The testing protocol contains more information.

Overview

The Ames test uses bacteria cultures to determine whether a substance causes mutations. Because there is a significant overlap between substances that are mutagens and those that are carcinogens, a quick and inexpensive mutagenicity test is useful as an initial screening for possible carcinogens.

Methodology

1. Bacteria Strains

   The Ames test relies on specific strains of S. typhimurium, namely:

   • TA97
   • TA98
   • TA100
   • TA102
   • TA104
   • TA1535
   • TA1537
   • TA1538

   In addition to the S. typhimurium strains, the test may also use the E. coli strain WP2 uvrA pKM101.

2. Principle of Assay

   Each of the bacterial strains has a mutation that prevents it from making an amino acid (a protein building block) required for normal cell growth. The S. typhimurium strains are unable to manufacture histidine; the E. coli strain cannot manufacture tryptophan. The tester strains will not be able to grow without amino acid supplementation, unless the mutated gene is changed back to the correct DNA sequence (reversion mutation). Being exposed to a mutagenic chemical increases the chance that the mutant gene will be restored to the correct sequence, which allows the bacteria to grow and form colonies. Each substance under study is tested in a variety of bacterial strains with different types of altered DNA sequences in order to provide a comprehensive assessment of the mutagenic potential of the substance.

3. Metabolic Conversion

   Some non-mutagenic substances are converted to mutagenic substances when metabolized by the liver; in contrast, some substances that are mutagenic in their natural state are converted to non-mutagens by the liver. All substances are tested in their natural state, as well as in the presence of liver enzymes that can produce this metabolic conversion of the test substance. Extracts of rat, hamster, or mouse liver enzymes (S9 mix) are used for this purpose.

4. Preparing the Cultures

   Multiple sets of cultures are prepared using a range of doses and different amounts of liver enzymes for each bacterial strain. Each culture is prepared in a test tube containing a suspension of one bacterial strain and either an S9 mix or plain buffer solution. Then the test substance is added. Negative control cultures are established using the same ingredients, but without the test substance. Positive control cultures are established using known mutagens active in particular bacterial strains and metabolic conditions (for example, with or without S9). Once prepared, each culture is incubated for 20 minutes at 37° C. Agar is then added to the cultures, and the contents of each tube are poured onto the surface of a Petri dish that was prepared with standard bacterial culture medium. The plates are then incubated, usually for two days.

5. Variations

   Depending on the substance to be tested, the exposure and incubation processes may be modified. These variations include:

   • Elimination of the 20-minute incubation period in a test tube prior to plating (this method is referred to as the standard plate incorporation method)
   • Enzymatic reduction of azo and diazo substances (this process occurs in the intestine or liver of animals) using a chemical called flavin mononucleotide (FMN) or intestinal bacterial preparations from rats
   • Testing in sealed chambers such as desiccators for volatile and gaseous chemicals

Analysis

To validate the test, a trained investigator compares the number of colonies on the chemical-treated plates to the number of colonies on the negative control plates. If the substance under test is mutagenic, the chemical-treated plates will have a much greater number of colonies than the negative control plates.

• A positive response is a reproducible, dose-related increase in mutant colonies in any single strain, with or without the addition of S9 metabolic enzymes. While there is no minimum percentage of increase required for a result to be considered positive, a twofold increase in mutant colonies in a treated plate is usually considered to be a positive (mutagenic) response.
• An equivocal response is any increase that is not reproducible, not dose-related, or not high enough in magnitude to be considered positive.
• A negative response occurs when no increases in mutant colonies are seen in the cultures treated with the test chemical, compared with the control.

The testing protocol contains more information.

Overview

The comet assay examines the ability of substances to cause DNA damage in cells from a variety of different tissues, such as liver, stomach, lung, or brain.

There are multiple forms of DNA damage, including DNA adducts (addition of a chemical structure to a DNA base), oxidative damage to DNA, and breaks in one or both strands. The comet assay can detect single and double strand breaks, DNA adducts, and DNA crosslinks, as well as breaks along the DNA strand that are produced during the normal processes of DNA repair or replication. Thus, the comet assay provides a snapshot of the balance between DNA damage and DNA repair at the time the tissue was harvested.

The DNA damage detected by the comet assay can resolve in several ways. The damage is most often rapidly and correctly repaired. Alternately, a cell that sustains DNA damage may die without passing the damage on to daughter cells following cell division. However, if the damage is not repaired or is repaired incorrectly, the daughter cells may inherit the damage as mutations or chromosome breaks, which are found in many human diseases including cancer. The outcome of the detected DNA damage is unknown.

The comet assay provides the advantage of being able to look at chemical effects on DNA in tissues other than bone marrow, which is the main source of cells used to examine chemical-induced chromosome damage and mutation induction in animals (see Micronucleus Assay).

Methodology

Animals (rats or mice) are treated with a test substance once or multiple times, and DNA damage is usually evaluated 3–6 hours after the final treatment.

