For biocompatibility tests that require genotoxicity, we offer multiple test types, including Ames Mutagenicity, Chromosomal Aberration, Mouse Lymphoma and Mouse Micronucleus, depending on your product type and data requirements. These tests comply with the OECD and ISO guidelines as one of three in vitro test levels for genotoxicity.
The Ames Mutagenicity test is used to determine the potential mutagenic activity of an extract from a medical device / material. The Bacterial Reverse Mutation Test (Ames test) is performed as part of the genotoxicity battery of tests to determine if leaks from a medical device / material are mutagenic.
The Chromosomal Aberration test is used to scan medical devices / supplies to determine if they are causing structural chromosomal abnormalities in Chinese Hamster Ovary (CHO) cells. The test is performed on devices / materials with permanent patient contact, prolonged patient contact, or blood contact. The Chromosomal Aberration test complies with OECD and ISO guidelines as one of three levels of in vitro testing for genotoxicity.
Mouse Lymphoma testing often uses mammalian cell cultures to identify gene mutations, change in chromosome structure and number, and other gene toxicities caused by medical devices, material or extracts. These tests are used to ensure that devices that have long-term contact with the body do not reveal genotoxicity that could lead to gene mutations with cancer potential.
The Mouse Micronucleus assay is used to evaluate the potential of a medical device to induce micronucleus formation in immature polychromatic erythrocytes found in the bone marrow of adult mice.
Ames Mutagenicity
The Ames Mutagenicity test uses several Salmonella typhimurium strains selected for their susceptibility to mutation due to:
Test strains also require histidine for growth due to a mutation in the gene that controls histidine production. Ames tests are performed by mixing an extract of the test substance with the test organism in a soft agar solution containing only a small amount of histidine. Histidine allows the inoculated test organism to enter a limited number of compartments, but is insufficient to allow normal growth.
However, if the strain is subjected to a reverse mutation (spontaneous or induced by the test substance or a positive control material), the organism no longer requires histidine to grow and can produce a visible colony or reverser. Mutations to the test organism only in the histidine gene region cause the test organism to reverse mutation into an organism, which no longer requires histidine.
Test strains are selected to detect various types of mutagen. The test strains used are TA97A, TA98, TA100, TA102 and TA1535. A spot test is also performed in which extracts of the test product are stained on a plate containing a small amount of histidine-containing test organism. As the test substance spreads through the agar, it becomes more diluted. This allows the detection of mutagens that cause some inhibition of the tester strains. Tests are done with S-9 activation or without S-9 activation. The S-9 activation system is designed to simulate mammalian liver enzyme systems and is used to detect substances undergoing metabolic activation from non-mutagenic forms.
The Chromosomal Aberration test is performed by exposing the CHO cells to the test substance extract. A bleed medium is included in the assay as a negative control.
CHO cells are seeded into 75 cm 2 cell culture flasks and incubated with cell culture medium + 10% serum until 40-60% confluence.
CHO cells are exposed to the test product extract in the presence and absence of a metabolic activation system. The metabolic activation system is a mixture of S9 homogenate, isocitrate and NADP. Positive controls used are Cyclophosphamide with metabolic activation and Mitomycin C without metabolic activation.
Before cell harvest, cells are examined for cytotoxicity. Vials are scored according to the degree of distinguishable morphological cytotoxicity on a relative scale of 10993 - 5 according to AAMI / ANSI / ISO 87-0 and USP <4>. The average of the results from the three bottles is averaged to give a final cytotoxicity score.
At the end of the exposure and expression periods, cells are arrested at the metaphase with coltazide. The cells are then removed from the vials and treated with a hypotonic solution and fixative. Cells are deposited on microscope slides. The chromosomes are stained with Giemsa stain and covered with Permount®. The cells are then examined microscopically for chromosomal abnormalities.
Biocompatibility In the genotoxicity test, mouse lymphoma cells are used to determine whether a test material is capable of inducing point mutations or clastogenic (chromosomal breakage) in a cultured mammalian cell line. In this case, the target gene, thymidine kinase (TK), is a dispensable gene. Loss of TK activity indicates that a mutation has occurred. Since TK is involved in a rescue pathway derived from DNA metabolism, cells can synthesize thymidine de novo. Therefore, loss of TK does not render the cell inanimate.
Mutants can be selected and mutant frequencies can be derived by including the thymidine analogue (trifluorothymidine or TFT) in the culture medium of cells after exposure to the test material. Regular cells with intact TK include the analog and template; Mutant cells (lacking TK activity) survive, form colonies and quantify.
In the Mouse Micronucleus test, a medical device is removed in a polar and non-polar environment. Test product extract positive controls and negative controls are administered at 20 mL / kg in both male and female mice. Bone marrow is then harvested 24 and 48 hours after dosing and examined for the presence of micronuclear polychromatic erythrocytes (PCEs). The presence of PCEs is an indication of the mutagenic substance leaking from the device.
EUROLAB, with its highly expert team and internationally accredited laboratories, Genotoxicity Test It performs all test, analysis, measurement, evaluation and report generation services completely and reliably.
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