Virginia C. Dunkel

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

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.

Histopathological Evaluation of Liver, Pancreas, Spleen, and Heart from Iron-Overloaded Sprague-Dawley Rats*1,2

The effects of increasing dietary levels of Fe on the histopathology of liver, pancreas, spleen, and heart were examined in a rat model for iron overload. Sprague-Dawley rats were fed diets containing 35, 350, 3,500, or 20,000 μg Fe/g, and, after 12 wk, there was a direct correlation between increased liver nonheme Fe and lipid peroxidation measured by the lipid-conjugated diene assay. Histopathological examination of tissues revealed the following: (a) hepatocellular hemosiderosis in all groups of rats, with a dose-related accumulation of cytoplasmic Fe-positive material predominantly in hepatocytes located in the periportal region (Zone 1), (b) myocardial degeneration and necrosis (cardiomyopathy) with hemosiderin in interstitial macrophages or in myocardial fibers of animals with heart damage, (c) splenic lymphoid atrophy affecting the marginal zone of the white pulp and hemosiderin deposition in the sinusoidal macrophages, and (d) pancreatic atrophy with loss of both the endocrine and exocrine pancreatic tissue in those animals receiving 3,500 and 20,000 μg Fe/g of diet. The toxic effects of Fe overload in this rat model include cellular apoptosis or necrosis in heart, spleen, and pancreas and, when coupled with the findings on lipid peroxidation, suggests that oxidative stress is involved in the pathogenesis of the lesions.

Mutagenic activity of 27 dyes and related chemicals in the Salmonella/microsome and mouse lymphoma TK+/− assays

A total of 27 dyes and related chemicals were tested for mutagenicity in both the Salmonella typhimurium plate-incorporation and FMN-modified assays as well as the mouse lymphoma TK+/- assay. Half of the compounds tested were monoazo dyes (14); the remainder consisted of disazo (3), aminotriphenylmethane derivatives (4), and other miscellaneous (6) color compounds. The results obtained in this study are compared with data from dyes of the same batch tested in other laboratories in the Salmonella plate-incorporation assay and in both in vitro and in vivo/in vitro UDS assays. Agreement of results from the various assays that could be compared (excluding results that were equivocal or indeterminate) ranged from 80 to 91%. Sufficient data were available to provide an overall index of in vitro activity for 15 chemicals; of these, 14 compounds could be compared to and agreed with reports of their carcinogenic potential in the literature.

Mutagenic activity of some coffee flavor ingredients

The mutagenicity of 4 coffee flavor ingredients (chlorogenic acid, caffeic acid, pyrazine, and trigonelline) was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK +/- assay. Two of the compounds, pyrazine and trigonelline, were negative in both assays. The other two compounds, caffeic acid and chlorogenic acid, were positive in the mouse lymphoma assay but negative in the Salmonella assay.

Genome-Linked Toxic Responses to Dietary Iron Overload

Genome-related differences to Fe overload between and within rodent species were evaluated in the present study. Male B6C3F, mice, yellow and black C5YSF1 mice, and Fischer 344 (F344) rats were fed AIN-76A diets containing 35 (control), 1,500, 3,500, 5,000, or 10,000 μg carbonyl Fe/g for 12 wk. No effects on body weight gain were observed in the B6C3F, and black C5YSF, mice, whereas at all doses of Fe above the control, weight gain was reduced in yellow C5YSF1 mice and F344 rats. At the 10,000 μg Fe/g dose, 9 of 12 rats died, but there was no mortality among the mice. In all animals, there was a dose-related increase in liver nonheme Fe, and the Fe was stored in hepatocytes predominantly in the periportal region. There was significant hypertrophy of the hepatocytes in both B6C3F, mice and F344 rats fed the 10,000 μg Fe/g diet. PCNA assays showed significant stimulatory effects of the high dose of Fe on hepatocyte proliferation in the F344 rats and the C5YSF, mice but not in the B6C3F, mice. In the rat, there was pancreatic atrophy with loss of both endocrine and exocrine tissue. Morphometric evaluation of pancreas showed fewer β cells in B6C3F, and yellow C5YSF1 mice but not in the black C5YSF 1 mice. There were fewer islets in the yellow C5YSF1 mice, and total and mean islet areas were smaller than in the control mice. Rats in the 10,000 μg Fe/g dose group had markedly exacerbated dose-dependent nephropathy and changes in glomerular and tubular epithelium associated with Fe accumulation. The rats also showed degeneration of the germinal epithelium of the testis, formation of multinucleated giant cells, and lack of mature sperm.

