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

doi:10.1016/0165-1218(87)90056-5

Mutation Research/Genetic Toxicology

Volume 189, Issue 3, November 1987, Pages 223-261

https://doi.org/10.1016/0165-1218(87)90056-5 Get rights and content

Abstract

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.

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The mutagenicity of Direct Black 38, Sudan I, and Para Red were evaluated in the in vivo MutaMouse assay and the in vitro MutaMouse primary hepatocyte (PH) assay. Direct Black 38 is an International Agency for Research on Cancer (IARC) Group 1 carcinogen and a prototypical benzidine-based azo compound that requires azo-reduction to yield a DNA-reactive metabolite. Sudan I and Para Red are structurally related azo compounds that have been detected as illegal contaminants in foods. Sudan I is an in vivo mutagen, and both it and Para Red are known to be mutagenic in vitro. Sudan I is oxidized by hepatic and/or bladder enzymes to yield a mutagenic metabolite, but little is known about Para Red. In the present study, Direct Black 38 elicited a significant mutagenic response in the bone marrow, glandular stomach, small intestine and colon in vivo, and in PHs in vitro. Sudan I elicited a weak positive response in the bone marrow and a marginally significant treatment effect in the bladder (p = 0.059); it did not elicit a significant response in PHs in vitro. Para Red elicited a positive response in the colon, as well as in PHs in vitro, albeit at a cytotoxic concentration. The findings are well aligned with the known mechanisms of action of Direct Black 38 and Sudan I; they suggest that intestinal azo-reduction plays an important role in the activation of Para Red. The MutaMouse pH results illustrate the ability of this assay to detect chemicals requiring azo-reduction; however, they also demonstrate a gap in applicability domain, as MutaMouse PHs elicit a negative response following exposure to Sudan I. Elucidation of the mechanisms underlying this gap will require further study.
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In addition, Sudan I also gave positive results in Salmonella Typhimurium mutagenicity tests with S9 activation, a post-mitochondrial fraction prepared from the livers of rats [31]. Sudan II (C.I. Solvent Orange 7) is a dimethyl derivative of Sudan I. Early studies showed that Sudan II induced mutation of Salmonella Typhimurium TA 1538 in the presence of a rat liver preparation [32] and gave positive results in both Salmonella assays (the standard plate-incorporation assay and the FMN preincubation modification of the Salmonella assay) [33]. Sudan III (C.I. Solvent Red 23) is a diazo dye and approved for use in drug and cosmetics.
Sudan azo dyes are banned for food usage in most countries, but they are illegally used to maintain or enhance the color of food products due to low cost, bright staining, and wide availability of the dyes. In this report, we examined the toxic effects of these azo dyes and their potential reduction metabolites on 11 prevalent human intestinal bacterial strains. Among the tested bacteria, cell growth of 2, 3, 5, 5, and 1 strains was inhibited by Sudan I, II, III, IV, and Para Red, respectively. At the tested concentration of 100 μM, Sudan I and II inhibited growth of Clostridium perfringens and Lactobacillus rhamnosus with decrease of growth rates from 14 to 47%. Sudan II also affected growth of Enterococcus faecalis. Growth of Bifidobacterium catenulatum, C. perfringens, E. faecalis, Escherichia coli, and Peptostreptococcus magnus was affected by Sudan III and IV with decrease in growth rates from 11 to 67%. C. perfringens was the only strain in which growth was affected by Para Red with 47 and 26% growth decreases at 6 and 10 h, respectively. 1-Amino-2-naphthol, a common metabolite of the dyes, was capable of inhibiting growth of most of the tested bacteria with inhibition rates from 8 to 46%. However, the other metabolites of the dyes had no effect on growth of the bacterial strains. The dyes and their metabolites had less effect on cell viability than on cell growth of the tested bacterial strains. Clostridium indolis and Clostridium ramosum were the only two strains with about a 10 % decrease in cell viability in the presence of Sudan azo dyes. The present results suggested that Sudan azo dyes and their metabolites potentially affect the human intestinal bacterial ecology by selectively inhibiting some bacterial species, which may have an adverse effect on human health.
Evaluation of potential genotoxicity of five food dyes using the somatic mutation and recombination test
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Erythrosine’s genotoxicity and mutagenicity are under discussion provided by some equivocal results in some different cytogenetic tests. In bacterial reversion assays (Auletta et al., 1977; Brown et al., 1978; Haveland-Smith et al., 1981; Lin and Brusick, 1986; Cameron et al., 1987) were negative results, whereas in strains D7 and XV185-14C of S. cerevisiae, gene conversions and reverse mutation were positive (Matula and Downie, 1984). In Ames test erythrosine gave negative results in a range of 2 mg/plate (Lakdawalla and Netrawali, 1988a,b) to 10 mg/plate (Lin and Brusick, 1986).
In this study, different concentrations of five food dyes (amaranth, patent blue, carminic acid, indigotine and erythrosine) have been evaluated for genotoxicity in the Somatic Mutation and Recombination Test (SMART) of Drosophila melanogaster. Standard cross was used in the experiment. Larvae including two linked recessive wing hair mutations were chronically fed at different concentrations of the test compounds in standard Drosophila Instant Medium. Feeding ended with pupation of the surviving larvae. Wings of the emerging adult flies were scored for the presence of spots of mutant cells which can result from either somatic mutation or somatic recombination. For the evaluation of genotoxic effects, the frequencies of spots per wing in the treated series were compared to the control group, which was distilled water. The present study shows that carminic acid and indigotine demonstrated negative results while erythrosine demonstrated inconclusive results. In addition 25 mg mL⁻¹ concentration of patent blue and 12.5, 25 and 50 mg mL⁻¹ concentrations of amaranth demonstrated positive results in the SMART.
A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins
