BIOCOMPATIBILITY TESTING

Medical devices and their component materials may leach compounds or have surface
characteristics that may produce undesirable effects when used clinically. The selection and
evaluation of materials and devices intended for use in humans requires a structured program of
assessment to establish biocompatibility and safety. Current regulations, whether in accordance
with the U.S. Food and Drug Administration (FDA), the International Organization for
Standardization (ISO), or the Japanese Ministry of Health and Welfare (JMHW), require that
manufacturers conduct adequate safety testing of their finished devices through pre-clinical and
clinical phases as part of the regulatory clearance process.

The ISO, JMHW and FDA guidelines provide a general testing framework to aid manufacturers in
the assessment of device biocompatibility. The number and type of specific safety tests required
to assess product safety and compliance are dependent on the individual characteristics of the
device, its component materials, and its intended clinical use.

Within the general safety testing framework, it remains the responsibility of the device manufac-
turer to select and justify the specific tests most appropriate for the establishment of product
safety and compliance with ISO, JMHW and FDA requirements. It is recommended that testing
be performed to comply with GLP regulations. WuXi AppTec, with many years of experience in
biocompatibility testing, provides exceptional expertise to assist medical device manufacturers in
designing thorough, well-constructed testing programs to satisfy regulatory requirements.

NOTE: All samples for biocompatibility testing should be sent to WuXi AppTec’s St. Paul facility.
As applicable, samples should be submitted after exposure to the Sponsor’s sterilization process.

IN THIS SECTION

• Cytotoxicity Testing
Agarose Overlay
MEM Elution
Growth Inhibition Assay
Direct Contact Cytotoxicity
Cytotoxicity Testing To Meet JMHW Regulations

• Genotoxicology / Mutagenicity Testing

Bacterial Reverse Mutation Tests
In Vitro Chromosomal Aberration Assay
In Vitro Chromosomal Aberration Assay in CHO Cells (to meet JMHW regulations)
In Vitro Mouse Lymphoma Assay
In Vivo Mouse Micronucleus Screen
In Vivo Mouse Micronucleus Test

• Hemocompatibility Testing
NIH and ASTM Hemolysis Tests
Complement Activation
Partial Thromboplastin Time
Platelet and Leukocyte Counts
Thrombosis (In Vivo)

[CONTINUED NEXT PAGE]

BIOCOMPATIBILITY B-1
 IN THIS SECTION
[CONTINUED]

• Implantation Testing
USP / ISO Intramuscular Implantation
ISO Subcutaneous Implantation
Intramuscular Implant Screen Test
Long-Term Implant Test in Rabbits With Additional Chronic Data Collection
Intramuscular or Subcutaneous Implantation Screen Test (Custom)

• Irritation/Intracutaneous Reactivity Testing

USP / ISO Intracutaneous Irritation Test
ISO Vaginal Mucosal Irritation Test
Ocular Irritation Test [Topical]
Intraocular Irritation Test
ISO Primary Skin Irritation Test

• Pyrogenicity Testing (In Vivo)

USP Rabbit Pyrogen Test
ISO Rabbit Pyrogen – Materials Mediated

• Sensitization Testing
Murine Local Lymph Node Assay (LLNA)
Guinea Pig Maximization for Sensitization [Magnusson-Kligman]
ISO Maximization Sensitization Test
Repeated Patch Dermal Sensitization Test

• Subacute Toxicity Testing

14-day Subacute Toxicity Test

• Systemic (Acute) Toxicity Testing

Acute Systemic Toxicity Test (USP and ISO)

• Chronic Toxicity and Carcinogenicity Testing

• Finished Product Release Testing

USP Safety Test in Mice

For USP Physicochemical testing, see the “Chemistry” section of this catalog.

Additional test methods are available. Contact your WuXi AppTec Account Manager for more information.

REFERENCE INFORMATION Pages B-24 – B-28

• Biocompatibility Testing Flow Chart

• Testing Requirements Under GLP Regulations
• Required Biocompatibility Tests for Classifications of Plastics (USP)
• Device Categories
• Initial Evaluation Tests for Consideration [Chart based on ISO 1993-1 and FDA G95-1 Guidelines]
• “Quick Guide” to Sample Requirements for Biocompatibility Tests [Chart]

B-2 BIOCOMPATIBILITY
 CYTOTOXICITY TESTING

These tests involve the exposure of substances extracted from test material to one of two cell culture
lines. Cell cultures are extremely sensitive to minute quantities of leachable chemicals and readily
display characteristic signs of toxicity in the presence of potentially harmful leachables. The tests
are frequently used during product planning stages to qualify the use of a material and as a periodic
check for routinely used materials to ensure that no shift in quality has occurred. Cytotoxicity in vitro
testing is also required in testing the biocompatibility of materials. Typical testing programs will
utilize the ISO test method to meet international regulatory requirements. The USP test method is
performed to meet the FDA’s U.S. regulatory requirements. The screening test method is performed
to characterize materials or to evaluate new materials against established ones.

AGAROSE OVERLAY
SAMPLE REQUIREMENTS 1 mL liquid (sterile) • 3 samples of 1 cm2 solid (sterile)
TURNAROUND TIME 7 days (non-GLP) • 14 days (GLP)

L-929 mouse fibroblast cells are overlayed with a permeable agar film. A solid 140150
sample or liquid saturated disc is then placed in triplicate containers on the agar
surface. Cells are examined at 24 hours for signs of toxicity. (ISO) Agarose Overlay – L-929
Mouse Fibroblast Cells

L-929 mouse fibroblast cells are overlayed with a permeable agar film. A solid 140100
sample or liquid saturated disc is then placed in duplicate containers on the agar
surface. Cells are examined at 24 hours for signs of toxicity. (USP) Agarose Overlay – L-929
Mouse Fibroblast Cells

L-929 mouse fibroblast cells are overlayed with a permeable agar film. A solid 140000
sample or liquid saturated disc is then placed in one single container on the agar
surface. Cells are examined at 24 hours for signs of toxicity. (Screening) Agarose Overlay –
L-929 Mouse Fibroblast Cells

MRC-5 human embryonic lung cells are overlayed with a permeable agar film. A 140220
solid sample or liquid saturated disc is then placed in triplicate containers on the
agar surface. Cells are examined at 24 hours for signs of toxicity. (ISO) Agarose Overlay – MRC-5
Human Embryonic Lung Cells

MRC-5 human embryonic lung cells are overlayed with a permeable agar film. A 140200
solid sample or liquid saturated disc is then placed in duplicate containers on the
agar surface. Cells are examined at 24 hours for signs of toxicity. (USP) Agarose Overlay – MRC-5
Human Embryonic Lung Cells

MRC-5 human embryonic lung cells are overlayed with a permeable agar film. A 140175
solid sample or liquid saturated disc is then placed in one single container on the
agar surface. Cells are examined at 24 hours for signs of toxicity. (Screening) Agarose Overlay –
MRC-5 Human Embryonic Lung
Cells

BIOCOMPATIBILITY B-3
 CYTOTOXICITY TESTING

MEM ELUTION

SAMPLE REQUIREMENTS All samples should be sterile and cut into

5 x 0.3 cm sizes with total surface area as shown.

By Thickness By Weight By Weight

< 0.5mm thick >0.5mm thick (low density material)
30 cm2 15 cm2 1g 0.5 g

Tests are set up on Mondays and Thursdays.

Samples should arrive at least one day prior to testing (Friday and Wednesday).
Samples will not be set up on holidays.