Evaluation of the butter flavoring chemical diacetyl and a fluorochemical paper additive for mutagenicity and toxicity using the mammalian cell gene mutation assay in L5178Y mouse lymphoma cells

Diacetyl (2,3-butanedione) is a yellowish liquid that is usually mixed with other ingredients to produce butter flavor or other flavors in a variety of food products. Inhalation of butter flavoring vapors was first associated with clinical bronchiolitis obliterans among workers in microwave popcorn production. Recent findings have shown irreversible obstructive lung disease among workers not only in the microwave popcorn industry, but also in flavoring manufacture, and in chemical synthesis of diacetyl, a predominant chemical for butter flavoring. It has been reported that perfluorochemicals utilized in food packaging are migrating into foods and may be sources of oral exposure. Relatively small quantities of perfluorochemicals are used in the manufacturing of paper or paperboard that is in direct contact with food to repel oil or grease and water. Because of recent concerns about perfluorochemicals such as those found on microwave popcorn bags (e.g. Lodyne P208E) and diacetyl in foods, we evaluated both compounds for mutagenicity using the mammalian cell gene mutation assay in L5178Y mouse lymphoma cells. Lodyne P208E was less toxic than diacetyl and did not induce a mutagenic response. Diacetyl induced a highly mutagenic response in the L5178Y mouse lymphoma mutation assay in the presence of human liver S9 for activation. The increase in the frequency of small colonies in the assay with diacetyl indicates that diacetyl causes damage to multiple loci on chromosome 11 in addition to functional loss of the thymidine kinase locus.

Morphological transformation of BALB/3T3 mouse embryo cells in vitro by vomitoxin.

The transforming potential of vomitoxin, a trichothecene mycotoxin produced on cereal grains by fungi of the genus Fusarium, was assessed using mouse embryo BALB/3T3 A31-1-1 cells. Cells grown in Eagles basal medium with Earles salts supplemented with 7.5% foetal bovine serum were treated with highly purified vomitoxin, which was dissolved in distilled water and filter-sterilized. Assays were conducted using cells from three different passages at dose levels ranging from 0.1 to 1.6 microgram/ml. The treatment time was 48 hr and the highest dose levels tested produced approximately 10% survival as determined by in situ cell counts. Distilled water and 3-methylcholanthrene (5.0 micrograms/ml) were used as the vehicle and positive controls, respectively. Of the 20 dishes examined per dose group, the numbers of type III foci were 0-1 in the solvent control, 12-15 in the positive control and 0-9 in the treated groups. Comparison of the three assays showed that the level of response varied with passage number. Of the three passages of cells tested-passage numbers 6, 8 and 9 (p6, p8 and p9)--passage-9 cells produced the strongest positive effect.

Genotoxicity of 6 oxime compounds in the Salmonella/mammalian-microsome assay and mouse lymphoma TK+/− assay

To aid in the selection of chemical candidates for in vivo tests, the mutagenicity of 6 oxime compounds was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK +/- assay. All of the oximes were mutagenic in the mouse lymphoma assay in the absence of exogenous metabolic activation. Acetaldehyde oxime was also mutagenic in the presence of S9 activation. In contrast to these results, a positive response was noted only for 2-(hydroxyimino)-N-phenyl-acetamide oxime in strain TA1535 in the absence of activation in the Salmonella/microsome test.

Mutagenic activity of 5 thiazole compounds in the Salmonella/microsome and mouse lymphoma TK+/− assays

To aid in the selection of chemical candidates for in vivo tests, the mutagenicity of 5 thiazole compounds was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK+/- assay. Two of the compounds, 2-thiazolamine and 3-methyl-5-isothiazolamine, were positive in both assays; and one, thiazole, was negative. With the other 2 compounds the results were nonconcordant: 5-phenyl-2,4-thiazolediamine was negative in the Salmonella assay but positive in the mouse lymphoma assay, and C.I. Basic Red 29 was positive in the Salmonella assay while the response in the mouse lymphoma assay was considered equivocal.