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The purpose of the bacterial reverse mutation assay is to evaluate a chemical's genotoxicity by measuring its ability to induce reverse mutations at selected loci in several bacterial strains.The assay, commonly referred to as the Ames test, was originally developed by Dr. Bruce Ames and others in the early 1970’s. It is sensitive to a wide range of mutagenic and carcinogenic chemicals (1, 2, 3). This simple, quick and inexpensive genotoxicity assay is one of several genotoxicity assays required for product safety testing of a variety of products including drugs, medical devices, food additives, industrial chemicals and pesticides.

This assay measures genetic damage at the single base level in DNA by using five or more tester strains of bacteria. The Salmonella typhimurium and Escherichia coli strains used in the assay each have a unique mutation that has turned off histidine biosynthesis in Salmonella or tryptophan biosynthesis in E. coli (4, 5). Because of these original mutations, the bacteria require exogenous histidine or tryptophan to survive and will starve to death if grown without these essential nutrients (auxotrophy). The key to the assay is the bacteria can undergo a reverse mutation turning the essential gene back on permitting the cell to grow in the absence of either histidine or tryptophan (prototrophy). Each bacterial strain was created by a specific type of mutation - either a base-pair substitution or frame-shift mutation. Because a reverse, compensating mutation usually must occur by the same mutagenic mechanism, mechanistic toxicology information is also available from Ames assay results based on the pattern of which strain(s) reverted.

The standard Ames assay designs include preliminary toxicology tests or combined toxicity and mutation tests followed by a definitive mutation assay. In both toxicity and mutation tests, tester strains are combined with S9 mix or buffer, test or control article, a trace of histidine or tryptophan and molten agar. The bacteria use the trace histidine or tryptophan to undergo several cell divisions, but will stop growing once they have run out, leaving a characteristic “background lawn” that decreases in density with increasing toxicity. After 48 hours, only those cells that have undergone a reverse mutation turning the essential gene back on have survived, producing mutant colonies. The background lawn density is scored followed by counting the number of revertant colonies. Mutation results are reported as revertants per plate. Figure 1A shows a vehicle control with strain TA 100 and figure 1B shows a positive control plate with TA 100.

Fig.1: Ames Bacterial Reverse Mutation Assay strain TA 100
A: Vehicle Control	B: Positive Control

Each strain has a characteristic spontaneous background rate and positive control response pattern. Assay evaluation criteria for a positive response look for a dose-related increase of revertant colonies in test article treated cells over concurrent vehicle controls.

Special modifications to the Ames assay

The bacterial reverse mutation assay is remarkable in its flexibility and adaptability to unique testing requirements. Many types of test materials have been successfully tested in the Ames assay - often with custom modifications. The following is a list of several custom modifications available to the Ames assay:

• Gasses and volatile liquids
  There are a number of approaches for dosing Ames assay plates that involve the metered exposure of compressed gasses or volatile liquids.
• Chemical class-specific modifications
  Experience has shown that some chemical classes respond poorly or not at all under standard assay conditions. Specific modifications include use of the “preincubation” method of dosing for some chemicals, use of different species or enzyme induction method for S9 or use of reductive S9 conditions for diazo compounds. Some chemicals with known structure or activity may require the use of a specific strain such as strain TA 102 with cross-linking agents. In these cases, experience is essential in designing and conducting a successful assay.
• Environmental samples
  Often biological monitoring of environmental samples provides rapid identification of hazard. Wastewater can be collected using resin exchange columns and extracts testing in the Ames assay. Fugitive gaseous emissions from industrial plants and combustion sources can be sampled and tested.
• Batch monitoring
  Specific products or raw materials know to have a specific problem can be monitored by running just the one bacterial strain and/or activation condition that picks up the problem.
• Medical devices and biomaterials
  Of special concern for some products is what can be extracted or leached from materials implanted in the body or used to store products. Designs are available to evaluate the mutagenicity of polar and nonpolar extracts of such materials
• Human monitoring
  Methods are available for the evaluation of mutagenicity of extracts of human urine. This approach can help monitor humans for exposure to mutagens in occupational and clinical situations.
• Photomutagenicity
  Chemicals may be photoactivated to reactive, mutagenic forms. Modifications are available to co-treat the bacterial cells with test article and photo-illumination.

Salmonella / Escherichia coli Reverse Mutation Assay