140320 Solid materials are extracted in cell culture medium and the extracts are then
placed in triplicate containers of L-929 mouse fibroblast cells. Cells are
(ISO) MEM Elution – examined at 24, 48 and 72 hours for signs of toxicity.
L-929 Mouse Fibroblast Cells TURNAROUND TIME 7 days (non-GLP) • 21 days (GLP)

140300 Solid materials are extracted in cell culture medium and the extracts are then
placed in duplicate containers of L-929 mouse fibroblast cells. Cells are
(USP) MEM Elution – examined at 24 and 48 hours for signs of toxicity.
L-929 Mouse Fibroblast Cells
TURNAROUND TIME 6 days (non-GLP) • 20 days (GLP)

140270 Solid materials are extracted in cell culture medium and the extracts are then
placed in a single container of L-929 mouse fibroblast cells. Cells are examined
(Screening) MEM Elution – at 24 hours for signs of toxicity.
L-929 Mouse Fibroblast Cells
TURNAROUND TIME 5 days (non-GLP) • 19 days (GLP)

140350 When a test material is shown to be toxic by the MEM elution test, four 2-fold
dilutions are made to determine the toxic endpoint. Performance of the test will
MEM Endpoint Dilution – result in an estimation of the relative "strength" of the cytotoxic substance in the
material.
L-929 Mouse Fibroblast Cells
TURNAROUND TIME 7 days (non-GLP) • 21 days (GLP)

B-4 BIOCOMPATIBILITY
 CYTOTOXICITY TESTING

Solid materials are extracted in cell culture medium and the extracts are then 140420
placed in triplicate containers of MRC-5 human embryonic lung cells. Cells are
examined at 24, 48 and 72 hours for signs of toxicity. (ISO) MEM Elution –
TURNAROUND TIME 7 days (non-GLP) • 21 days (GLP) MRC-5 Human Embryonic Lung
Cells

Solid materials are extracted in cell culture medium and the extracts are then 140400
placed in duplicate containers of MRC-5 human embryonic lung cells. Cells are
examined at 24 and 48 hours for signs of toxicity. (USP) MEM Elution –
TURNAROUND TIME 6 days (non-GLP) • 20 days (GLP) MRC-5 Human Embryonic Lung
Cells

Solid materials are extracted in cell culture medium and the extracts are then 140370
placed in one container of MRC-5 human embryonic lung cells. Cells are examined
at 24 hours for signs of toxicity.
(Screening) MEM Elution –
TURNAROUND TIME 5 days (non-GLP) • 19 days (GLP) MRC-5 Human Embryonic Lung
Cells

When a test material is shown to be toxic by the MEM elution test, four 2-fold 140450
dilutions are made to determine the toxic endpoint. Performance of the test will
result in an estimation of the relative "strength" of the cytotoxic substance in the MEM Endpoint Dilution –
material.
MRC-5 Human Embryonic Lung
TURNAROUND TIME 7 days (non-GLP) • 21 days (GLP) Cells

GROWTH INHIBITION ASSAY

The purpose of the test is to evaluate the cytotoxic potential of test articles or test 140500
article extracts according to ISO method (9363-1:1994(E)). Solid materials are
extracted in cell culture medium and a series of dilutions of the test article extract Growth Inhibition Assay
are placed into triplicate containers of L-929 mouse fibroblast cells for 72 hours.
The protein levels of all samples will be determined by using the Lowry protein (ISO 9363)
determination method. The percentage of growth inhibition will then be determined.

SAMPLE REQUIREMENTS Sample requirements are the same as for MEM tests.

TURNAROUND TIME 7 days (non-GLP) • 21 days (GLP)

(Test set-up schedule is the same as for MEM tests.)

BIOCOMPATIBILITY B-5
 CYTOTOXICITY TESTING

DIRECT CONTACT CYTOTOXICITY

140250 A sample is placed in direct contact with L-929 mouse fibroblast cells. Cells are
examined at 24 hours for signs of toxicity.
Direct Contact Cytotoxicity –
SAMPLE REQUIREMENTS 3 samples of 1 cm2 solid (sterile)
L-929 Mouse Fibroblast Cells
TURNAROUND TIME 4 days (non-GLP) • 18 days (GLP)

140260 A sample is placed in direct contact with MRC-5 human embryonic lung cells.
Cells are examined at 24 hours for signs of toxicity.
Direct Contact Cytotoxicity –
SAMPLE REQUIREMENTS 3 samples of 1 cm2 solid (sterile)
MRC-5 Human Embryonic
Lung Cells TURNAROUND TIME 4 days (non-GLP) • 18 days (GLP)

CYTOTOXICITY TESTING TO MEET JMHW regulations

140470 This test evaluates the cytotoxic response of L-929 mouse fibroblast cell line
when exposed to the extract of the materials under test. It is designed to utilize
Extract Colony Assay the sensitivity typical of low cell density cultures to determine material or device
cell toxicity.
Cytotoxicity Test
2
SAMPLE REQUIREMENTS 2 g or 120 cm
TURNAROUND TIME 31 days (non-GLP) • 38 days (GLP)

B-6 BIOCOMPATIBILITY
 GENOTOXICOLOGY TESTING

Genotoxicology (mutagenicity) tests evaluate the ability of a material to cause mutation or gross
chromosomal damage. Any materials intended for implantation or long term exposure should be
evaluated for mutagenic properties. Unpolymerized materials, additives, trace monomers or oligomers
and biodegradative products can all be potential mutagens.

The International Organization for Standardization (ISO) 10993-3 outlines tests for genotoxicity,
carcinogenicity and reproductive toxicity. The ISO guidelines for genetic toxicity testing require three
tests: one for gene mutation (Bacterial Mutagenicity Test), one for chromosomal aberrations
(Chromosomal Aberration Assay) and one for DNA effects (Mouse Lymphoma Assay).

The FDA also requires three tests. The Bacterial Reverse Mutation and the in vitro Mouse Lymphoma
tests are the same as suggested by ISO. Currently, for the third test, some within the FDA are
recommending an in vivo test, such as the Mouse Micronucleus test. (See Page B-10.)

Extracts are prepared using solutions that will extract both hydrophilic (polar) and lipophilic (non-polar)
compounds which may be present in device materials. Generally, medical devices require the use of
one dose (the undiluted extract) for regulatory submission, unless significant toxicity is anticipated.

In addition to the tests listed on the following pages, other genotoxicology tests are available.
For more information, contact your WuXi AppTec Account Manager.

Sample Requirements for Genotoxicology Testing

All material samples should be cut into 5 x 0.3 cm sizes with total surface area as shown.
Note: For ISO, sample preparation complies with ISO10993-12.

EXTRACT OPTIONS: Polar Extracts (e.g., Normal Saline)

Non-polar Extracts (e.g., DMSO or Cottonseed Oil)

When ordering a test, specify one set of extraction conditions:

121°C/1hour 70°C/24 hours 50°C/72 hours 37°C/72 hours
[NOTE: 50°C/72 hours is recommended for genotoxicology testing.]

SAMPLE REQUIREMENTS FOR IN VITRO TESTING (PER EXTRACT)

By Thickness By Weight By Weight
(low density material)
<0.5mm thick >0.5mm thick
60 cm2 30 cm2 2g 1g

SAMPLE REQUIREMENTS FOR IN VIVO TESTING (PER EXTRACT)

By Thickness By Weight By Weight
(low density material)
<0.5mm thick >0.5mm thick
120 cm2 60 cm2 4g 2g

For testing that meets Japanese regulatory requirements, call laboratory regarding sample size.

