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.

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: 14 working 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 working days

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.

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: 6 weeks (per extract)

 

The Closed Patch Test (Buehler) involves multiple topical doses and is recommended for devices that will contact unbroken skin only.

Turnaround time: 7 weeks (per extract)

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.

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: 17 working days

 

The Primary Skin Irritation test is usually performed for topical devices that have external contact with intact skin. The test material or an extract is applied directly to intact sites on the skin of a rabbit. After a 4-hour exposure, the material is removed and the sites are scored for erythema and oedema.

Turnaround time: 17 working days

Genotoxicity Testing 

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: 35 working days

 

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: 35 working days

 

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: 20 working days

Systemic Toxicity Testing – ISO 10993-11, OECD, USP 

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. Two categories of systemic toxicity are evaluated for:

Acute (24 hours) and Pyrogenicity

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: Oral, Intraperitoneal, Intravenous or Dermal (4 weeks) 

 

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 – 17 working days

Continuing test – 17 working days