The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975b). The present paper is a revision of the methods. Two new tester strains, a frameshift strain (TA97) and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set. TA97 replaces TA1537. TA1535 and TA1538 are removed from the recommended set but can be retained at the option of the investigator. TA98 and TA100 are retained. We discuss other special purpose strains and present some minor changes in procedure, principally in the growth, storage, and preservation of the tester strains. Two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine. Some test modifications are discussed.

Revised methods for the salmonella mutagenicity test

Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/%28SICI%291098-2280%282000%2935%3A3%3C206%3A%3AAID-EM8%3E3.0.CO%3B2-J

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

The recent advances in nanotechnology and the corresponding increase in the use of nanomaterials in products in every sector of society have resulted in uncertainties regarding environmental impacts. The objectives of this review are to introduce the key aspects pertaining to nanomaterials in the environment and to discuss what is known concerning their fate, behavior, disposition, and toxicity, with a particular focus on those that make up manufactured nanomaterials. This review critiques existing nanomaterial research in freshwater, marine, and soil environments. It illustrates the paucity of existing research and demonstrates the need for additional research. Environmental scientists are encouraged to base this research on existing studies on colloidal behavior and toxicology. The need for standard reference and testing materials as well as methodology for suspension preparation and testing is also discussed.

Nanomaterials in the environment: Behavior, fate, bioavailability, and effects

The relationship between antioxidant activity and antimutagenicity of various tea extracts (green tea, pouchong tea, oolong tea, and black tea) was investigated. All tea extracts exhibited markedly antioxidant activity and reducing power, especially oolong tea, which inhibited 73.6% peroxidation of linoleic acid. Tea extracts exhibited a 65-75% scavenging effect on superoxide at a dose of 1 mg and 30 - 60% scavenging effect on hydrogen peroxide at a dose of 400 microgram. They scavenged 100% hydroxyl radical at a dosage of 4 mg except the black tea. Tea extracts also showed 50 - 70% scavenging effect on alpha, alpha-diphenyl-beta-picrylhydrazyl radical. The antioxidant activity and the scavenging effects on active oxygen decreased in the order semifermented tea > nonfermented tea > fermented tea. Tea extracts showed strong antimutagenic action against five indirect mutagens, i.e., AFB1, Trp-P-1, Glu-P-1, B[a]P, and IQ, especially oolong and pouchong teas. The antioxidant effect of tea extracts was well correlated to their antimutagenicity in some cases but varied with the mutagen and antioxidative properties.

Antioxidant activity of various tea extracts in relation to their antimutagenicity

The Ames Salmonella/microsome mutagenicity assay (Salmonella test; Ames test) is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances that can produce genetic damage that leads to gene mutations. The test employs several histidine dependent Salmonella strains each carrying different mutations in various genes in the histidine operon. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When the Salmonella tester strains are grown on a minimal media agar plate containing a trace of histidine, only those bacteria that revert to histidine independence (his(+)) are able to form colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner. The Ames test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs. The test is also used for submission of data to regulatory agencies for registration or acceptance of many chemicals, including drugs and biocides. International guidelines have been developed for use by corporations and testing laboratories to ensure uniformity of testing procedures. This review provides historical aspects of how the Ames was developed and detailed procedures for performing the test, including the design and interpretation of results.

http://people.uleth.ca/%7Eselibl/Biol3200/AmesTest/AmesTestreview.pdf

The Ames Salmonella/microsome mutagenicity assay

We have screened 14 solvents for compatibility with the Salmonella mutagenicity test and have found 12 to be satisfactory under the conditions specified. These 12 solvents are: dimethyl sulfoxide, glycerol formal, dimethyl formamide, formamide, acetonitrile, 95% ethanol, acetone, ethylene glycol dimethyl ether, 1-methyl-2-pyrrolidinone, p-dioxane, tetrahydrofurfuryl alcohol and tetrahydrofuran. The influence of the nutrient broth culture medium on the spontaneous mutation rate of the tester strains is also discussed.

Compatibility of organic solvents with the Salmonella/microsome test

A detection and classification system for mutagens has been developed that identifies the six possible base-pair substitution mutations. A set of six Salmonella typhimurium (TA7001 to TA7006) strains has been constructed, each of which carries a unique missense mutation in the histidine biosynthetic operon. In addition to the his mutation, these strains carry different auxiliary features that enhance the mutability of the target his mutation. These include the R factor pKM101, which has the SOS-inducible mucAB system; a deletion of the uvrB component of excision repair; and rfa mutations to increase the accessibility of bulky chemicals to the bacteria. Another set of strains (TA7041 to TA7046) contain a wild-type rfa gene. Reversion via the base substitution unique to each strain was verified by sequence analyses of > 800 revertants obtained from different types of mutagens. The strains have considerably lower spontaneous reversion frequencies and detect a variety of mutagens at a sensitivity comparable to the Salmonella tester strains TA100, TA102, and TA104. The low spontaneous frequency of reversion of a mixture of the six tester strains (approximately 10 revertants per plate) enables a single mutation assay with the mixture that is followed by classification of the type of mutation with the individual strains.

https://www.pnas.org/content/pnas/91/24/11606.full.pdf

Dorothy M. Maron

Papers

Revised methods for the salmonella mutagenicity test

Compatibility of organic solvents with the Salmonella/microsome test

Detection and classification of mutagens: a set of base-specific Salmonella tester strains.

6 – revised methods for the salmonella mutagenicity test

Detection of phosphate esters on chromatograms: An improved reagent