BIOCOMPATIBILITY B-7
 GENOTOXICOLOGY TESTING

BACTERIAL REVERSE MUTATION TESTS

190800 5 S. typhimurium The Ames Mutagenicity test is performed on extracts of solid materials. To
190802 4 S. typhimurium perform this test, five tester strains of bacteria (five Salmonella typhimurium or
+ 1 E. coli four Salmonella typhimurium and one Escherichia coli), in which specific
mutations can be induced, are exposed to the test material and to a number of
Ames Mutagenicity Screen – positive controls. This test can only be used for screening purposes.
Saline (SCI) Extract TURNAROUND TIME 24 days (non-GLP) • 31 days (GLP)

190860 5 S. typhimurium The ISO Bacterial Reverse Mutation test is performed according to ISO 10993-3
190862 4 S. typhimurium using OECD test method 471. Tester strains of bacteria (five Salmonella
+ 1 E. coli typhimurium or four Salmonella typhimurium and one Escherichia coli or five
Salmonella typhimurium and one Escherichia coli) are exposed to extracts of the
190863 5 S. typhimurium
+ 1 E. coli
test material in the presence and absence of an exogenous metabolic activation
system. One dose level of the test article per extract and both positive and
negative controls are used.
(ISO) Bacterial Mutagenicity
Test – Saline (SCI) and Dimethyl- TURNAROUND TIME 24 days (non-GLP) • 31 days (GLP)
sulfoxide (DMSO) Extracts

190760 5 S. typhimurium This Bacterial Reverse Mutation test is performed according to ISO 10993-3 using
190762 4 S. typhimurium OECD test method 471. Tester strains of bacteria (five Salmonella typhimurium or
+ 1 E. coli four Salmonella typhimurium and one Escherichia coli or five Salmonella
typhimurium and one Escherichia coli) are exposed to an extract of the test
190763 5 S. typhimurium
+ 1 E. coli
material in the presence and absence of an exogenous metabolic activation
system. One dose level of the test article extract and both positive and negative
controls are used.
Bacterial Mutagenicity Test –
One Extract TURNAROUND TIME 24 days (non-GLP) • 31 days (GLP)

190812 The purpose of this study is to evaluate the mutagenic potential of the test article
(or its metabolites) by measuring its ability to induce back mutations at selected
Bacterial Reverse Mutation Assay loci of four strains of Salmonella typhimurium and one strain of Escherichia coli in
(To Meet JMHW Regulations) the presence and absence of microsomal enzymes. For the assay, the test article
is extracted according to specialized procedures involving the use of solvents and
evaporation in order to obtain a residue for testing. When residues are obtained,
they are tested at five dose levels along with appropriate vehicle and positive
controls. Alternate methodologies are outlined in the study protocol for test articles
that do not yield a residue.
TURNAROUND TIME 28 days (non-GLP) • 45 days (GLP)

190810 Test materials yielding positive results on the Ames Mutagenicity plate
incorporation test should be repeated with the strains that showed positive results.
Ames Mutagenicity Confirmation The confirmation test is run with appropriate positive and negative controls and in
the presence and absence of metabolic activation.
Test
TURNAROUND TIME 24 days (non-GLP) • 31 days (GLP)

B-8 BIOCOMPATIBILITY
 GENOTOXICOLOGY TESTING

IN VITRO MAMMALIAN CELL TESTS

Mammalian cells are exposed to the test material or extract in the presence and 190820 1 extract
absence of metabolic activation and blocked in metaphase using a spindle poison.
Visualization of chromosomes is performed microscopically after hypotonic 190828 2 extracts
swelling, fixation and staining. Test code 190828 (two extracts) satisfies the ISO 190823 Full regulatory,
requirement for assessing in vitro chromosomal aberrations. 4 dose levels

TURNAROUND TIME 46 days (non-GLP) • 53 days (GLP) In Vitro Chromosomal

Aberration Assay

Mammalian cells are exposed to the test material or extract and blocked in 190827
metaphase using a spindle poison. Visualization of chromosomes is performed
microscopically after hypotonic swelling, fixation and staining the cells. For the
assay, the test article is extracted according to specialized procedures involving In Vitro Chromosomal
the use of solvents and evaporation in order to obtain a residue for testing. When Aberration Analysis in
residues are obtained, they are tested at five dose levels along with appropriate Chinese Hamster Ovary
vehicle and positive controls. Alternate methodologies are outlined in the study
protocol for test articles that do not yield a residue. (CHO) Cells
(To Meet JMHW Regulations)
TURNAROUND TIME 60 days (non-GLP) • 67 days (GLP)

Mouse Lymphoma cells are used to determine whether a test material has the 190851 1 extract
capacity to induce either point mutations or clastogenic (chromosomal breakage)
events in a cultured mammalian cell line. Mutants can be selected and mutant 190850 2 extracts
frequencies derived by including a thymidine analogue (trifluorothymidine, TFT) in 190855 Full regulatory,
the culture medium of cells after exposure to the test material. Test code 190850 4 dose levels
(two extracts) satisfies the ISO requirement for assessing in vitro DNA genotoxic
effects.
In Vitro Mouse Lymphoma
TURNAROUND TIME 34 days (non-GLP) • 41 days (GLP) Assay

BIOCOMPATIBILITY B-9
 GENOTOXICOLOGY TESTING

IN VIVO TESTS

190700 1 extract The mammalian in vivo micronucleus test is used for the detection of damage
induced to the chromosomes or the mitotic apparatus of erythroblasts by analysis
of erythrocytes sampled from bone marrow of mice. For this assay, male mice
In Vivo Mouse are injected with 1 dose level of the test article extract or positive or negative
control. Cells are collected at 24 and 48 hours after dosing.
Micronucleus Screen
TURNAROUND TIME 48 days (non-GLP) • 55 days (GLP)

190852 1 extract The mammalian in vivo micronucleus test is used for the detection of damage
induced to the chromosomes or mitotic apparatus of erythroblasts by analysis of
190853 2 extracts erythrocytes from bone marrow of treated mice. In this assay, male and female mice
190854 Full regulatory, are injected with 1 dose level of the test article extract or positive or negative control.
4 dose levels Cells are collected for analysis at 24 and 48 hours after dosing.
TURNAROUND TIME 48 days (non-GLP) • 55 days (GLP)
In Vivo Mouse
Micronucleus Test

B-10 BIOCOMPATIBILITY
 HEMOCOMPATIBILITY TESTING

HEMOLYSIS TESTING
An important measure of hemocompatibility is the hemolysis test, which measures the ability of a
material or material extract to cause red blood cells to rupture. Hemolysis testing should be
performed on all materials directly contacting the bloodstream, or any materials used to form a
fluid conduit to the bloodstream. The following tests are derived from well-established
studies/standards and are useful in evaluating a variety of materials intended to contact blood or
fluids entering the circulatory system. While both the NIH and ASTM methods are accepted by
regulatory agencies, the ASTM method is the preferred method for ISO-compliant testing.

This test is intended for materials that directly contact the bloodstream or 150100 NIH Method
compromised tissues. It is performed in triplicate and uses rabbit blood in direct
contact with the test material. The degree of hemolysis is measured 150300 ASTM Method
spectrophotometrically.

NIH Method Hemolysis Test –

Direct Contact Method
SAMPLE REQUIREMENTS 3 x 2 g or 3 x 0.2 g (light weight)
(No comparison product required)

TURNAROUND TIME 2 days (non-GLP) • 21 days (GLP)

Tests set up on Tuesday.

ASTM Method
2
SAMPLE REQUIREMENTS 3 x 12 cm or 3 x 2 g or 3 x 0.2 g (light weight)
(No comparison product required)

TURNAROUND TIME 2 days (non-GLP) • 21 days (GLP)

Tests set up on Thursday.

