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MAKMAL BIOSERASI
Universiti Kebangsaan Malaysia
BIOCOMPATIBILITY & TOXICITY TESTING**
Cytotoxicity Test – ISO 10993-5
Cytotoxicity is a rapid and standardized test that is very sensitive to evaluate a
significant quantity of harmful extractables (leachable substances) present in a
device material and the effect they may have on cellular components.
Test Scopes: MEM Elution (ISO), Agar Diffusion (ISO), and MTT Assay (ISO,with
appropriate provision from Mossmann, 1983).
MEM Elution assay uses extracting media and extraction conditions to test the
device according to actual use conditions or to exaggerate those conditions.
Extracts are usually titrated to yield a semi-quantitative measurement of cytotoxicity.
After extraction, the extracts are transferred onto a monolayer of cells and incubated
at 37oC. Following incubation, cells are examined microscopically for malformation,
degeneration and lysis. At least one type of cytotoxicity test should be performed on
each component of any device.
Turnaround time: 12 days
Agar Diffusion assay is appropriate for high density materials, such as elastomeric
closures. The method utilised a thin layer of nutrient-supplemented agar overlaid on
cultured cells. The test material (or its extract) is placed on top of the agar layer, and
the cells are incubated. A zone of malformed, degenerative or lysed cells under and
around the test material indicates cytotoxicity.
Turnaround time: 14 days
MTT Assay is used to evaluate the toxic characteristics of a material or substance.
In this study, yellow tetrazolium salt (MTT) is reduced in metabolically active cells to
form insoluble purple formazan crystals which are solubilized by the addition of a
solvent (dimethyl sulfoxide). Cell viability is quantified by colorimetric enumeration
whereby a low OD reading corresponds to low cell viability associated with a loss in
mitochondrial dehydrogenase activity. The study provides information on dose
response curve and inhibition concentration (IC) of a material or compound.
Turnaround time: 14 days
Key: ** The additional test scopes are in the process of obtaining accreditation.
1
Minimum Sample Required
Test Material / Test
Solid (thickness
<0.5 mm)
Solid (thickness
0.5 – 1.0 mm)
Solid (thickness
>1.0 mm)
Extraction
ratio
MEM Elution
Agar Diffusion
MTT Assay
6 cm2/ml
60 cm2
60 cm2
60 cm2
3 cm2/ml
30 cm2
30 cm2
30 cm2
3 cm2/ml
30 cm2
30 cm2
30 cm2
1.25 cm2/ml
12.5 cm2
12.5 cm2
12.5 cm2
E.g. Larger moulded
items
Solid (thickness >1.0
mm)
E.g. Elastomeric
closures
Irregular shaped
0.2 g/ml
2g
2g
solid device
Irregular shaped
porous devices (low0.1 g/ml
1g
1g
density materials)
Liquid
N/A
N/A
5 ml
- Note: Please provide extra for archiving and any unforeseen circumstances.
2g
1g
10 ml
Skin Sensitization Test – ISO 10993-10
Sensitization or hypersensitivity tests look for adverse reactions in animals by
exposing the animal to the material or by using extracts from the device or materials
and injecting and/or topically applying them to the animal. Sensitization reactions are
graded according to scores for erythematous and oedematous lesions on the skin.
Test Scopes: Guinea Pig Maximization Test (GPMT) and Closed Patch (Buehler).
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. The test material is mixed with complete Freund’s adjuvant (CFA) to
enhance the skin sensitization response.
Turnaround time: 7 – 10 weeks
The Closed Patch Test (Buehler) involves multiple topical doses and is
recommended for devices that will contact unbroken skin only.
Turnaround time: 8-10 weeks
2
Minimum Sample Required
Test Material /
Test
Solid (thickness
<0.5 mm)
Solid (thickness
0.5 – 1.0 mm)
Solid (thickness
>1.0 mm)
Extraction ratio
GPMT*
Buehler*
6 cm2/ml
600 cm2
600 cm2
3 cm2/ml
400 cm2
300 cm2
3 cm2/ml
400 cm2
300 cm2
1.25 cm2/ml
150 cm2
125 cm2
0.2 g/ml
36 g
20 g
E.g. Larger
moulded items
Solid (thickness
>1.0 mm)
E.g. Elastomeric
closures
Irregular shaped
solid device
Solid
Buehler
700 cm2
(i.e. any flat
surface)
N/A
0.2 g/ml
60 g
20 g
60 g
E.g. powder, tube
Irregular shaped
porous devices
0.1 g/ml
30 g
10 g
N/A
(low-density
materials)
Liquid
N/A
50 ml
N/A
60 ml
- Note: Please provide extra for archiving and any unforeseen circumstances.
* Where extraction is applied, polar & non-polar extraction is performed. GPMT involves
extraction procedure during intradermal induction phase, and if the test material cannot be
patched directly, in the topical induction and challenge phase. As for the Buehler Test,
extraction will be performed for test material that cannot be directly patched.
Skin Irritation Test – ISO 10993-10
This test evaluates the reaction to a single, repeated or continual exposure from
device materials that have the potential to produce skin irritation. The test does not
involve immunological mechanism.
