four other time points. The
Materials Biocompatibility Matrix recommends this test for all blood contact
devices. It may also be appropriate for any other device that contacts internal
tissues.

The Material Mediated Pyrogen test evaluates the potential of a material to cause a

pyrogenic response, or fever, when introduced into the blood. Lot release testing for
pyrogenicity is done in vitro using the bacterial endotoxin (LAL) test. It must be
validated for each device or material. However, for assessing biocompatibility, the
rabbit pyrogen test is preferred. The rabbit test, in addition to detecting bacterial
endotoxins, is sensitive to material-mediated pyrogens that may be found in test
materials or extracts.

Subchronic Toxicity
Tests for subchronic toxicity are used to determine potentially harmful e ects from
longer-term or multiple exposures to test materials and/or extracts during a period
of up to 10% of the total lifespan of the test animal (e.g. up to 90 days in rats). Actual
use conditions of a medical device need to be taken into account when selecting an 
animal model for subchronic toxicity. Appropriate animal models are determined on
a case-by-case basis.

Pacific BioLabs o ers two standard protocols for subchronic testing that are
appropriate for many devices. Both are done in mice. One
uses intraperitoneal administration of an extract of the device or device
material. The other uses an intravenous route of administration. Implant tests are
o en performed for di erent durations appropriate to assess subchronic toxicity of
devices and device materials.

Subchronic tests are required for all permanent devices and should be considered
for those with prolonged contact with internal tissues.

Genotoxicity
Genotoxicity evaluations use a set of in vitro and in vivo tests to detect mutagens,
substances that can directly or indirectly induce genetic damage directly through a
variety of mechanisms. This damage can occur in either somatic or germline cells,
increasing the risk of cancer or inheritable defects. A strong correlation exists
between mutagenicity and carcinogenicity.

Genotoxic e ects fall into one of three categories: point mutations along a strand of
DNA, damage to the overall structure of the DNA, or damage to the structure of the
chromosome (which contains the DNA). A variety of tests have been developed to
determine if damage has occurred at any of these levels. These assays complement
one another and are performed as a battery.   

The most common test for mutagenicity, the Ames test, detects point mutations by
employing several strains of the bacteria Salmonella typhimurium, which have been
selected for their sensitivity to mutagens. The Mouse Lymphoma and the HGPRT
assays are common procedures using mammalian cells to detect point mutations.
The Mouse Lymphoma assay is also able to detect clastogenic lesions in genes
(chromosome damage). Assays for DNA damage and repair include both in
vitro and in vivo Unscheduled DNA Synthesis (UDS). Cytogenetic assays allow direct
observation of chromosome damage. There are both in vitro and in vivo methods,
including the Chromosomal Aberration and the Mouse Micronucleus assays. 

ISO 10993-1 specifies an assessment of genotoxic potential for permanent devices

and for those with prolonged contact (>24 hours) with internal tissues and blood.
Extracorporeal devices with limited contact (<24 hours) may require a genotoxicity
evaluation. Generally, devices with long-tem exposure require an Ames test and
two in vivomethods, usually the Chromosomal Aberration and Mouse Micronucleus
tests. Devices with less critical body contact may be able to be tested using only the
Ames test.


When selecting a battery of genotoxicity tests, you should consider the
requirements of the specific regulatory agency where your submission will be
made. Because of the high cost of genotoxicity testing, Pacific BioLabs strongly
recommends that you consult your FDA reviewer before you authorize testing.

Implantation Tests
Implant studies are used to determine the biocompatibility of medical devices or
biomaterials that directly contact living tissue other than skin (e.g. sutures, surgical
ligating clips, implantable devices, etc.). These tests can evaluate devices, which, in
clinical use, are intended to be implanted for either short-term or long-term
periods.  Implantation techniques may be used to evaluate both absorbable and
non-absorbable materials. To provide a reasonable assessment of safety, the
implant study should closely approximate the intended clinical use.

The dynamics of biochemical exchange and cellular and immunologic responses

may be assessed in implantation studies, especially through the use of
histopathology. Histopathological analysis of implant sites greatly increases the
amount of information obtained from these studies. 

Hemocompatibility
Materials used in blood contacting devices (e.g. intravenous catheters, hemodialysis
sets, blood transfusion sets, vascular prostheses) must be assessed for blood
compatibility to establish their safety. In practice, all materials are to some degree
incompatible with blood because they can either disrupt the blood cells (hemolysis)
or activate the coagulation pathways (thrombogenicity) and/or the complement
system.

The hemolysis assay is recommended for all devices or device materials except

those which contact only intact skin or mucous membranes.  This test measures the
damage to red blood cells when they are exposed to materials or their extracts, and
compares it to positive and negative controls. 

