Dietary Supplements A Framework for Evaluating Safety (2005) / Chapter Skim
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7 Categories of Scientific Evidence--In Vitro Data
7 Categories of Scientific Evidence--In Vitro Data
Pages 217-234
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From page 217... ...
The first section of this chapter describes the unique power of in vitro studies and considerations for interpreting them. The next section describes various types of in vitro data that may be available, with descriptions of 1In this report, in vitro assays are considered validated when their results have been proven to predict a specific effect in animals and/or humans with reasonable certainty (not necessarily universally accepted or without detractors)
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From page 218... ...
These systems include isolated organs, isolated cells, microorganisms, subcellular organelles, and molecular entities such as enzymes, receptors, transport proteins, isolated membranes, and genes or gene fragments. A primary advantage of conducting in vitro studies is that their reductionist approach allows insight into a compound's mechanisms of action that might be more difficult to obtain in a "wholeanimal study." The control possible with in vitro experiments enables examination of effect on the target process or structure in isolation from confounding factors.
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From page 219... ...
Knowledge of a dietary supplement ingredient's pharmacokinetics and in vivo metabolism, if available from animal or human studies, will allow the most appropriate interpretation of the relevance of compound concentrations used in in vitro experiments to amounts ingested by humans. GENERAL TYPES OF IN VITRO ASSAYS As described above, in vitro assays are valuable because they allow scientists to answer questions that otherwise may not be answerable due to various constraints with whole-animal and human studies.
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, changing the action potential duration in cardiac cells. In the pharmaceutical world, concern about drugs that may induce this syndrome has led to the development of wellaccepted in vitro assays to identify drugs that may exert this adverse effect on potassium channels (Roche et al., 2002)
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From page 221... ...
Assay Types by Experimental System The previous paragraphs organized in vitro assays by the effects assessed: effects on enzymes, ion channels, and nucleic acids. These and other in vitro tests may be conducted in a variety of experimental systems, such as isolated tissues or in isolated cells, each of which has its own considerations.
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From page 222... ...
Another approach is to add substrates to cell cultures that replace and/or activate metabolic enzymes, thus producing reactive metabolites that simulate in vivo metabolism of a given chemical/dietary supplement. VALIDATED IN VITRO ASSAYS In vitro studies vary in their value as predictors of harm.
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From page 223... ...
Although the regulatory situation is such that completion of these or other in vitro tests is not required for dietary supplement ingredients, the scientific value of these assays in predicting adverse effects in humans is as valuable in assessing the safety of dietary supplements as it is in assessing the safety of other substances. For example, specific types of in vitro tests are used by the Environmental Protection Agency (EPA)
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Many in vitro studies can be useful for this purpose. SERIOUSNESS OF HARM PREDICTED BY THE ASSAY In addition to considering the correlation of the particular in vitro assay with a particular adverse health outcome, it is of utmost importance to consider the seriousness of the outcome when evaluating the level of concern warranted by results of in vitro studies.
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In vitro information such as that described in the left column warrants lower levels of concern, while information described in the middle column warrants concern, but additional information may be required to warrant conclusion that a risk exists. ANNEX 7-1 USE OF GENETIC TOXICOLOGY INFORMATION Genetic toxicology is the study of the ability of substances to cause selective damage to the DNA of living organisms (or RNA in the case of RNA viruses)
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This is the type of mutation that occurs in the heritable human disease sickle-cell anemia. Agents that cause these kinds of genetic effects are detected using assays that detect changes in specific genes, such as the Ames assay in Salmonella bacteria or the mouse lymphoma TK+/- assay in mammalian cells in vitro.
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Because of the importance of genetic alterations in cancer development, and the observation that many carcinogens were also mutagenic, the original focus of genetic toxicology on evaluating potential hazards to the human gene pool shifted in the early 1970s to the use of genotoxicity assays as rapid screens for potential carcinogens (Ames et al., 1973, 1975; Bridges, 1976)
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. Some programs require a mammalian gene mutation assay in addition to the typical Ames bacterial gene mutation assay, and the test battery may include an assay for induction of DNA repair.
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, but tests held in such high regard by a governmental agency do, on their own, indicate a potential for significant or unreasonable risk if results are not mitigated by other information. In addition to mutagenicity tests, there is the opportunity to examine agents of interest in a battery of other in vitro tests that measure cytotoxicity, structural chromosomal aberrations, DNA damage, developmental toxicity, estrogenicity, and cell transformation (there are many other specific tests that could be used and these examples should not be viewed as inclusive)
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Specifically, the guidelines for specific toxicity studies suggest the following short-term tests for genetic toxicity: · Bacterial Reverse Mutation Test, · In Vitro Mammalian Chromosome Aberration Test, · In Vitro Mouse Lymphoma TK+/- Gene Mutation Assay, and · In Vivo Mammalian Erythrocyte Micronucleus Test. FDA considers that positive results from these tests are sufficient to
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In fact, dietary supplement ingredients may sometimes be ingested in higher amounts than food additives. If these tests, or similarly accepted tests, are positive for dietary supplement ingredients or products, then it is scientifically justifiable to consider that a risk exists unless there is mitigating information.
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2001. Applications of gene arrays in environmental toxicology: Fingerprints of gene regulation associated with cadmium chloride, benzo(a)
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picolinate pro duces chromosome damage in Chinese hamster ovary cells.
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1990. Evaluation of four in vitro genetic toxicity tests for predicting rodent carcinogenicity: Confirming of earlier results with 41 additional chemicals.
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