Test Scopes: Intracutaneous Irritation (ISO), Primary Skin Irritation (ISO)
The Intracutaneous Irritation test uses extracts of a test material and blank
injected intradermally. Injection sites are scored for erythema and oedema. This
procedure is recommended for devices that will have externally communicating or
internal contact with the body or body fluids. It can reliably detect the potential for
local irritation due to chemicals that may be extracted from a device material.
Turnaround time: 3 – 4 weeks
3
The Primary Skin Irritation test is usually performed for topical devices that have
external contact with intact or breached skin. The test material or an extract is
applied directly to intact and abraded sites on the skin of a rabbit. After a 24-hour
exposure, the material is removed and the sites are scored for erythema and
oedema.
Turnaround time: 3 – 4 weeks
Minimum Sample Required
Test Material / Test
Solid (thickness <0.5
mm)
Solid (thickness
0.5 – 1.0 mm)
Solid (thickness >1.0
mm)
Extraction ratio
2
E.g. Larger moulded
items
Solid (thickness >1.0
mm)
Intracutaneous*
PSI*
2
6 cm /ml
120 cm
3 cm2/ml
60 cm2
3 cm2/ml
60 cm2
1.25 cm2/ml
25 cm2
E.g. Elastomeric closures
Irregular shaped solid
0.2 g/ml
6g
device
Irregular shaped porous
device
0.1 g/ml
4g
(low-density materials)
Liquid
N/A
10 ml
- Note: Please provide extra for archiving and any unforeseen circumstances.
* Where extraction is applied, polar & non-polar extraction is performed
30cm2
4g
2g
5 ml
Genotoxicity Testing – (in vitro)
Testing Scopes: Ames Reverse Mutation (TG OECD 471), and Micronucleus Assay
(TG OECD 487), and COMET Assay (Singh et al).
The Ames Reverse Mutation test applies bacterial cells to determine gene
mutations caused by medical devices, materials, or their extracts. Tester strains of
Salmonella typhimurium and one Escherichia coli are exposed to a test material
extract in the presence and absence of a metabolic activation system (S9).
Appropriate positive and negative controls are used to validate the test. Mutagenicity
is evaluated by comparing the number of revertant colonies observed in treated
cultures to those in untreated cultures, whereby a compound is judged mutagenic if a
2-fold or greater increase is observed in a treated culture.
Turnaround time: 7 weeks
4
The in vitro Micronucleus Assay is used to detect chemicals or materials that
induce DNA acentric fragments (chromosome fragments lacking a centromere) or
whole chromosomes that are unable to migrate with the rest of the chromosomes
during the anaphase of cell division. The number of micronuclei (MNi) detected in at
least 3000 mononucleated cells is scored and the frequency of MNi determined.
Turnaround time: 7 weeks
The Comet Assay or single-cell gel electrophoresis (SCGE) is used to measure
deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells resulting from the
effects of medical device extract treatment. Cells embedded in agarose on a
microscope slide are lysed with detergent and high salt to form nucleoids containing
supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH
results in structures resembling comets, observed by fluorescence microscopy; the
intensity of the comet tail relative to the head reflects the number of DNA breaks.
Evaluation of the DNA “comet” tail shape and migration pattern allows for
assessment of DNA damage.
Turnaround time: 4 weeks
Minimum Sample Required
Test Material /
Test
Solid (thickness
<0.5 mm)
Solid (thickness
0.5 – 1.0 mm)
Solid (thickness
>1.0 mm)
Extraction
ratio
Ames Reverse
Mutation
Micronucleus
Assay
COMET Assay
6 cm2/ml
300 cm2
60 cm2
60 cm2
3 cm2/ml
150 cm2
30 cm2
30 cm2
3 cm2/ml
150 cm2
30 cm2
30 cm2
1.25 cm2/ml
62.5 cm2
12.5 cm2
12.5 cm2
E.g. Larger
moulded items
Solid (thickness
>1.0 mm)
E.g. Elastomeric
closures
Irregular shaped
0.2 g/ml
1 0g
5g
solid device
Irregular shaped
porous devices
0.1 g/ml
5g
1g
(low-density
materials)
Liquid
N/A
10ml
20 ml
- Note: Please provide extra for archiving and any unforeseen circumstances.
5
5g
1g
20 ml
Systemic Toxicity Testing – ISO 10993-11, OECD, USP & ICH (in vivo)
Systemic toxicity tests evaluate the potential adverse effects of a medical device on
the body’s organs and tissues which are remote from the site of contact. Five
categories of systemic toxicity are evaluated for:
Acute (24 hours), Subacute (14 to 28 days) Subchronic (90 days), Chronic (6 months
or longer), and Pyrogenicity.
Test Scopes: Acute Systemic Toxicity (ISO, OECD 402 & 423), Subacute Toxicity
(ISO), Subchronic Toxicity (ISO), Chronic Toxicity (OECD, ICH) and Pyrogenicity
Test (USP).
