At the Washington International Workshop on Genotoxicity Test Procedures (March 25-26, 1999), the current methodologies and data for the in vitro micronucleus test were reviewed. From this, guidelines for the conduct of specific aspects of the protocol were developed. Because there are a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at this time. Agreement was achieved on the following topics: Cells. The choice of cells is flexible, yet the choice of cell type should be justified and take into consideration doubling time, spontaneous frequency of micronuclei, and genetic background. Slide preparation. A fixation method that preserves the cytoplasm and cytoplasmic boundaries, and minimizes clumping should be used. Use of fluorescent DNA-specific dyes is encouraged for better detection of small micronuclei. Analysis. Micronuclei should have a diameter less than one-third of the main nucleus, and should be clearly distinguishable from the main nucleus. In the cytokinesis-block method, binucleated cells selected for analysis should have two clearly distinguishable main nuclei. Cells where the main nucleus(ei) is undergoing apoptosis should not be scored for micronuclei because the assumed micronuclei may have been the result of nuclear fragmentation during the apoptotic process. Toxicity. Cytotoxicity can be measured by various methods including cell growth, cell counts, nucleation (i.e., percent binucleated), division/proliferation index, confluence. A majority of the group recommended that the highest concentration should induce at least 50% cytotoxicity (by whatever measure is selected). Cytochalasin B. There is much debate regarding the use of cytochalasin B. For human lymphocytes, the use of cytochalasin B (6 microg/ml [lymphocytes cultured from whole blood cells] and 3-6 microg/ml [isolated lymphocyte cultures]) is recommended. For cell lines, because there were no definitive data showing a clear advantage or disadvantage of the use of cytochalasin B for a variety of chemicals, the majority opinion of the group was that at this time, the use of cytochalasin B for cell lines is considered optional. Further studies (many chemicals of a variety of potencies, tested both with and without cytochalasin B) are clearly needed to resolve this issue. Number of doses. At least three concentrations should be scored for micronuclei. Treatment/harvest times. At this time, there are not enough data to define the most appropriate treatment/harvest times. Following the principles of the in vitro metaphase assay (with or without metabolic activation), it was agreed that there was a need for a short treatment followed by a recovery time in the absence of test chemical, there was a need for a long treatment (maybe with and without recovery time), and ideally, treatment should cover cells in different cell cycle stages.

The genetic toxicity of atrazine, a member of the s-triazine herbicides, was reviewed with the objective of classifying the chemical. Atrazine has been subjected to a broad range of genetic tests with predominantly negative results. Some publications, specifically those measuring dominant lethality in mice and bone marrow clastogenicity in rodents, reported conflicting results across two or more independent tests. Two approaches were employed to evaluate and interpret the results. The first approach attempts to classify each type of genetic endpoint as positive or negative and resolve test conflicts by critical assessment of the study and detailed data. This is the more traditional “expert judgment” approach to hazard assessment. The second approach employs a computer-assisted weight-of-evidence method of data analysis. This approach does not require resolution of conflicts but uses all data sets to arrive at a classification of hazard. The first approach was able to resolve some conflicts but not all. Use of the “expert judgement” results in an equivocal conclusion and classification. Use of the weight-of-evidence method resulted in a conclusion that atrazine does not pose a mutagenic hazard. The weight-of-evidence scheme is proposed to be a more practical and relevant approach for assessing complex data sets.

Journal Article Nucleotide sequence of Salmonella typhimurium nitroreductase gene Get access Masahiko Watanabe, Masahiko Watanabe Division of Genetics and Mutagenesis, Biological Safety Research Center, National Institute of Hygienic Sciences1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Motoi Ishidate, Jr, Motoi Ishidate, Jr Division of Genetics and Mutagenesis, Biological Safety Research Center, National Institute of Hygienic Sciences1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Takehiko Nohmi Takehiko Nohmi Division of Genetics and Mutagenesis, Biological Safety Research Center, National Institute of Hygienic Sciences1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 18, Issue 4, 25 February 1990, Page 1059, https://doi.org/10.1093/nar/18.4.1059 Published: 25 February 1990 Article history Received: 05 January 1990 Published: 25 February 1990

Nitration of benzo[a]pyrene (BaP) with HNO3 (d = 1.38) produced a mixture of dinitroBaPs (1,6- and 3,6-isomers) and mononitroBaPs (1-, 3- and 6-isomers). Pure 1,6-dinitroBaP and 3,6-dinitroBaP were obtained by the reduction of the dinitroBaPs mixture with NaSH to yield the separable products 1-amino-6-nitroBaP and 3-amino-6-nitroBaP, followed by conversion to dinitroBaPs via the the diazonium salts. The half-wave potentials (E1/2) corresponding to the one-electron reduction of dinitroBaPs were measured and the relationship of these values to the mutagenicity is discussed.