This test is intended for materials through which fluids pass before entry into the 150200 NIH Method
body. It is performed in triplicate and uses saline to extract leachable substances.
150500 ASTM Method
The material is removed and rabbit blood is added to the extract. The degree of
hemolysis is measured spectrophotometrically.

Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours

37°C/72 hours 50°C/72 hours Hemolysis Test –
Extract Method
NIH Method

SAMPLE REQUIREMENTS 3 x 2 g or 3 x 0.2 g (light weight)

(No comparison product required)

TURNAROUND TIME 2 days (non-GLP) • 21 days (GLP)

Tests set up on Tuesday.

ASTM Method
2
SAMPLE REQUIREMENTS 3 x 12 cm or 3 x 2 g or 3 x 0.2 g (light weight)
(No comparison product required)

TURNAROUND TIME 2 days (non-GLP) • 21 days (GLP)

Tests set up on Thursday.

HEMOCOMPATIBILITY TESTING CONTINUED NEXT PAGE

BIOCOMPATIBILITY B-11
 HEMOCOMPATIBILITY TESTING

The following tests are offered for evaluating the interaction of biomaterials, polymers and medical
devices with circulating blood. The assays are designed to be compliant with ISO 10993-4.
Additional studies are available. For more information, contact your Account Manager.
Note: Testing a sponsor-supplied comparison product is recommended when requesting these assays to aid in
clarifying interpretation of test results.

150600 C3a Assay The activation of complement resulting from the use of a medical device has been
associated with many adverse clinical findings. An enzyme immunoassay is used
150620 SC5b-9 Assay to screen for complement component(s) in human serum that has been incubated
150630 C3a and SC5b-9 with the test article. Elevated levels of complement components C3a and SC5b-9
indicate activation of the complement system. Both the C3a and SC5b-9 assays
Complement Activation are available.

––––––––––––––––––– SAMPLE REQUIREMENTS PER ASSAY:

2 2
6 cm (0.5 mm thick) or 3 cm (0.5 mm thick) or 0.2 g
An additional fee will apply for test articles with a surface area greater than required.
150600 C3a Assay
150620 SC5b-9 Assay TURNAROUND TIME 14 days (non-GLP) • 21 days (GLP)
150630 C3a and SC5b-9

Complement Activation Including

Sponsor-Supplied Comparison
Product

155200 The PTT assay is a general screening test for the detection of coagulation
abnormalities in the intrinsic coagulation pathway. The test determines the time it
Partial Thromboplastin Time takes citrated human plasma to form a clot when it is exposed first to the test
material, then to calcium chloride and finally to partial thromboplastin. (Partial
(PTT) thromboplastin is a phospholipid suspension extracted from rabbit brain cephalin.)
Test results report the “partial thromboplastin time,” which is the time it takes the
–––––––––––––––––– recalcified citrated plasma to clot once the partial thromboplastin has been added.
Shortening of the PTT following contact with a material under standardized
155205 conditions indicates activation of the contact phase of blood coagulation.
2
SAMPLE REQUIREMENTS 3 x 4 cm
Partial Thromboplastin Time
(PTT) Including Sponsor- TURNAROUND TIME 14 days (non-GLP) • 21 days (GLP)
Supplied Comparison Product

155600 Platelet and leukocyte counts are evaluated before and after exposure to the test
material in human blood. Counts are evaluated for changes that may indicate
activation, adhesion, aggregation, or lysis.
Platelet and Leukocyte Counts
2
SAMPLE REQUIREMENTS 3 x 12 cm
––––––––––––––––––
TURNAROUND TIME 14 days (non-GLP) • 21 days (GLP)
155605

Platelet and Leukocyte Counts

Including Sponsor-Supplied
Comparison Product

B-12 BIOCOMPATIBILITY
 HEMOCOMPATIBILITY TESTING

Test article (e.g., tubing or catheter) is implanted in the jugular veins of two (2) dogs. 800520
Usually the test article is implanted in the jugular vein on one side and a sponsor-
supplied comparative control article on the contralateral side for up to 3 days. The Thrombosis (In Vivo)
test article and implant sites are removed and examined for the presence of thrombi,
and the vein is examined for patency (occlusion). These observations are
augmented with photographs.

SAMPLE REQUIREMENTS 2 test products and 2 control products (min. length 10 cm)

TURNAROUND TIME 32 days (non-GLP) • 39 days (GLP)

BIOCOMPATIBILITY B-13
 IMPLANTATION TESTING
These tests assess the local effects of material or finished product on contact with living tissue.
Using a needle or surgical procedure, a sample is implanted into the tissue site appropriate for the
intended use of the device. After the selected duration of contact, the tissue sites are evaluated for
gross changes and – if requested –histopathology.

900100 1 wk implantation duration Biomaterial is implanted intramuscularly into rabbits to assess the reaction of the
900200 2 wk implantation duration surrounding tissue. The implants remain in the muscle for the sponsor-designated
900300 4 wk implantation duration time period. If the animals exposed to the test article do not show significant signs
900400 8 wk implantation duration of irritation above that observed in the concurrent test control, the test article
900500 12 wk implantation duration passes the test. Scoring of the sample and control reactions will be by gross
observation. Histopathologic evaluation and photomicrographs (gross and
USP Intramuscular Implantation histologic) will also be provided at the request of the sponsor. (Additional fees
apply.)
(2 Rabbits)
SAMPLE REQUIREMENTS
–––––––––––––––––– The sample used in the test is approx. 1 mm by 10 mm dimension. The sponsor
can submit the sample in this form or the lab will sub-divide a larger sample.
902100 1 wk implantation duration Somewhat larger samples can be surgically implanted. (Additional fees apply.) A
902200 2 wk implantation duration minimum of 15 implant samples must be submitted. The sponsor can submit the
902300 4 wk implantation duration sample pre-sterilized or the lab will sterilize according to sponsor directions.
902400 8 wk implantation duration
902500 12 wk implantation duration [Note: For ISO, sample preparation complies with ISO10993-12.]

TURNAROUND TIME
ISO Intramuscular Implantation
(3 Rabbits) GLP: Implant Gross With Histopathology
1 wk 21 days 50 days
2 wk 28 days 57 days
4 wk 42 days 71 days
8 wk 70 days 99 days
12 wk 98 days 127 days

Non-GLP: Subtract 7 days from GLP TATs shown above.

901310 1 wk implantation duration A portion of the material or device is implanted subcutaneously into rabbits to
901320 2 wk implantation duration assess the reaction of the surrounding tissue. The implants remain for the
901340 4 wk implantation duration sponsor-designated time period. If the animals exposed to the test article do not
901380 8 wk implantation duration show significant signs of irritation above that observed in the concurrent test control
901420 12 wk implantation duration sites, the test article passes the test. The sample and control tissue sites will be
evaluated grossly. Histopathologic evaluation and photomicrographs (gross and
histologic) will also be provided at the request of the sponsor. (Additional fees
ISO Subcutaneous Implantation apply.)
(3 Rabbits) SAMPLE REQUIREMENTS Representative portions of the device. (Minimum 15)
[Note: For ISO, sample preparation complies with ISO10993-12.]

TURNAROUND TIME

GLP: Implant Gross With Histopathology

1 wk 21 days 50 days
2 wk 28 days 57 days
4 wk 42 days 71 days
8 wk 70 days 99 days
12 wk 98 days 127 days

Non-GLP: Subtract 7 days from GLP TATs shown above.

