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What bacterial strains do you assess and why?
Cyprotex offer the Ames test as an early stage assessment of genotoxicity using a miniaturised screening version which requires less compound and evaluates two of the most common mutations, TA98 and TA100.
TA98 (frameshift mutation) and TA100 (base-pair substitution) are two common strains of Salmonella typhimurium strains assessed in Ames testing. Both strains have:
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rfa mutations, a defective lipopolysaccharide layer that makes bacteria more permeable to larger molecules
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uvrB mutations, which eliminate excision repair of DNA damage
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the pKM101 plasmid, which increases error-prone repair of DNA damage
Why is it important to investigate mutagenicity of compounds?
Compounds that have been demonstrated to induce genetic mutations, chromosomal breaks, and/or rearrangements are considered genotoxic and have the potential to cause cancer in humans. In some cases it is not the compounds themselves that are genotoxic, but impurities generated in the manufacturing process; thus it is important to identify and test these impurities as well as the compounds.
Why should I perform the Ames MPF™ Mutagenicity Assays rather than the standard plate incorporation test?
The Ames MPF™ / Ames II Mutagenicity Assays are liquid microplate modifications of the standard Ames test which offer a higher speed format, easier handling, and the possibility of automated plating and plate reading. The assays are fast and efficient, show good correlation with the standard Ames test, and consume less test compound and consumables than the standard Ames test.
Please provide an overview of the Cyprotex Ames MPF™ mutagenicity assay.
Compounds are tested at the highest concentration at which they are soluble in aqueous solutions, or at 2 mg/mL. Actively growing cultures of TA98 and TA100 are exposed to serial dilutions of the compound for 90 minutes in medium containing sufficient histidine to support two cell divisions, then diluted into a histidine-free medium containing an indicator of reversion. Small aliquots are dispensed into 48 wells of multiwell plates in triplicate. The cells are incubated for 48 hours, by which time cells that have undergone reversion will grow into colonies. Metabolism by the colony changes the pH of the medium in that well, which changes colour. The number of wells containing revertant colonies are counted for each dose of compound and compared to a zero dose (vehicle) control. An increase in the number of revertants relative to the zero dose control indicates that the compound is mutagenic.
What controls are included in the Ames assay?
Positive controls are known mutagens 2-nitrofluorene + 4-nitroquinolone-N-oxide for the assay without S9 and 2-aminoanthracene for the assay with S9. The negative control is 4% DMSO.
What are the regulatory requirements for genotoxicity assessment?
The general features of a standard test battery for regulatory genotoxicity studies include the assessment of mutagenicity in a bacterial reverse gene mutation test (e.g., Ames test) and the assessment of genotoxicity in mammalian cells in vitro and/or in vivo.
Two options are proposed in the 2011 ICH guidance S2(R1)9:
Option 1
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A test for gene mutation in bacteria.
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A cytogenetic test for chromosomal damage (the in vitro metaphase chromosome aberration test or in vitro micronucleus test), or an in vitro mouse lymphoma Tk gene mutation assay.
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An in vivo test for genotoxicity, generally a test for chromosomal damage using rodent hematopoietic cells, either for micronuclei or for chromosomal aberrations in metaphase cells.
Option 2
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A test for gene mutation in bacteria.
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An in vivo assessment of genotoxicity with two different tissues, usually an assay for micronuclei using rodent hematopoietic cells, and a second in vivo assay. Typically this would be a DNA strand breakage assay in liver, unless otherwise justified.
For compounds that give negative results, the completion of either option of the standard test battery, performed and evaluated in accordance with current recommendations, will usually provide sufficient assurance of the absence of genotoxic activity and no additional tests are warranted. Compounds that give positive results in the standard test battery might, depending on their therapeutic use, need to be tested more extensively.
In cases where compounds are highly toxic to bacteria (e.g., some antibiotics), the bacterial reverse mutation (Ames) test should still be carried out, just as cytotoxic compounds are tested in mammalian cells, because mutagenicity can occur at lower, less toxic concentrations. In such cases, any one of the in vitro mammalian cell assays should also be done, i.e., Option 1 should be followed.
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