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 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

1. The in vivo alkaline comet (single cell gel electrophoresis) assay (hereafter called simply the comet assay) is used for the detection of DNA strand breaks in cells or nuclei isolated from multiple tissues of animals, usually rodents, that have been exposed to potentially genotoxic material(s). The comet assay has been reviewed and recommendations have been published by various expert groups (1) (2) (3) (4) (5) (6) (7) (8) (9) (10). This Test Guideline is part of a series of Test Guidelines on genetic toxicology. A document presented as an Introduction to the Test Guidelines on genotoxicity (11) can also be referred to and provides succinct and useful guidance to users of these Test Guidelines.

Oecd guideline for testing of chemicals

A new tripartite, harmonised guideline is proposed in the Quality topic area on the “Comparability of Biotechnological / Biological Products Subject to Changes in their Drug Substance and /or Drug Product Manufacturing Process”. For establishing these comparisons, it is necessary to address both product and process aspects which, while in some instances region-specific, are uniform in their principles and are necessary to support changes in manufacturing processes yielding products defined within the scope of Q6B. When the Quality aspects have been addressed, experts from the Safety and Efficacy groups will be invited to consider issues in the preclinical and in the clinical areas, as appropriate.

/pdf/international-conference-on-harmonisation-of-technical-ykgvo7gt3a.pdf

International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use

Caffeine is probably the most frequently ingested pharmacologically active substance in the world. It is found in common beverages (coffee, tea, soft drinks), in products containing cocoa or chocolate, and in medications. Because of its wide consumption at different levels by most segments of the population, the public and the scientific community have expressed interest in the potential for caffeine to produce adverse effects on human health. The possibility that caffeine ingestion adversely affects human health was investigated based on reviews of (primarily) published human studies obtained through a comprehensive literature search. Based on the data reviewed, it is concluded that for the healthy adult population, moderate daily caffeine intake at a dose level up to 400 mg day(-1) (equivalent to 6 mg kg(-1) body weight day(-1) in a 65-kg person) is not associated with adverse effects such as general toxicity, cardiovascular effects, effects on bone status and calcium balance (with consumption of adequate calcium), changes in adult behaviour, increased incidence of cancer and effects on male fertility. The data also show that reproductive-aged women and children are 'at risk' subgroups who may require specific advice on moderating their caffeine intake. Based on available evidence, it is suggested that reproductive-aged women should consume </=300 mg caffeine per day (equivalent to 4.6 mg kg(-1) bw day(-1) for a 65-kg person) while children should consume </=2.5 mg kg(-1) bw day(-1).

Effects of caffeine on human health.

The Salmonella assay has been in use for almost 15 years and can be defined as a routine test for mutagenicity and for predicting potential carcinogenicity It detects the majority of animal carcinogens and consequently plays an important role in safety assessment The test is also routinely used as the frontline screen for environmental samples (complex mixtures) isolated from air, water and food This role will continue to remain an area of growth as or because sample volumes associated with these testing areas are generally very limited and more extensive testing is generally impossible While this test, like all others, has some limitations, it is recommended that it be regularly included in all genetic testing batteries

The Salmonella typhimurium/mammalian microsomal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

A total of 63 chemicals were tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, and Escherichia coli WP2 uvrA in a four-laboratory study. Sixty of the chemicals had been tested for carcinogenicity by the National Cancer Institute or the National Toxicology Program. All chemicals were tested for mutagenicity without metabolic activation and with liver S-9 preparations from uninduced and Aroclor 1254-induced F344 rats, B6C3F1 mice, and Syrian hamsters. The intra- and interlaboratory reproducibility of the Salmonella assay with regard to the overall judgment of mutagenic or nonmutagenic was good. The results in the E coli strain, however, exhibited a high degree of variability between laboratories. With one or two exceptions, the mutagens were detected with S-9 preparations from all three species. The uninduced liver S-9 preparations did not activate any chemicals to mutagens that were not also activated by induced S-9, but some chemicals were detected as mutagens only when induced S-9 was used. A positive mutagenic response in Salmonella was predictive of carcinogenicity 69% of the time; when equivocal carcinogens and borderline mutagens were included, the predictivity increased to 83%. Conversely, 76% of the carcinogens were mutagens. When the equivocal carcinogens were included, the proportion dropped to 75%. Relatively few chemicals (18%) were mutagenic in E coli. Not all the carcinogens induced tumors in both rats and mice, and the species-specific carcinogenicity could not be predicted from the S-9-specific mutagenicity.

Reproducibility of microbial mutagenicity assays: II. Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli.

The Salmonella/microsome test developed by Ames and his coworkers has been widely used in the evaluation of chemicals for genotoxic potential. Although the value of this assay is well recognized, there have been no comprehensive studies on the interlaboratory reproducibility of the method using a standardized protocol. A program was therefore initiated to compare the results obtained in four laboratories from testing a series of coded mutagens and nonmutagens using a standardized protocol. Additional objectives of this study were to compare male Fisher 344 rat, B6C3F1 mouse, and Syrian hamster liver S-9 preparations for the activation of chemicals; to compare Aroclor 1254-induced liver S-9 from all three species with the corresponding non-induced liver S-9's; and to compare the response of Escherichia coli WP-2 uvrA with the Salmonella typhimurium tester strains recommended by Ames. Since a primary use of in vitro microbial mutagenesis tests is the identification of potential carcinogens by their mutagenicity, the authors decided to compare the animal species and strains used by the National Cancer Institute/National Toxicology Program (NCI/NTP) for animal carcinogenicity studies.

Reproducibility of microbial mutagenicity assays: I. Tests with Salmonella typhimurium and Escherichia coli using a standardized protocol

A workshop describing the mutagenic effects of low pH and high osmotic levels on cultured mammalian cells was held at the 16th Annual Meeting of the Environmental Mutagen Society in Las Vegas, Nevada, 1985. Data presented in this summary report were generously provided by the five invited speakers.

David Brusick

Papers

The Salmonella typhimurium/mammalian microsomal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

Reproducibility of microbial mutagenicity assays: II. Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli.

Reproducibility of microbial mutagenicity assays: I. Tests with Salmonella typhimurium and Escherichia coli using a standardized protocol

Genotoxic effects in cultured mammalian cells produced by low pH treatment conditions and increased ion concentrations.

Genotoxicity under extreme culture conditions