B-14 BIOCOMPATIBILITY
 IMPLANTATION TESTING

This implantation test is most applicable for biomaterials that are exposed to the body 902599
for an extended duration. This test is used to access not only the local tissue effects
but also any long-term systemic effects of the implanted material. A sample device or
biomaterials are implanted into the muscle of rabbits for the sponsor-designated time Long-Term Intramuscular
period. Blood and body weights will be collected at specified intervals from each
rabbit throughout the study. Target organs will be collected at termination and
Implant Test in Rabbits
evaluated microscopically. All implant sites will be observed for gross reaction and With Additional
microscopic evaluation. The results will be based on the overall assessment, Chronic Data Collection
including statistical analysis. Gross and histologic photomicrographs will also be (5-8 Rabbits)
provided at the request of the sponsor. (Additional fees apply.)

SAMPLE REQUIREMENTS
The sample size best suited for this test is 10 mm by 1 mm (length : diameter). The
sponsor can submit the sample in this form or the lab will subdivide a larger sample.
Somewhat larger samples may be surgically implanted. (Additional fees apply.) The
client should discuss the number of samples during protocol development.

TURNAROUND TIME Inquire

WuXi AppTec’s professional staff can assist in the design and implementation of Custom Implant Studies
studies to investigate medical device biocompatibility issues. These studies help
determine whether device surface characteristics, polymeric composition and physical
geometry may effect local tissue responses such as inflammation, tissue ingrowth, Intramuscular or
vascularization and fibroplasia. Gross and histologic photomicrographs can also be Subcutaneous Implant
provided at the request of the sponsor.
Screen Test
For other custom implant studies, such as bone, intracranial and intraperitoneal implants,
contact your Account Manager.

Histopathology assessment is conducted for muscle or subcutaneous tissues if part 800250

of the protocol. (Additional fees apply.)
Histopathology Assessment
Note: ISO guidelines assume histopathology will be conducted.

TURNAROUND TIME Inquire

Surgical implantation may be requested by client or may be required for larger 910100
sample sizes. (Additional fees apply.)
Surgical Implantation

BIOCOMPATIBILITY B-15
 IRRITATION TESTING

Irritation (reactivity) tests assess the localized reaction of tissues to device materials or extracts.
For ocular, dermal and mucosal tissue contact, the appropriate test is selected. For breached
tissue and blood contact, the intracutaneous test is usually selected and uses only extracts. The
dermal irritation test usually involves direct contact with the test material. The mucosal irritation test
can involve either direct contact or use of extracts. The ocular tests usually use extracts. Extracts
are prepared using solvents that will extract either hydrophilic (polar) or lipophilic (non-polar)
compounds which may be present in the device materials.

900600 An extract of the device or biomaterial is prepared in up to four (4) standard USP
extraction solutions and is injected intracutaneously into rabbits to assess the
USP Intracutaneous irritancy of extractable compounds that may exist in the biomaterial. The animals are
observed for dermal reactions over a 72-hour period. If the animals exposed to the
Irritation Test test article extract do not show significant signs of irritation above those observed in
(2 rabbits per extract or the concurrent test control groups, the test article passes the test. Gross
pair of extracts) photography may be requested. (Additional fees apply.)

EXTRACT OPTIONS • Normal Saline • 5% Ethanol in Saline

-------------------------- • Cottonseed Oil • Polyethylene Glycol

Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours

910700 50°C/72 hours 37°C/72 hours

SAMPLE REQUIREMENTS
ISO Intracutaneous (Per Extract) By Thickness By Weight
By Weight Liquids
Irritation Test <0.5mm thick >0.5mm thick (lightweight)
(3 rabbits per extract or 24 cm2 12 cm2 0.8 g 0.4 g 3 mL
pair of extracts)
Note: For ISO, normal saline and/or cottonseed oil extracts are
recommended. Sample preparation complies with ISO10993-12.

TURNAROUND TIME 24 days (non-GLP) • 31 days (GLP)

910790 Test material coming in direct or indirect contact with mucosal tissue can be
assessed as to its irritation potential by repeated instillation of an extract into rabbit
vaginas. Acute irritation is evaluated by gross observation and histopathology of the
ISO Vaginal Mucosal Irritation vaginal mucosa and submucosa. Usually an application is performed on each of five
days unless longer treatment is indicated due to the use of the device.
Test
(6 rabbits per extract) EXTRACT OPTIONS • Normal Saline • Cottonseed Oil

Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours

50°C/72 hours 37°C/72 hours

SAMPLE REQUIREMENTS
(Per Extract) By Thickness
By Weight
By Weight
Liquids
< 0.5mm thick >0.5mm thick (lightweight)
5 x 60cm2 5 x 30 cm2 5x2g 5x1g 30 mL

Note: For ISO, normal saline and/or cottonseed oil extracts are
recommended. Sample preparation complies with ISO10993-12.

TURNAROUND TIME 47 days (non-GLP) • 54 days (GLP)

B-16 BIOCOMPATIBILITY
 IRRITATION TESTING

Three rabbits receive a 0.1 mL dose of a polar (normal saline) or nonpolar 910810
(cottonseed oil) extract of the test material into one eye of each rabbit. After a 24-
hour exposure, the eye is flushed and the conjunctiva, cornea and iris are evaluated
for up to 72 hours for acute irritation or injury. Test materials should be prescreened Ocular Irritation Test
using the Cytotoxicity Agarose Diffusion Test. [Topical]
EXTRACT OPTIONS • Normal Saline • Cottonseed Oil (3 rabbits per extract)

Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours

50°C/72 hours 37°C/72 hours
SAMPLE REQUIREMENTS
(Per Extract) By Thickness
By Weight
<0.5mm thick >0.5mm thick
60 cm2 30 cm2 2g
Note: For ISO, sample preparation complies with ISO10993-12.

TURNAROUND TIME 28 days (GLP)

A 0.15 mL dose of test material extract is injected with a fine-gauge needle into the 910820
anterior chamber of one eye. At the same time, a vehicle control is injected in the
same manner into the opposing eye. Over the 3-day exposure of the test, the
reaction to the injected substance is evaluated using a slit-lamp microscope and the Intraocular Irritation Test
degree of reaction is scored using a standard scoring system. The test material (6 rabbits)
passes the test if the test article extract does not produce irritation to a significantly
greater degree than the control material. Test materials should be prescreened
using a cytotoxicity test.

EXTRACT Balanced Salt Solution (BSS)

Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours

50°C/72 hours 37°C/72 hours
SAMPLE REQUIREMENTS
By Thickness
By Weight
<0.5mm thick >0.5mm thick
60 cm2 30 cm2 2g

Note: For ISO, sample preparation complies with ISO10993-12.

TURNAROUND TIME 28 days (GLP)

This test is performed to assess the potential for topical irritation from acute 910699
exposure or use of the device material. The material is applied to intact skin of three
(3) rabbits and left in contact for 4 to 24 hours. An estimate of irritation, erythema
(redness) and edema (swelling) is made during the next 72 hours. Histopathologic ISO Primary Skin Irritation
evaluation and photomicrographs (gross and histologic) will also be provided at the Test
request of the sponsor. Additional fees apply.
(3 rabbits)
Note: Sponsor specifies contact duration on test request form.

SAMPLE REQUIREMENTS At least 7 one-square-inch portions of the test material.

5 mL of liquid.

TURNAROUND TIME 25 days (non-GLP) • 32 days (GLP)

NOTE: For additional irritation tests, contact your WuXi AppTec Account Manager.

BIOCOMPATIBILITY B-17
 PYROGENICITY TESTING (IN VIVO)

Pyrogenicity tests determine the potential of materials, extracts, and/or a finished device to induce a
pyrogenic (fever) response.

Note: Bacterial endotoxin (the most commonly encountered type of pyrogen) can be readily detected and
quantified using the in vitro Limulus Amebocyte Lysate (LAL) Test. For information on LAL tests, see this catalog’s
“Endotoxin (LAL)” section.