In vitro microassay for the screening of teratogens was investigated on cancer chemotherapeutic agents sterigmatocystins and benzimidazoles using human embryonic palatal mesenchymal (HEPM) cells. Five thousand cells were inoculated into each well of 96-well microtiter plates, and cultivated for 24 hr, after which the media were changed with new ones that contained various amounts of chemicals; after cultivation for an additional 72 hr, the media were discarded, and cells attached to the tissue plate were fixed and stained with Giemsa's solution; the cell number then was counted by colony counter with three readings for each well. For the metabolic activation, the liver S9 obtained from rats pretreated with phenobarbital and 5,6-benzoflavone and cofactors (S9 mix) were added directly to the HEPM cell cultures along with chemicals. After 6 hr, the cultures were exchanged with a fresh medium and incubated for a further 72 hr. The final IC50 (the concentration that inhibits growth 50%) concentration-finding run had 7 to 11 concentration points (mean, three to four wells). Concentrations of the cancer chemotherapeutic agents that inhibited growth by 50% ranged from 0.001 to 10 micrograms/ml. Sterigmatocystins indicated strong inhibition; among three derivatives, O-acetyl sterigmatocystin was the most potent inhibitor. Benzimidazoles also exhibited an inhibitory action on HEPM cell growth; nitro and chloro groups at the 5 position in 2-(2-pyridyl)benzimidazole were found to be potent substituents. As for the activation of cyclophosphamide in the HEPM cell culture, IC50 was decreased to 1.0 ug/ml by the incubation with S9 mix for 6 hr under our experimental conditions, and sterigmatocystin was found to be activated by S9 mix.(ABSTRACT TRUNCATED AT 250 WORDS)

An experiment was performed on Wistar rats to examine the possible carcinogenic effects of charred fish meat. Each group (of 30 male rats each) was fed one of the following diets: the standard diet mixed with 50% charred fish meat (Group 1), the standard diet mixed with 25% charred fish meat (Group 2), the standard diet alone (Group 3), or the 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) treatment in drinking water (Group 4); this last group served as positive control. The animals were observed for two years. No significant incidences of tumors were recorded. However, there were statistically significant incidences of nonspecific pathological lesions of the glandular stomach, such as erosion, regeneration, and hyperplastic epithelium. Also, an atypical epithelium of the stomach was found incidentally in two animals (Groups 1 and 2); this might suggest a premalignant condition.

The induction of chromosomal aberrations in a superoxide-generating system using xanthine oxidase and hypoxanthine was investigated in cultured Chinese hamster cells. The production of chromosomal aberations in this system was inhibited by the addition of cytochrome C. This finding indicates that the generation of superoxide was the primary requirement for induction of chromosomal aberrations. On the other hand, superoxide dismutase showed no effect on the frequency of chromosomal aberrations, whereas catalase was effective in preventing the aberrations. It is conceivable, therefore, that the induction of chromosomal aberrations in the superoxide-generating system may be directly or indirectly due to hydrogen peroxide formed in the cultured medium as a result of the spontaneous dismutation reaction of superoxide.

Short-term tests in vitro as well as in vivo using mammalian cells are reliable and useful screening tools for the chemicals which may have any carcinogenic potential and/or genetic hazard. Chromosomal aberration test in vitro combined with or without a metabolic activation appeared to be a suitable method for mass screening level and it could cover a few carcinogens which gave a false negative in microbial mutation assays like Ames test. The clastogenic activity was generally well correlated with the mutagenic potential in the Ames test, if the positive data were compared and quantitatively evaluated. The results obtained from in vitro systems, however, did not always reflect those obtained from in vivo system. Therefore whole animal tests such as bone marrow cytogenetics, micronucleus test, spot test and others were further recommended as the secondary system to confirm the data obtained from in vitro primary tests. Technological problems of the method as well as qualitative or quantitative evaluation on the screening data were discussed on the basis of our experiments which had been carried out on more than 500 different chemicals including well-known carcinogens, such as food additives, medicinal drugs, pesticides, house-hold materials and other industrial chemicals.