Coagulation assays measure the e ect of the test article on human blood

coagulation time. They are recommended for all devices with blood contact.
The Prothrombin Time Assay (PT) is a general screening test for the detection of
coagulation abnormalities in the extrinsic pathway.
The Partial Thromboplastin Time Assay (PTT) detects coagulation abnormalities in
the intrinsic pathway.

The most common test for thrombogenicity is the in vivo method. For devices

unsuited to this test method, ISO 10993-4 requires tests in each of four categories:
coagulation, platelets, hematology, and complement system.

Complement activation testing is recommended for implant devices that contact
circulatory blood. This in vitro assay measures complement activation in human
plasma as a result of exposure of the plasma to the test article or an extract. The
measure of complement actuation indicates whether a test article is capable of
inducing a complement-induced inflammatory immune response in humans.

Other blood compatibility tests and specific in vivo studies may be required to
complete the assessment of material-blood interactions, especially to meet ISO
requirements. 

DEVICES OR COMPONENTS WHICH CONTACT CIRCULATING BLOOD AND THE

CATEGORIES OF APPROPRIATE TESTING

External Communicating Devices

Test Category

Device Examples
Complement
Thrombosis Coagulation Platelets Hematology
System

Atherectomy devices xa

Blood monitors x xa

Blood storage and

administration equipment,
x x xa
Blood collection devices,
Extension sets

Extracorporeal membrane
oxygenator systems
Haemodialysis/haemofiltration
x x x x x
equipment,
Percutaneous circulatory
support devices

Catheters, guidewires,
intravascular endoscopes,
Intravascular ultrasound, laser
x x xa
systems,
Retrograde coronary perfusion
catheters.

Cell savers x x xa

Devices for absorption of x x x x 

specific substances from blood
 Donor and therapeutic
x x x x
apheresis equipment

a- Hemolysis testing only

Implant Devices

Test Category
Device
examples Complement
Thrombosis Coagulation Platelets Hematology
System
Annuloplasty x xa
rings,
mechanical
heart valves
Intra-aortic
balloon x x x x x
pumps
Total artificial x x
hearts,
ventricular-
assist devices
Embolization xa
devices
Endovascular x xa
gra s
Implantable x xa
defibrillators
and
cardioverters
Pacemaker x xa
leads
Leukocyte x x xa
removal filter
Prosthetic
(synthetic)
vascular gra s
and patches, x xa
including
arteriovenous
shunts

Stents x xa
 Tissue heart x xa
valves
Tissue
vascular gra s
and patches, x xa
including
arteriovenous
shunts
Vena cava x xa
filters

a – Hemolysis testing only

Carcinogenesis Bioassay
These assays are used to determine the tumorigenic potential of test materials
and/or extracts from either a single or multiple exposures, over a period consisting
of the total lifespan of the test system (e.g. two years for rat, 18 months for mouse,
or seven years for dog).

Carcinogenicity testing of devices is expensive, highly problematic, and

controversial. Manufacturers can almost always negotiate an alternative to full scale
carcinogenicity testing of their devices.

Reproductive And Developmental

Toxicity
These studies evaluate the potential e ects of test materials and/or extracts on
fertility, reproductive function, and prenatal and early postnatal development. They
are o en required for devices with permanent contact with internal tissues.

Pharmacokinetics
Pharmacokinetic or ADME (Absorption/Distribution/Metabolism/Excretion) studies
are used to investigate the metabolic processes of absorption, distribution,
biotransformation, and elimination of toxic leachables and potential degradation
products from test materials and/or extracts. They are especially appropriate for
bioabsorbable materials or for drug/device combinations. Our toxicology team is

happy to work with you in setting up the appropriate PK or ADME study for your
product. 

Preclinical Safety Testing

The objectives of preclinical safety studies are to define pharmacological and
toxicological e ects not only prior to initiation of human studies but throughout
clinical development. Both in vitro and in vivo studies can contribute to this
characterization. Pacific BioLabs has extensive experience in designing and running
successful preclinical safety studies. 

Histopathology Services
Implant studies are o en the most direct evaluation of device biocompatibility. The
test material is placed in direct contact with living tissue. A er an appropriate
period, the implant site is recovered and examined microscopically for tissue
reaction. The histopathologist can detect and describe many types of tissue and
immune system reactions.

Similarly, in subchronic and chronic studies, various organs and tissues are
harvested at necropsy and evaluated microscopically for toxic e ects. Many of these
studies also call for clinical chemistry analysis of specimens or serum samples form
the test animals.

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