900750 The test articles are prepared in a sterile solution, which is injected intravenously into
three (3) rabbits to assess pyrogenicity. The animals are observed over a 3-hour period
for an increase in body temperature. If the animals exposed to the solution do not show
USP Rabbit Pyrogen Test significant increase in body temperature, the test article passes the test. If any single
(3 rabbits) animal of the three has a temperature increase above the acceptable range, the test can
be continued with 5 additional animals at client’s request. (See below.)
SAMPLE REQUIREMENTS
Transfusion / Infusion Assemblies and Similar Devices:
10 device assemblies to represent the lot under test.
Blood and Tissue Contact Devices:
10 devices to represent the lot under test.

TURNAROUND TIME 22 days (non-GLP) • 29 days (GLP)

900770 An extract of the test article is prepared in a sterile saline solution and injected
intravenously into three (3) rabbits to assess pyrogenicity. The animals are observed over
a 3-hour period for an increase in body temperature. If the animals exposed to the test
ISO Rabbit Pyrogen – article extract do not show significant increase in body temperature, the test article passes
Materials Mediated the test. If any single animal of the three has a temperature increase above the
(3 rabbits) acceptable range, the test can be continued with 5 additional animals at client’s request.
(See below.)
EXTRACT OPTION Normal Saline

Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours

50°C/72 hours 37°C/72 hours
SAMPLE REQUIREMENTS
By Thickness By Weight
By Weight
<0.5mm thick >0.5mm thick (lightweight)

900 cm2 450 cm2 30 g 15 g

TURNAROUND TIME 22 days (non-GLP) • 29 days (GLP)

900755 See USP Rabbit Pyrogen and ISO Rabbit Pyrogen – Materials Mediated tests above.
Continuation of testing is at client's request. (Additional fees apply.)
Rabbit Pyrogen Test
EXTRACT OPTION Normal Saline
Continuation
(5 rabbits) EXTRACTION CONDITIONS Same as original test
SAMPLE REQUIREMENTS
By Thickness By Weight
By Weight
<0.5mm thick >0.5mm thick (lightweight)

1350 cm2 675 cm2 45 grams 22.5 g

TURNAROUND TIME 22 days (non-GLP) • 29 days (GLP)

B-18 BIOCOMPATIBILITY
 SENSITIZATION TESTING

Sensitization tests estimate the potential for contact sensitization of devices through the testing of
appropriate materials or extracts.

The Murine Local Lymph Node Assay (LLNA) is quickly becoming the standard methodology for
sensitization studies. This assay offers greater sensitivity and specificity than the Guinea pig
assays, and – when combined with interpretation based on statistical data analysis between test
and negative control groups – it has become the model for determination of delayed-type
hypersensitivity.

In this assay, the ability of a material to potentially elicit a delayed-type 190856 Standard Assay
hypersensitivity response is evaluated by the ability to cause mitotic proliferation of
lymphocytes within the draining auricular lymph nodes.
Murine Local Lymph Node
The Standard Assay (3 groups) Assay (LLNA)
The standard assay utilizes four groups of mice – test article, positive control and
negative control (n=15 total). Adult female CBA strain mice are treated topically
with solutions or extracts applied to the dorsum of the ears bilaterally. The
response is compared to appropriate concurrent positive and negative controls.
Measurement of the degree of cell proliferation is quantified by incorporation of
3
H-thymidine into DNA of replicating lymph node lymphocytes. Interpretation is
based on a statistically significant difference in the stimulation index (SI) between
the test and negative control groups. Assay performance requires that the positive
control's SI be greater than 3.0.

EXTRACT OPTIONS • Normal Saline • Dimethyl Sulfoxide/Polyethylene Glycol

Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours

50°C/72 hours 37°C/72 hours

SAMPLE REQUIREMENTS
(Per Extract)
By Thickness By Weight
By Weight Liquid
<0.5mm thick >0.5mm thick (lightweight)

3 x 30 cm2 30 x 15 cm2 3x1g 3 x 0.5 g 3 mL

TURNAROUND TIME 25 days (non-GLP) • 32 days (GLP)

SENSITIZATION TESTING CONTINUED NEXT PAGE

BIOCOMPATIBILITY B-19
 SENSITIZATION TESTING

900850 Guinea pigs are exposed to the extract twice within a 2-week period (Inductions I and
II). The animals are re-exposed (challenged) 10-14 days after Induction II by placing
fresh extract in contact with previously unexposed skin. Over a 72-hour period, the
ISO Maximization animals are observed for signs of a delayed allergic response when compared to a
Sensitization Test control group. If the test results are equivocal, a re-challenge can be conducted within
7-10 days of the initial challenge.

NUMBER OF ANIMALS 10 test • 5 irritant controls • 5 negative controls

EXTRACT OPTIONS • Normal Saline • Cottonseed Oil

Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours

50°C/72 hours 37°C/72 hours
SAMPLE REQUIREMENTS
By Thickness By Weight
By Weight Liquid
<0.5mm thick >0.5mm thick (lightweight)

3 x 60 cm2 3 x 30 cm2 3x2g 3x1g 15 mL

Note: For ISO, sample preparation complies with ISO10993-12.

TURNAROUND TIME 49 days (non-GLP) • 56 days (GLP)

900899 Guinea pigs are patched with the test material 3 times per week for 3 weeks during the
Induction Phase. After 2-week recovery period, animals are topically challenged with
similar patches of test material to assess for delayed contact sensitization.
Repeated Patch Dermal
Sensitization Test NUMBER OF ANIMALS 10 test / 5 controls
2
[Buehler method modified SAMPLE REQUIREMENTS Solid Sheets: 106 x 1 in
for medical devices] Liquid: 50 mL

TURNAROUND TIME 53 days (non-GLP) • 60 days (GLP)

B-20 BIOCOMPATIBILITY
 SUBACUTE TOXICITY TESTING

Subacute toxicity is assessed after single or multiple exposures to extracts of device materials. The
exposure period is longer than typical acute toxicity tests, but not exceeding 10% of animal lifespan.
Subacute studies involve expanded evaluations and can include systemic changes, local irritation,
body weight, blood values and tissue changes as part of the protocol. The length of time for the
test and the parameters evaluated will depend on the end use of the device. WuXi AppTec will
assist you in the test program design.

Multiple extracts of a device are prepared with standard polar and/or nonpolar 14-Day Subacute Toxicity Test
vehicles and injected into 6 male and 6 female mice or rats over a 14-day period.
Two similar control groups are also injected with control vehicle. The animals are
observed during the 14-day period for signs of toxicity and are subjected to a gross 800560
observation at study termination. The test article passes if the test parameters Subacute Intravenous Toxicity
(weight, survival, clinical observations and gross necropsy) are not significantly in Mice (5 repeat dose)
different from the concurrent control animal parameters. Other parameters
evaluated include clinical chemistry and hematology. 800570
Subacute Intraperitoneal Toxicity
Tests 800560, 800570, 800540 and 800550 are recommended for medical devices in Mice (5 repeat dose)
categorized as “prolonged contact” (24 hours to 30 days).
800565
Tests 800565, 800575, 800545 and 800555 are recommended for medical devices Subacute Intravenous Toxicity
categorized as “permanent contact” (more than 30 days). in Mice (14 repeat dose)

