BIOCOMPATIBILITYLasMed

Technical University of Lodz, POLAND

Institute of Applied Radiation Chemistry, Technical University of Lodz, POLANDWroblewskiego 15, 90-924 Lodz, Poland, tel: +4842 6313184

Radoslaw Wach, Alicja Olejnikwach@mitr.p.lodz.pl; olejnik@mitr.p.lodz.pl

http://www.mitr.p.lodz.pl/biomat

Biocompatibility

Is the product safe for people?

Are there any unwanted biological effects?• local e.g. cell death, irritation• systemic effects e.g. sensitisation, toxicity,

genotoxicity

Biological evaluation ISO 10993

The International Standards Organization (ISO) developed document:

ISO 10993 “Biological Evaluation of Medical Devices”

a set of harmonised standards for evaluating thebiocompatibility of a medical device prior to a clinical study;

ISO 10993 consists of the following partsISO 10993-1:2009 Part 1: Evaluation and testingISO 10993-2:2006 Part 2: Animal welfare requirementsISO 10993-3:2003 Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicityISO 10993-4:2002 Part 4: Selection of tests for interactions with bloodISO 10993-5:2009 Part 5: Tests for in vitro cytotoxicityISO 10993-6:2007 Part 6: Tests for local effects after implantationISO 10993-7:2008 Part 7: Ethylene oxide sterilization residualsISO 10993-9:2009 Part 9: Framework for identification and quantification of potential degradation

productsISO 10993-10:2010 Part 10: Tests for irritation and delayed-type hypersensitivityISO 10993-11:2006 Part 11: Tests for systemic toxicityISO 10993-12:2007 Part 12: Sample preparation and reference materialsISO 10993-13:2010 Part 13: Identification and quantification of degradation products from polymeric

medical devicesISO 10993-14:2001 Part 14: Identification and quantification of degradation products from ceramicsISO 10993-15:2000 Part 15: Identification and quantification of degradation products from metals and

alloysISO 10993-16:2010 Part 16: Toxicokinetic study design for degradation products and leachablesISO 10993-17:2002 Part 17: Establishment of allowable limits for leachable substancesISO 10993-18:2005 Part 18: Chemical characterization of materialsISO/TS 10993-19:2006 Part 19: Physico-chemical, morphological and topographical characterization

of materialsISO/TS 10993-20:2006 Part 20: Principles and methods for immunotoxicology testing of medical devices

Categorization of medical devicesthe nature of body contact• non-contact devices• surface-contacting devices (skin, mucosal membranes,

breached or compromised surface)• external communicating devices (blood path – indirect,

tissue/bone/dentin, circulating blood)• implant devices (tissue/bone, blood)

the duration of body contact• limited exposure - use up to 24 h• prolonged exposure - use exceed 24 h but not 30 days• permanent contact - use or contact exceeds 30 days

Biological tests – initial and supplementary evaluation test (according with ISO 10993-1)

Advantages of in vitro tests

Controlled testing conditionsLack of systemic effectsReduction of variability between experimentsTesting is fast (and cheap)Small amount of test material is requiredLimited amount of toxic waste is producedHuman cells and tissues can be usedTransgenic cells carrying human genes can be usedReduction of testing on animals

Limitations of in vitro testsGeneral toxic effects cannot be assessed (e.g. weight reduction)In vivo dose-responses cannot be obtained (for human risk assessment)Systemic effects cannot be evaluatedInteractions between tissues and organs cannot be testedPharmacokinetics cannot be evaluatedSpecific organ sensitivity cannot be assessedChronic effects cannot be tested

Selection of portion from a deviceIf a device cannot be tested as a whole, each individual material in the final product shall be represented proportionally in the test sample;

The test sample of devices with surface coatings shall include both coating material and the substrate;

Composite materials shall be tested as finished materials;

The test sample shall be chosen to maximize the exposure of the test system to the components of a device that are known to have a potential for a biological response;

Preparation of extractsExtraction conditions based on common practices are as follows:

(37±1)°C for (24±2)h or(37±1)°C for (72±2)h

Extraction using both polar and non-polar solvents:• polar medium: water, physiological saline; culture media

without serum;• non-polar medium: freshly refined vegetable oil (e.g.