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In vitro genotoxicity testing needs to include tests in both bacterial and mammalian cells, and be able to detect gene mutations, chromosomal damage and aneuploidy. This may be achieved by a combination of the Ames test (detects gene mutations) and the in vitro micronucleus test (MNvit), since the latter detects both chromosomal aberrations and aneuploidy. In this paper we therefore present an analysis of an existing database of rodent carcinogens and a new database of in vivo genotoxins in terms of the in vitro genotoxicity tests needed to detect their in vivo activity. Published in vitro data from at least one test system (most were from the Ames test) were available for 557 carcinogens and 405 in vivo genotoxins. Because there are fewer publications on the MNvit than for other mammalian cell tests, and because the concordance between the MNvit and the in vitro chromosomal aberration (CAvit) test is so high for clastogenic activity, positive results in the CAvit test were taken as indicative of a positive result in the MNvit where there were no, or only inadequate data for the latter. Also, because Hprt and Tk loci both detect gene-mutation activity, a positive Hprt test was taken as indicative of a mouse-lymphoma Tk assay (MLA)-positive, where there were no data for the latter. Almost all of the 962 rodent carcinogens and in vivo genotoxins were detected by an in vitro battery comprising Ames + MNvit. An additional 11 carcinogens and six in vivo genotoxins would apparently be detected by the MLA, but many of these had not been tested in the MNvit or CAvit tests. Only four chemicals emerge as potentially being more readily detected in MLA than in Ames + MNvit – benzyl acetate, toluene, morphine and thiabendazole – and none of these are convincing cases to argue for the inclusion of the MLA in addition to Ames + MNvit. Thus, there is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames + MNvit.
Cytogenetic evaluation and DNA interaction studies of the food colorants amaranth, erythrosine and tartrazine
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Citation Excerpt :
Erythrosine’s genotoxicity and mutagenicity are under discussion provided by some equivocal results in some different cytogenetic tests. In bacterial reversion assays (Auletta et al., 1977; Brown et al., 1978; Cameron et al., 1987; Haveland-Smith et al., 1981; Lin and Brusick, 1986) were negative results, whereas in strains D7 and XV185-14C of S. cerevisiae, gene conversions and reverse mutation were positive (Matula and Downie, 1984). In Ames test erythrosine gave negative results in a range of 2 mg/plate (Lakdawalla and Netrawali, 1988) to 10 mg/plate (Lin and Brusick, 1986).
Food coloring agents, amaranth, erythrosine and tartrazine have been tested at 0.02–8 mM in human peripheral blood cells in vitro, in order to investigate their genotoxic, cytotoxic and cytostatic potential. Amaranth at the highest concentration (8 mM) demonstrates high genotoxicity, cytostaticity and cytotoxicity. The frequency of SCEs/cell was increased 1.7 times over the control level. Additionally, erythrosine at 8, 4 and 2 mM shows a high cytotoxicity and cytostaticity. Finally, tartrazine seems to be toxic at 8 and 4 mM. No signs of genotoxicity were observed. Reversely, tartrazine showed cytotoxicity at 1 and 2 mM. Furthermore, spectroscopic titration studies for the interaction of these food additives with DNA showed that these dyes bind to calf thymus DNA and distinct isosbestic points are observed clearly suggesting binding of the dyes to DNA. Additionally DNA electrophoretic mobility experiments showed that these colorants are obviously capable for strong binding to linear dsDNA causing its degradation. PCR amplification of all DNA fragments (which previously were pre-treated with three different concentrations of the colorants, extracted from agarose gel after separation and then purified), seems to be attenuated with a manner dye concentration-dependent reflecting in a delayed electrophoretic mobility due to the possible binding of some molecules of the dyes. Evaluation of the data and curves were obtained after quantitative and qualitative analysis of the lanes of the gel by an analyzer computer program. Our results indicate that these food colorants had a toxic potential to human lymphocytes in vitro and it seems that they bind directly to DNA.
New considerations regarding the risk assessment on Tartrazine. An update toxicological assessment, intolerance reactions and maximum theoretical daily intake in France
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Tartrazine is an artificial azo dye commonly used in human food and pharmaceutical products. Since the last assessment carried out by the JECFA in 1964, many new studies have been conducted, some of which have incriminated tartrazine in food intolerance reactions. The aims of this work are to update the hazard characterization and to revaluate the safety of tartrazine. Our bibliographical review of animal studies confirms the initial hazard assessment conducted by the JECFA, and accordingly the ADI established at 7.5 mg/kg bw. From our data, in France, the estimated maximum theoretical intake of tartrazine in children is 37.2% of the ADI at the 97.5th percentile. It may therefore be concluded that from a toxicological point of view, tartrazine does not represent a risk for the consumer. It appears more difficult to show a clear relationship between ingestion of tartrazine and the development of intolerance reactions in patients. These reactions primarily occur in patients who also suffer from recurrent urticaria or asthma. The link between tartrazine consumption and these reactions is often overestimated, and the pathogenic mechanisms remain poorly understood. The prevalence of tartrazine intolerance is estimated to be less than 0.12% in the general population. Generally, the population at risk is aware of the importance of food labelling, with the view of avoiding consumption of tartrazine. However, it has to be mentioned that products such as ice creams, desserts, cakes and fine bakery are often sold loose without any labelling.

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^∗: Present address: SITEK Research Laboratories, 12111 Parklawn Drive, Rockville, MD 20852 (U.S.A.).

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

Abstract

Mutation Res.

Mutation Res.

Mutation Res.

Mutation Res.

Mutation Res.

Mutation Res.

Cancer Lett.

Mutation Res.

Food Chem. Toxicol.

Mutation Res.

Mutation Res.

Mutation Res.

Dyes Pigm.

Dyes Pigm.

Mutation Res.

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