800575
EXTRACT OPTIONS • Normal Saline • Cottonseed Oil Subacute Intraperitoneal Toxicity
in Mice (14 repeat dose)
Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours
50°C/72 hours 37°C/72 hours 800540
Subacute Intravenous Toxicity
SAMPLE REQUIREMENTS
in Rats (5 repeat dose)
By Thickness By Weight
800560 • 800570 By Weight
<0.5mm thick >0.5mm thick (lightweight)
800565 • 800575 800550
800540 • 800550 5 x 30 cm2 5 x 15 cm2 5x1g 5 x 0.5 g Subacute Intraperitoneal Toxicity
in Rats (5 repeat dose)

By Thickness
By Weight
By Weight 800545
800545 • 800555 <0.5mm thick >0.5mm thick (lightweight)
Subacute Intravenous Toxicity
14 x 30 cm2 14 x 15 cm2 14 x 1 g 14 x 0.5 g in Rats (14 repeat dose)

Note: For ISO, sample preparation complies with ISO10993-12. 800555

Subacute Intraperitoneal Toxicity
TURNAROUND TIME Inquire in Rats (14 repeat dose)

OPTIONS
Additional fees apply for the following services. For further information, contact your The above tests include:
Account Manager.
Clinical Chemistry for 14-Day
800210 Histopathology for 14-Day Subacute Testing Subacute Testing
Hematology for 14-Day
800215 Tissue Storage Subacute Testing

NOTE: For special subacute toxicity testing involving dermal, mucosal or oral dosing programs, contact your Account Manager.

See Page B-23 for a note on Chronic Toxicity and Carcinogenicity testing.

BIOCOMPATIBILITY B-21
 SYSTEMIC (ACUTE)
TOXICITY TESTING

Acute systemic toxicity tests estimate the potential harmful systemic effects from a single exposure
to polar or nonpolar extracts of device materials.

900700 An extract of the device or biomaterial is prepared in up to four (4) standard USP
extraction solutions and injected into mice (10 per extract) to assess the toxicity of
extractable compounds that may exist in the biomaterial. The animals are observed
USP Acute Systemic over a 72-hour period. If the animals exposed to the test article extract do not show
Toxicity Test signs of toxicity greater than the concurrent control groups, the test article passes
the test.

------------------------ EXTRACT OPTIONS • Normal Saline • 5% Ethanol in Saline

• Cottonseed Oil • Polyethylene Glycol
901770
Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours
50°C/72 hours 37°C/72 hours
ISO Acute Systemic
Toxicity Test SAMPLE REQUIREMENTS
(Per Extract) By Thickness
By Weight
By Weight
< 0.5mm thick >0.5mm thick (lightweight)

48 cm2 24 cm2 1.6 g 0.8 g

Note: For ISO, normal saline and/or cottonseed oil extracts are
recommended. Sample preparation complies with ISO10993-12.

TURNAROUND TIME 24 days (non-GLP) • 31 days (GLP)

B-22 BIOCOMPATIBILITY
 OTHER BIOCOMPATIBILITY TESTS

CHRONIC TOXICITY
CARCINOGENICITY

These tests are often long-term studies that can extend for a period of up to two years or longer. If the
device involves new chemistry that (from material characterization and exposure assessments) indicate
a high enough risk, one or more of these studies may be necessary. If this is the case, contact your
Account Manager for assistance in designing an appropriate long-term study.

FINISHED PRODUCT RELEASE

This assay is designed to serve as a safety evaluation for lot release. A single unit 800510
or device is extracted with sterile saline solution and injected intravenously into five
mice to assess the acute toxicity of the device. The animals are observed over a
48-hour period. If the animals survive the injection with no signs of toxicity, the USP Safety Test in Mice
device passes the test.
(5 mice)
EXTRACT Normal Saline

Specify one set of extraction conditions: 121°C/1hour 70°C/24 hours

50°C/72 hours 37°C/72 hours

SAMPLE REQUIREMENTS One (1) device to represent the lot under test.

TURNAROUND TIME 22 days (non-GLP) • 29 days (GLP)

A comprehensive range of additional finished product

release testing is available, including
Rabbit Pyrogen tests [see Page B-18] and
Bacterial Endotoxin (LAL) tests [see “Endotoxin” section].

Please contact your WuXi AppTec Account Manager for more information.

BIOCOMPATIBILITY B-23
 BIOCOMPATIBILITY TESTING FLOW CHART

Material Characterization / Risk Assessment

Flow Chart for the Selection of Toxicity Tests for 510(k)s

FROM: May 1, 1995 FDA General Program Memorandum - #G95-1 / Attachment C

B-24 BIOCOMPATIBILITY
 TESTING UNDER GLP REGULATIONS

Quality Assurance-Audited Testing Requirements

Under Good Laboratory Practices (GLP) Regulations

• Protocol Development
• Study Director
• Testing Communications
• QA Master Schedule
• Test Schedule Notification
• Test Article Control
• QA Inspections
• Audited Final Report
• Raw Data Archive

NOTE: For GLP testing, clients will receive notification

as to the date they can expect to receive their final reports.

BIOCOMPATIBILITY TESTS FOR CLASSIFICATIONS OF PLASTICS (USP)

PLASTICS CLASSES
Acute Systemic Toxicity [Mice]
Extract of sample in: I II III IV V VI
Sodium chloride injection • • • • • •
1 in 20 solution of alcohol in sodium chloride injection • • • • •
Polyethylene glycol 400 • • •
Vegetable oil (cottonseed oil) • • • •
Intracutaneous Irritation Test [Rabbit]
Extract of sample in: I II III IV V VI
Sodium chloride injection • • • • • •
1 in 20 solution of alcohol in sodium chloride injection • • • • •
Polyethylene glycol 400 • •
Vegetable oil (cottonseed oil) • • •
Intramuscular Implantation Test [Rabbit]
Length of test: I II III IV V VI
5-day • •
7-day •
NOTE: These tests are for material qualification, not for regulatory submission.

BIOCOMPATIBILITY B-25
 DEVICE CATEGORIES

DEVICE CATEGORIES BY NATURE OF CONTACT

Medical devices fall into one of four categories based on the nature of patient contact:

1) Non-Contact Devices
Devices that do not directly or indirectly contact the patient are not required to undergo biocompatibility
testing.

2) Surface Devices
• Contacting Intact Skin
(e.g., electrodes, external prostheses, fixation tapes, compression bandages)

• Contacting Mucous Membranes

(e.g., contact lenses, urinary catheters, colonoscopes, endotracheal tubes)

• Contacting Breached or Compromised Surfaces

(e.g., wound dressings, occlusive patches, healing devices)

3) External Communicating Devices

• Contacting Blood Path Indirectly
(e.g., solution administration sets, I.V. extension sets, blood transfusion sets)

• Contacting Tissue, Bone or Dentin

(e.g., laparoscopes, arthroscopes, draining systems, dental cements, skin staples)

• Contacting Circulating Blood

(e.g., intravenous and delivery catheters, temporary pacemaker electrodes, dialyzers, dialysis tubing,
hemadsorbants, immuno-adsorbants)

4) Implant Devices
• Contacting Tissue and/or Bone
(e.g., orthopedic pins and plates, pacemakers, breast implants, replacement tendons, ligation clips,
drug supply devices)

• Contacting Blood
(e.g., pacemaker electrodes, heart valves, vascular grafts, ventricular assist devices, internal drug
delivery devices, stents)

DEVICE CATEGORIES BY DURATION OF CONTACT

Devices generally are placed in one of three categories based on expected duration of contact with patient.

