The field of toxicology frequently uses short-term tests, known as bioassays, to determine the presence and relative concentrations of toxicants in various biological samples. One of the most well know and frequently used bioassays is the Ames Test. Others include the various MicrotoxÔ tests, MutatoxÔ, AMAX IIÔ, as well as the numerous invertebrate, fish and amphibian tests for aquatic toxicity. Despite their widespread use, many bioassays serve as imperfect data retrieval systems for the detection of toxic agents for several reasons: the tests are costly, time-consuming, poorly suited for field applications, yield imprecise results, and are scientifically complex. Moreover, most bioassays have had limited utility in ascertaining the toxicity of complex particulate samples because suspected toxicants must be extracted from contaminated sediments before testing can begin.

ETL’s Tetramitus Assay measures the effects of toxic agents on the flagellate phenotype of Tetramitus rostratus, a protozoan organism. When a sample concentrate or particle suspension containing toxic agents is added to a Tetramitus culture, cell division is inhibited in direct proportion to the concentration of individual or multiple toxic agents. The Tetramitus Assay measures the dose-response relationship, thus indicating the presence and degree of toxicity in that sample. Because Tetramitus flagellates have a gullet and are particle feeders, the assay allows for the measurement of whole particle toxicity without prior extraction or solvent substitution procedures. This feature is a significant advancement in the field of toxicity testing and is but one of many that distinguishes the Tetramitus Assay from other bioassays.

 ETL has focused its research on applying the Tetramitus Assay to water testing, where the assay has significant environmental implications. Standard monopollutant water testing measures only the levels of individual toxicants and compares this amount with levels deemed acceptable by various regulatory agencies. The Tetramitus Assay is particularly important because it measures the total toxicity of a sample, thereby reflecting the synergistic or antagonistic effects of multiple toxicants. The test provides a comprehensive screening for cytotoxicity, thereby minimizing the need for expensive tests for specific toxicants. The assay’s whole particle analysis capability is a noteworthy breakthrough in the water testing field because particles contained in sediment contain higher levels of toxicity than the dissolved components of the water sample itself. In addition to its application of analyzing the toxicity of water samples, ETL plans to develop the protocol for use of the assay in other biological (food, urine, etc.) and air samples. 

The Tetramitus Assay has many important characteristics that will enable it to achieve a leadership position in the field of toxicity testing. The assay’s whole particle testing capability is unique amongst bioassays. The Tetramitus Assay, which can detect toxicants at low concentrations, is sensitive to a broad range of mutagens, including aminobiphenyl (main carcinogen in cigarette smoke), 2-aminoflourene (a nitroaromatic similar to many dyes), benzo[A]pyrene (a combustion by-product), and MX (predominant mutagen found in chlorinated water). Dose-response curves are also demonstrated with compounds positive for Salmonella strains TA97, TA98, TA100 ad TA102. In a test with 31 coded samples there were no false positives or negatives (on the basis of Ames Test values). Dose-response curves for three mutagens (aminobiphenyl, cadmium and para-phenylenediamine) measured three years apart, showed excellent agreement. Furthermore, repeat assays for 2-aminoflourene using populations of flagellates growing at two vastly different initial growth rates, were in close agreement. These results demonstrate the robustness of the Tetramitus Assay. Because of the Tetramitus Assay’s positive response to diverse types of DNA damaging agents, there is strong circumstantial evidence that the Tetramitus Assay is, in fact, measuring genotoxicity. The technology behind the Tetramitus Assay is scientifically simple. Very little technical training is required to run the test and minimal scientific equipment is needed. Further, results are achieved rapidly, the test is cost-effective and environmentally friendly. ETL has developed software which facilitates the statistical analysis of the results of the Tetramitus Assay.

Most of the data collected to date on the Tetramitus Assay has been compared to Ames Test values. The Ames Test, still considered by many to be the gold standard of bioassays and toxicological testing, is slow, expensive and scientifically complex. While the Tetramitus Assay performs very well when compared to the Ames Test, the competitors of this technology are the several rapid, inexpensive, benchtop toxicology tests now on the market, such as those offered by Azur Environmental, Xenometrix Corporation and others. The Tetramitus Assay seems to enjoy many advantages over other test systems:

• The Tetramitus Assay is inexpensive to perform. It can be run for a fraction of the cost of other benchtop tests. The primary expense associated with the Tetramitus Assay is the cost of the labor necessary to conduct the test.

• The Tetramitus Assay requires no specialized preparations or test kits, only inexpensive commercially available reagents and media. Results are obtained within 4-24 hours. While this is competitive with Microtox™, most other bioassays take considerably longer. The testing protocol is simple and can be mastered by a technician with only a few hours of training.

• The Tetramitus Assay measures the toxicity of whole particles by flagellate ingestion via the gullet. Most other bioassays require the extraction of toxicants, frequently using solvents, before testing of a particle sample can begin. The efficacy of this extraction process, as well as any solvent’s impact on the accuracy of test results, are unclear. This aspect of the Tetramitus Assay, the ability to perform true and reproducible whole particle testing, is a significant competitive advantage over other systems.

• The Tetramitus Assay does not require the addition of an exogenous activation mixture (Rat Liver S9) necessary for many other toxicity screens, because Tetramitus has its own activating enzymes. Accordingly, the Tetramitus Assay does not require the use of laboratory animals. This aspect of the Tetramitus Assay gives it significant ethical advantages over those bioassays and test systems that still use animals and animal products.

• The Tetramitus Assay is sensitive to a broad range of toxicants. Many other test systems still require the use of multiple strains of the indicator organisms or the use of different test kits for the detection of different classes of compounds.

• The Tetramitus Assay is not affected by dyes or other coloration in water samples, unlike other test systems.

The market for this technology is expansive. The Tetramitus Assay makes toxicity screening quick and inexpensive for water utilities, state and federal regulatory agencies, watershed projects, environmental testing companies, individual homeowners and any industry required to monitor effluent discharge. This technology addresses many goals in new state and federal clean water legislation, as it provides an inexpensive means of self-regulation and monitoring for industry. Moreover, the EPA recently announced a policy shift in favor of in vitro toxicity testing, and away from in vivo toxicity testing. The market for environmental technology, which is expanding rapidly, is but one small area of the Tetramitus Assay’s application. The potential use of this technology in the pharmaceutical, chemical, cosmetic and hazardous waste industries may well expand beyond its extensive environmental testing applications.