Special considerations for conducting genotoxicity tests with protein materials

• Published in: Mutagenesis 1995-09, Vol.10 (5), p.393-398
• Main Authors: Kirkland, David J., Kim, Norman N.
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Cells, Cultured; Chemical mutagenesis; Drug Contamination; Escherichia coli; Escherichia coli - drug effects; Humans; Mammals; Medical sciences; Mutagenicity Tests - economics; Mutagenicity Tests - instrumentation; Mutagenicity Tests - methods; Proteins - isolation & purification; Proteins - toxicity; Recombinant Proteins - economics; Recombinant Proteins - isolation & purification; Recombinant Proteins - toxicity; Research Design; Salmonella typhimurium; Salmonella typhimurium - drug effects; Solutions; Toxicology
• ISSN: 0267-8357; 1464-3804
• DOI: 10.1093/mutage/10.5.393

Standard genotoxicity tests are often inappropriate for testing new biological entities, in particular for recombinant proteins which are nature-identical. Arguments that these may contain mutagenic impurities are not substantiated; however, we have produced evidence that such impurities would be detected amidst a vast excess of protein. Concerns that human patients receiving therapy may be at risk from higher-than-physiological levels of proteins are also somewhat theoretical. However, it is apparent that genotoxicity testing will be required for these products for the time being, even if pragmatic approaches reduce the battery of in vitro tests to Ames and chromosomal aberrations only, and reduce the top dose in vivo to 1000 × the human therapeutic dose. There is a number of physical and chemical properties of proteins that demand special approaches to methodology if the tests are to produce accurate results. The potential for adsorption to certain forms of glass and plastic means special care must be taken in dissolving and diluting test solutions; adherence to filters means special low protein binding, non-pyrogenic filters should be used for sterilisation of test solutions, where this is necessary; freeze-dried powders aliquotted in multiple vials should be dissolved in minimal solvent and cascaded from vial to vial rather than trying to empty the solid contents for bulk weighing. As proteins are often supplied in solution, in order to achieve sufficiently high test concentrations, it may be necessary to resuspend test bacteria/cells in the test solutions for short periods of time before centrifuging and resuspending in selective or growth media. The possibility of release of histidine or tryptophan from the test article that could enhance the frequency of spontaneous Salmonella typhimurium and Escherichia coli mutants in the Ames test may need to be controlled by washing bacteria after a liquid treatment phase. Finally, mammalian cell treatments may need to be conducted in the absence of serum (and therefore be restricted to a few hours) in order of avoid degradation of test article by serum proteases.