A) Limited [≤ 24 hours]
B) Prolonged [> 24 hours and ≤ 30 days]
C) Permanent [> 30 days]

B-26 BIOCOMPATIBILITY
 INITIAL EVALUATION TESTS FOR CONSIDERATION

BIOCOMPATIBILITY SAFETY TESTING

[Based on ISO 10993-1 AND FDA G95-1 Guidelines]

BIOLOGICAL EFFECT
DEVICE CATEGORIES 4
Initial Other

Subacute (Subchronic) Toxicity

Contact
Duration

Hemocompatibility
Systemic Toxicity

Chronic Toxicity
Carcinogenicity
A – Limited

Sensitization

Genotoxicity
Implantation
Cytotoxicity
[≤ 24 hrs]

Irritation
Body Contact
B – Prolonged
[>24 hrs to ≤30 days]

C – Permanent
[>30 days]

A ● ● ●
Skin B ● ● ●
C ● ● ●
A ● ● ●
SURFACE Mucosal
Membranes B ● ● ● ◊ ◊ ◊
DEVICES
C ● ● ● ◊ ● ● ◊ ◊
Breached or A ● ● ● ◊
Compromised B ● ● ● ◊ ◊ ◊
Surfaces ◊ ◊ ◊
C ● ● ● ● ●
A ● ● ● ● ●
Blood Path,
Indirect 3 B ● ● ● ● ◊ ●
C ● ● ◊ ● ● ● ◊ ● ● ●
EXTERNAL 1
Tissue /Bone/ A ● ● ● ◊
COMMUNICATING Dentin B ● ● ● ● ● ● ●
DEVICES Communicating C ● ● ● ● ● ● ● ● ●
A ● ● ● ● ◊ 2
●
Circulating
Blood 3 B ● ● ● ● ● ● ● ●
C ● ● ● ● ● ● ● ● ● ●
A ● ● ● ◊
Tissue / Bone B ● ● ● ● ● ● ●
IMPLANT C ● ● ● ● ● ● ● ● ●
DEVICES A ● ● ● ● ● ● ●
Blood 3 B ● ● ● ● ● ● ● ●
C ● ● ● ● ● ● ● ● ● ●
807

1 ● – ISO Evaluation Tests for Consideration

“Tissue” includes tissue fluids and subcutaneous spaces.
2
For all devices used in extracorporial circuits. ◊ – Additional tests that the FDA considers may be applicable
3
Pyrogenicity / Materials Mediated should be considered.
4 For reproductive and biodegradation tests, contact your
Supplemental tests for consideration. WuXi AppTec Account Manager.

BIOCOMPATIBILITY B-27
 SAMPLE REQUIREMENTS FOR
BIOCOMPATIBILITY TESTING
This chart is designed as a convenient quick guide for some of our most commonly ordered tests (and does not list all available tests).
Sample requirements are based on the recommended minimum per extraction vehicle.
If you have questions, before submitting samples, contact Client Services at AppTec’s St. Paul facility: 651-675-2000 or 888-794-0077.
> 0.5 thickness Irregularly Shaped
< 0.5 thickness (60cm²/20mL ratio) (4g/20mL ratio)
Membranes
Test Name (120cm²/20mL ratio)
[tubing wall, slab, [powder, pellets, foam, non-
(2g/20mL ratio) Liquids
[film, sheet, tubing wall] (low-density materials)
molded items] absorbent molded items]

CYTOTOXICITY
ISO/USP MEM Elution Using L-929 Cells 1 x 30cm² 1 x 15cm² 1 x 1g 1 x 0.5g 3mL
Extract Colony Assay (JMHLW) 120cm² or 2g
ISO/USP Agarose Overlay Using L-929 Cells 3 pieces (1cm x 1cm each) 1mL
HEMOCOMPATIBILITY / BLOOD COMPATIBILITY
Hemolysis Test (NIH Method) Direct Contact Method NA NA 3 x 2g 3 x 0.2g 10mL
Hemolysis Test (NIH Method) Extract Method NA NA 3 x 2g 3 x 0.2g 10mL
Hemolytic Toxicity Test (JMHLW) 3 x 90cm² 3 x 45cm² 3 x 3g 3 x 1.5g NA
ASTM Hemolysis Direct Contact Method NA 3 x 12cm² 3 x 2g 3 x 0.2g 10mL
ASTM Hemolysis Extract Method NA 3 x 12cm² 3 x 2g 3 x 0.2g 10mL
Complement Activation C3a Assay and SC5b-9 Assay 1 x 6cm² 1 x 3cm² 1 x 0.2g 1 x NA 1 x NA
Partial Thromboplastin Time 3 x 4cm² (test samples) and 3 x 4cm² (comparison samples)
Platelet and Leukocyte Count 3 x 12cm² (test samples) and 3 x 12cm² (comparison samples)
In Vitro Hemocompatibility Assay 3 x 12cm² (test samples) and 3 x 12cm² (comparison samples)
Thrombosis (In Vivo) – 2 Dog 2 Test and 2 Comparison samples
GENOTOXICOLOGY
Bacterial Mutagenicity Test (Ames Assay) 2 x 24cm² 2 x 12cm² 2 x 0.8g 2 x 0.4g 4mL
Bacterial Reverse Mutation Assay (JMHLW) 5g *
In Vitro Chromosome Aberration Assay 2 x 42cm² 2 x 21cm² 2 x 1.4g 2 x 0.7g 8mL
In Vitro Chromosome Aberration Analysis (JMHLW) 5g *
In Vitro Mouse Lymphoma Assay 2 x 42cm² 2 x 21cm² 2 x 1.4g 2 x 0.7g 7mL
In Vivo Mouse Micronucleus Assay 2 x 120cm² 2 x 60cm² 2 x 4g 2 x 2g 18mL
IRRITATION / STIMULATION
ISO/USP/JMHLW Intracutaneous Reactivity Test 2 x 24cm² 2 x 12cm² 2 x 0.8g 2 x 0.4g 3mL
ISO Vaginal Mucosal Irritation Test 10 x 60cm² 10 x 30cm² 10 x 2g 10 x 1g 30mL
ISO Primary Skin Irritation Test 7 pieces (2.5cm x 2.5cm each) 5mL
Primary Skin Stimulatory Test (JMHLW) 2 x 48cm² 2 x 24cm² 2 x 1.6g 2 x 0.8g NA
PYROGENICITY
Materials Mediated Rabbit Pyrogen (also JMHLW) 900cm² 450cm² 30g 15g 150mL
SENSITIZATION
Murine Local Lymph Node Assay [LLNA] 6 x 30cm² 6 x 15cm² 6 x 1g 6 x 0.5g 3mL
ISO Maximization Sensitization (Guinea Pig) Test 6 x 60cm² 6 x 30cm² 6 x 2g 6 x 1g 15mL
Maximization Sensitization Test (JMHLW) 12g *
Repeated Patch Dermal Sensitization Test – Buehler 105 pieces (2.5cm x 2.5cm each) 50mL
SYSTEMIC TOXICITY
ISO/USP/JMHLW Acute Systemic Injection Test 2 x 48cm² 2 x 24cm² 2 x 1.6g 2 x 0.8g 7mL
Subacute [Subchronic] Toxicity – 5 Dose Exposure 5 x 30cm² 5 x 15cm² 5 x 1g 5 x 0.5g Inquire
Subacute [Subchronic] Toxicity –14 Dose Exposure 14 x 30cm² 14 x 15cm² 14 x 1g 14 x 0.5g Inquire
IMPLANTATION
ISO/JMHLW Intramuscular/Subcutaneous Implantation Test 15 pieces (~10mm x 3mm)
USP Intramuscular/Subcutaneous Implantation Test 12 pieces (~10mm x 3mm)
CHEMISTRY
USP Physicochemical Test for Plastics 10g or 600cm²
FTIR 5g
* Sample amounts will increase if residue is obtained.
BIOCOMPATIBILITY B-28