cottonseed or sesame oil);• additional media: ethanol/water, ethanol/saline,

polyethylene glycol 400 (diluted to a physiological osmotic pressure), dimethyl sulfoxide and culture media with serum;

Standard surface areas and extract liquid volumes (according to ISO 10993-12)

Thickness mm

Extraction ratio (surface area or mass/volume)

±10%

Forms of material

<0,5

6 cm2/ml

film, sheet, tubing wall

0,5 to 1,0

3 cm2/ml

tubing wall, slab, small molded items

items -

>1,0

1.25cm2/ml

larger molded item(s)

Irregularly shaped

solid devices 0.2 g/ml

powder, pellets, foam, non-absorbent,

moulded items

Irregularly shaped porous devices (low-density materials) 0.1 g/ml membranes

NOTE - While there are no standardized methods available at present for testing absorbents and hydrocolloids, the following is a suggested protocol: Determine the "absorption capacity" of the material, i. e. the amount of extract liquid absorbed per gram of the material. The test sample shall be 0,1 g/ml beyond the absorptive capacity of the material.

Cytotoxicity - ISO 10993-5

This in vitro test evaluates the potential of the materials/devices or their extracts to cause damage to cells in culture;

Different methods of establish cytotoxicity• qualitative (morphological estimation)• quantitative (e.g. NRU, MTT assay, LDH assay)

cell line L929

Haemocompatibility - ISO 10993-4

These tests evaluate, using an appropriate model or system, the effects of blood-contacting medical devices or materials on blood or blood components;

Blood interactions - thrombosis, coagulation, platelets, haematology and complement system;

Haemolysis tests determine the degree of red blood cell lysisand the release of haemoglobin caused by medical devices, materials and/or their extracts in vitro.

red cells

lymphocyte T

platelets

monocyte

red cells

lymphocyte T

platelets

monocyte

Pyrogenicity test or LAL test - ISO standard 10993-11

Pyrogenicity is the ability of a chemical agent or other substance to produce a febrile response. Pyrogenic responses may be material-mediated, endotoxin-mediated or mediated by other substances, such as components of gram-positive bacteria and fungi;

The rabbit pyrogen test – in vivo – suitable for detect all kind of pyrogenicity;

LAL testLimulus Amebocyte Lysate Test

The LAL test - in vitro – evaluates the potential of medical device extracts to cause lysate from horseshoe crab amebocyte to clot;The LAL test is not suitable for determining the pyrogenicity of these non-endotoxin substances;Test on final product (extracts from medical devices);3 items - approximately 3 weeks;

Horseshoe crab (Limulus polyphemus) Limulus Amebocyte Lysate Test Kit

Genotoxicity - ISO 10993-3

tests use mammalian or non-mammalian cell culture allow to determine gene mutations, changes in chromosome structure and number, and other DNA or gene toxicities caused by medical devices or their extracts;• a test for gene mutations in bacteria – Ames test (OECD

471) – detects point of mutation;• In vitro mammalian cell gene mutation test in mouse

lymphoma cells (OECD 476) – detects mutations and aberration;

• In vitro mammalian chromosome aberration test in human lymphocytes (OECD 473) – detects visible aberrations;

Approximately 4 months – 3 items (depends on devices size)/test;

BiodegradationDegradation is a process of lost of mechanical properties and/or chemical integrity through chemical changes in the material induced by environmental condition;

There are separate standards, which describe methods for identification and quantification of potential degradation products from polymers (ISO 10993-13), ceramics (ISO 10993-14) and metals and alloys (ISO 10993-15);

Biodegradation of polymers (ISO 10993-13)

Methods evaluate degradation of polymers –• real-time degradation (37±1ºC)• accelerated degradation (70±1ºC)

Accelerated degradation of polymer - screening methods(no degradation products - the real-time degradation is not necessary);Evaulation: mass reduction, molecular weight, FTIR, UV of leachables, morphology of surface and cross-section

LasMedTechnical University of Lodz, POLAND

Institute of Applied Radiation Chemistry, Technical University of Lodz, POLANDWroblewskiego 15, 90-924 Lodz, Poland, tel: +4842 6313164

Radoslaw Wach, Alicja Olejnikwach@mitr.p.lodz.pl; olejnik@mitr.p.lodz.pl

http://www.mitr.p.lodz.pl/biomat