Acute Toxicity tests are generally the first tests conducted. The test provides data
on the relative toxicity likely to arise from a single or brief exposure with the test
material. Standardized tests are available for oral, dermal, intravenous and
intraperitoneal exposures in animals.
Turnaround time:
Limit dose – 6 weeks
Main test – 10 weeks
Subacute, Subchronic, and Chronic Toxicity testing determines the systemic
effect of repeated doses of material/substance or their extracts. The test sample is
administered to groups of animals for 14 or 28 days (subacute), 90 days
(subchronic) and 6 month or longer (chronic). Animals are observed daily for signs of
toxicity with endpoints including changes clinical features, body and organ weights,
necropsy, hematology, clinical chemistry, and histopathology.
Animal Species: Mouse or Rat
Route of Administration: Oral, Intravenous or Intraperitoneal.
Turnaround time:
Subacute (14 days) – 10 weeks
Subacute (28 days) – 12 weeks
Subchronic (90 days) – 5 – 6 months
Chronic (6-9 months) – 9 – 12 months
The Rabbit Pyrogen test evaluates the potential of a material to cause a pyrogenic
response, or fever, when introduced into the blood. The rabbit test is sensitive to
material-mediated pyrogens that may be found in test materials or extracts. The test
is done by injecting the extract of test material intravenously into the ear veins of the
animal and observed for elevated body temperature. An elevated body temperature
greater than 3.8oC for 8 rabbits indicates a positive pyrogenic effect.
Turnaround time:
Main test – 3 weeks
Continuing test – 5 weeks
6
Minimum Sample Required for Systemic Toxicity Tests
Test Material / Test
Extraction
ratio
0.2 g/ml
Pyrogenicity Test*
Main test
Continuing test
24 g
40 g
Solid
Irregular shaped porous device
0.1 g/ml
12 g
(low-density materials)
Liquid
N/A
120 ml
- Note: Please provide extra for archiving and any unforeseen circumstances.
* Where extraction is applied, polar & non-polar extraction is performed
7
20 g
200 ml
Animal: Rat
Solid *
No
1
2
3
4
Test / Test material
Acute
Limit test
Dermal
Toxicity –
Main test
Limit Test
Acute I/P Toxicity
Acute I/V Toxicity
Dose
Acute Oral
Toxicity (TG
OECD 423)
Solid
(thickness
>1.0 mm)
Solid
(thickness
>1.0 mm)
Irregular
shaped
solid device
(g)
Irregular
shaped
porous
device
(lowdensity
materials)
(g)
E.g. Larger
moulded
items
(cm2)
E.g.
Elastomeric
closures
(cm2)
N/A
7
N/A
7
N/A
240
480
N/A
100
200
10
16
32
N/A
8
16
10
40
80
N/A
N/A
N/A
N/A
6,720
N/A
N/A
N/A
N/A
2,800
0.0105
0.105
0.63
4.2
448
N/A
N/A
N/A
N/A
224
0.0105
0.105
0.63
4.2
2240
5,600
896
448
4480
18,000
2,880
1,440
21,600
108,000
17,280
8,640
86,400
Solid
(thickness
<0.5 mm)
(cm2)
Solid
(thickness
0.5 – 1.0 mm)
(cm2)
N/A
N/A
N/A
N/A
480
960
N/A
240
480
N/A
N/A
N/A
N/A
13,440
N/A
N/A
N/A
N/A
6,720
(mg/kg)
5
50
300
2000
14 days
Subacute I/P
Toxicity
28 days
26,880
13,440
13,440
(ISO)
Subchronic
6
I/P Toxicity
90 days
86,400
43,200
43,200
(ISO)
Chronic
7
6 months
518,400
259,200
259,200
(ISO)
I/P – Intraperitoneal I/V – Intravenous
- Note: Please provide extra for archiving and any unforeseen circumstances.
* Where extraction is applied, polar & non-polar extraction is performed.
5
Liquid
(ml)
8
No
1
2
Test / Test material
Solid
(thickness
<0.5 mm)
(cm2)
Animal: Mouse
Solid*
Solid
Solid
(thickness
(thickness
Solid
>1.0 mm)
>1.0 mm)
(thickness
0.5 – 1.0 mm)
E.g. Larger
E.g.
(cm2)
moulded
Elastomeric
items
closures
(cm2)
(cm2)
60
60
25
60
60
25
1680
1680
700
Acute I/P Toxicity
120
Acute I/V Toxicity
120
Subacute I/P
14 days
3360
3
Toxicity
28 days
6720
3360
3360
(ISO)
Subchronic
4
I/P Toxicity
90 days
32400
16200
16200
(ISO)
Chronic
5
6 months
129,600
64,800
64,800
(ISO)
I/P – Intraperitoneal I/V – Intravenous
- Note: Please provide extra for archiving and any unforeseen circumstances.
* Where extraction is applied, polar & non-polar extraction is performed.
9
4
4
56
Irregular
shaped
porous
device
(lowdensity
materials)
(g)
2
2
28
1400
112
56
1120
6750
1080
540
5,400
27,000
4,320
2,160
21,600
Irregular
shaped
solid device
(g)
Liquid
(ml)
10
10
560