Genotoxicity hazard assessment of Caramel Colours III and IV.

Results from a battery of short-term tests in vitro and in vivo used to assess the genotoxicity of caramel colours are presented and discussed in relation to reports from the literature. No evidence of genotoxicity was found in the Salmonella plate incorporation test using five standard strains or in the Saccharomyces cerevisiae gene conversion assay using strain D4, either with or without S-9 for activation. A weak clastogenic effect for a sample of Caramel Colour III in CHO cells was abolished in the presence of S-9. Two samples of Caramel Colour IV were not clastogenic in CHO cells. Salmonella pre-incubation tests without S-9 also failed to reveal any mutagenic activity for any of the caramel colours tested. The Caramel Colour III sample that showed clastogenic activity in CHO cells in vitro did not induce micronuclei when evaluated in a mouse bone marrow assay. These results are in general agreement with reports in the literature regarding the genotoxicity of caramel colours, and support the conclusion that caramel colours do not pose a genotoxic hazard to humans.

False positive result for a peptide drug in the gene conversion assay with Saccharomyces cerevisiae strain D7.

A battery of mutagenicity tests was performed with nafarelin, an agonist analogue of luteinizing hormone releasing hormone (LHRH) containing tryptophan (Trp) and histidine (His). Included were the Ames assay and the gene conversion assay with yeast strain D7. Both tests were negative without S9 activation, and the Ames test was negative with S9, but the yeast test was positive with S9 activation. Since the yeast test is based on conversion of cells to Trp independence, release of Trp by metabolism of the drug could account for the positive result. The test was repeated using Trp instead of the drug. The result was positive even at the lowest Trp concentration. In another experiment with the drug, amino acid analysis of the incubation mixture revealed the presence of Trp but no detectable His. Since the Ames test is based on mutation to His-independent cells, these data are completely consistent with the negative result in the Ames test and the false positive result in the yeast test. These data suggest the need for caution in interpreting the results from mutagenicity assays with peptide drugs.

Genetic toxicology evaluation of commercial beers. I. Development of procedures for the preparation of extracts from commercial beer products.

Commercial beer was subjected to an investigation in order to establish standard conditions for preparing organic solvent extracts to be used in short-term genetic screening assays. Test samples for use in the evaluation were prepared by mixing several brands of commercially available beer into a composite pool which was then spiked with the mutagen, 2-nitrofluorene. The composite sample was then concentrated using varying ratios of beer to XAD-2 resin in a 1.5 cm X 30 cm column. Dry-weight analyses indicated that significant amounts of residue could be trapped by XAD-2 resin. Columns were sequentially eluted by methylene chloride, acetone and methanol followed by evaporation of the solvents under nitrogen gas. Residues from commercial products were not mutagenic, but mutagenic activity could be detected in residues from spiked beer, yielding nearly 90% of the expected biological activity in S. typhimurium TA98. A standard method amenable to processing large volumes of beer products was devised for application to other projects.

Genetic toxicology evaluation of commercial beers. II. Mutagenic activity of commercial beer products in Salmonella typhimurium strains TA98, TA100 and TA102.

5 concentrated extracts of commercial beers were prepared using XAD-2 resin. The residues were subjected to evaluation for mutagenic activity in Salmonella typhimurium strains TA98, TA100 and TA102. The tests were conducted using preincubation protocols including provisions for S9 metabolic activation. Although the extracts did produce moderate toxicity to the Salmonella organisms used in the assays, none of the residues were found to induce mutation up to their maximum testable concentrations.

Genotoxicity of theobromine in a series of short-term assays.

Theobromine (3,7-dimethylxanthine) was evaluated for genotoxic activity in a series of in vitro assays. Theobromine was not mutagenic in the Ames assay up to a maximum concentration of 5000 micrograms/plate either with or without S9 activation. The compound also failed to induce significant levels of chromosome aberrations in CHO cells (with and without S9 activation) or transformation in Balb/c-3T3 cells. At the maximum tolerated concentration theobromine increased the frequency of TK-/- mutants in mouse lymphoma L5178Y cells. Increased frequencies were observed both with and without S9 activation and they were reproducible in 2 independent experiments. Statistically significant increases in SCEs were obtained in human lymphocytes and in CHO cells under nonactivation test conditions. The spectrum of results in this battery of tests indicate that theobromine treatment results in the expression of genotoxic potential in some assays and the observed activity appears qualitatively and quantitatively similar to that of caffeine, a closely related methylxanthine.