The animal tissue is rapidly minced in a buffer solution, combined with agarose, and layered onto slides. The slides are placed in a solution that removes the cellular and nuclear membranes and cellular proteins, leaving behind the nuclear DNA. The slides are then incubated in an alkaline solution (pH > 13) to convert alkali-labile sites into strand breaks and allow the DNA to unwind. Next, the slides are subjected to an electrical field in a tank filled with a buffer solution. Broken DNA, having a negative charge, migrates out of the area of the nucleus toward the positively charged anode. This forms a comet-like shape with the nucleus at the "head" and the migrated DNA as the "tail." All steps of the method are performed using ice-cold solutions and buffers. After this process, the DNA is stained with a fluorescent dye and cells are analyzed using Comet Assay IV Imaging Software.

Analysis

For each cell, DNA migration is analyzed using an automated software system attached to a microscope. The extent of damage is characterized as the percent tail DNA (how much DNA migrated to the tail, compared to the total DNA as a percentage). At least 100 cells per sample are evaluated; current guidelines for the assay recommend analysis of at least 150 cells per sample. The data is statistically analyzed to determine if there is a significant increase in percent tail DNA in chemical-exposed animals compared to the controls (animals not exposed).

Dose-related responses, as well as significant dose groups, are considered during the evaluation of the data. For a positive test, both a dose response and at least one significant dose group are required. If only one of these measures is present, the test is considered to be equivocal. The absence of either condition results in a negative test. The final conclusions for comet assays are determined by considering the results of statistical analyses, reproducibility of any observed effects, and the magnitude of the effects. Ultimately, both biological and statistical significance are considered when reaching a final conclusion.

The testing protocol contains more information. Detailed explanations of the micronucleus assay methods and cell scoring are also available.

Overview

The erythrocyte micronucleus test examines the ability of substances to cause chromosome damage in developing red blood cells inside bone marrow. A micronucleus (literally, "little nucleus") is a biomarker of structural chromosome damage (e.g., breaks) and changes in chromosome number (e.g., chromosome loss). If a chromosome is broken or fails to migrate properly with other chromosomes during cell division, the broken piece or lost chromosome will form a small (micro) nucleus of its own in one of the two daughter cells produced. Detection of chromosomal damage is important because it has been linked to birth defects, infertility, and certain diseases, including cancer.

The micronucleus test is performed in rapidly dividing cell populations, and bone marrow is a site of rapid blood cell division. Both bone marrow and peripheral blood samples can be examined for the presence of micronuclei in red blood cells.

Methodology

Animals are treated with a test substance once or daily over a period of time, and the frequency of micronucleated cells is determined 24–48 hours after the final treatment, depending on the source of the cells (bone marrow or blood) that are examined and the treatment regimen. If treated animals show significantly higher frequencies of micronucleated red blood cells than untreated animals, the test substance is considered capable of causing chromosomal damage.

1. Bone Marrow Tests

   The test subjects are rodents (rats or mice) that are exposed to the test substance, usually by oral gavage or injection. Typically, one to three daily treatments of the test substance are administered over a range of doses; the highest dose tested is near the maximum tolerated dose. Groups of negative and positive control animals are included for each test. Bone marrow samples are obtained from all animals 24 hours after the last treatment, and red blood cells are examined for the presence of micronuclei.

2. Peripheral Blood Tests

   Micronucleus tests are performed on male and female rodents that are exposed to the test substance orally, dermally, or by inhalation in subchronic (e.g., 28 or 90 days) or short term (e.g., 48–72 hours) toxicity studies. 24 to 48 hours after the final exposure, peripheral blood samples are obtained and red blood cells are examined for the presence of micronuclei. Samples may be examined using standard slide scoring procedures or automated flow cytometric approaches. Since 2007, NTP has routinely relied on flow cytometry to evaluate the frequency of micronucleated cells in peripheral blood.

   Peripheral blood micronucleus assays are typically integrated into all NTP toxicity studies, and they also lend themselves to serial monitoring of micronucleus frequencies over time, since blood samples can be obtained without the need to sacrifice the animals.

Analysis

Blood or bone marrow samples from treated animals are compared to samples from untreated animals. With the slide-based approach for data acquisition, 1000–2000 cells were scored per animal. Using flow cytometric procedures to evaluate blood samples, 20,000 immature red blood cells and around one million mature red blood cells are examined per animal, resulting in a marked increase in the ability to detect small changes in the frequency of micronucleated red blood cells. The data is analyzed using statistical methods to determine if there is a significant increase in the frequency of cells containing micronuclei. Both dose-related responses and the magnitude of response for each independent dose group are considered during the evaluation of the data. For a positive test, both a significant dose response (trend) and at least one significant dose group are required. If only one of these measures is present, the test is determined to be questionable. The absence of either condition results in a negative test. The final conclusions for micronucleus tests are determined by considering the results of statistical analyses, reproducibility of any observed effects, and the magnitude of the effects. Ultimately, both the biological significance as well as statistical significance is considered when reaching a final conclusion.