Genotoxicity of cocoa in a series of short-term assays.

Cocoa powder was evaluated for genotoxic activity and found to be inactive in the Ames assay, the mouse lymphoma assay, cytogenetic assays measuring chromosome breakage and SCE, and a cell transformation assay using Balb/c-3T3 cells. Although pure theobromine has been shown to be active in some of these test procedures, the levels of this methylxanthine present in cocoa powder were insufficient to elicit responses in this battery of tests.

Mutagenic evaluations of four rubber accelerators in a battery of in vitro mutagenic assays.

The mutagenic/carcinogenic potential of four commercial accelerators were evaluated using a battery of in vitro assays. All of these compounds were mutagenic in one or more assays. Positive responses were noted in the Escherichia coli pol A+/pol A- DNA repair, mouse lymphoma L5178Y TK+/- forward mutation, BALB/3T3 cell transformation, and CHO cell chromosome aberration assays. In contrast to previous studies of accelerators, no mutagenic response was observed in the E coli WP2 uvrA- assay or in any of the Salmonella typhimurium strains tested. These studies have indicated that rubber accelerators should be regarded as potential human health hazards and that further in vitro and in vivo studies are needed to assess the potential genetic hazards of this large class of chemicals.

The activity of 2-(2',4'-diaminophenoxy)ethanol in 3 genetic toxicity bioassays.

2-(2',4'-Diaminophenoxy)-ethanol, a hair-dye ingredient was evaluated for genetic activity in vitro using urine collected from mice in an Ames test and in vivo using the mouse dominant-lethal assay and the mouse spot test for somatic mutation detection. All 3 studies were conducted using dermal application of the dye material to shaved skin. The applied dose levels ranged from 15 to 1500 mg/kg body weight. The results of these 3 studies were considered to be negative although urine analysis and spot-test data showed non-significant dose-related increases.

Structure--activity relationships in the mutagenicity of quinone methides of 7-hydroxyflavylium salts for Salmonella typhimurium.

Several synthetic 7-hydroxyflavylium salts related to apigeninidin, a natural 3-deoxyanthocyanidin, have been studied in the Ames mutagenicity test using strain TA1537 of Salmonella typhimurium. Under the neutral pH conditions of the test, these flavylium salts are deprotonated through ionization of the C7-OH (pK'a = 4.2-4.4) to form quinone methides. Only the quinone methides of 4-methyl-7-hydroxyflavylium chloride and 4'-methoxy-4-methyl-7-hydroxy-flavylium chloride showed mutagenicity. Responses of 4-8 times the background were observed at the higher doses (1000 micrograms/plate), both with and without metabolic activation. It was concluded that the induction of frameshift mutagenicity by this group of compounds is caused by those quinone methides that have non-ionic, stable polycyclic structures at neutral pH.

Effect of rye chromosome 2 substitution on kernel protein content of wheat.

Significant increases in the kernel protein content of lines of the bread wheat variety Chinese Spring, in which a pair of rye chromosome 2 substitutes for group 2 chromosomes of wheat, were observed in plants grown at three different locations. Fractionation of proteins on the basis of their solubility did not show any significant variation in the relative proportions of alcohol, salt, acid and alkali soluble proteins. Similarly, electrophoretic studies of saline, alcohol and acetic acid-urea soluble proteins did not reveal any addition or deletion of protein bands in comparison with the Chinese Spring control. Apparently, the substituted rye chromosomes do not contribute any proteins different from those already present in wheat. The electrophoretic data give further evidence of the homoeology of rye chromosome 2 with the group 2 chromosomes of wheat.