Mutagenicity of a new hair dye ingredient: 4-ethoxy-m-phenylenediamine.

An ingredient recently introduced in hair dyes, 4-ethoxy-m-phenylenediamine, is mutagenic in histidine-requiring strains of Salmonella typhimurium. Its mutagenic activity is similar to that of the hair dye ingredient is apparently replaced, 4-methoxy-m-phenylenediamine.

Tris(2,3-dibromopropyl) phosphate: mutagenicity of a widely used flame retardant.

Tris(2,3-dibromopropyl) phosphate, a widely used flame-retardant additive for textiles, is mutagenic to histidine-requiring strains of Salmonella typhimurium. Extracts of fabrics treated with this compound are also capable of inducing mutations in these bacterial strains.

Influence of microsomal and cytosolic fractions from rat, mouse, and hamster liver on the mutagenicity of dimethylnitrosamine in the Salmonella plate incorporation assay.

Dimethylnitrosamine (DMN) was mutagenic in the Salmonella plate incorporation assay (Ames test) at a level of 10 mumol/plate (3.7 mM) in the presence of hamster liver S-9. Mutagenicity of DMN at this level was not observed when the S-9 was derived from mouse or rat liver, although the mouse liver and hamster liver S-9 had similar DMN demethylase activities. Both mouse and rat liver S-9 inhibited the mutagenicity of DMN mediated by hamster liver S-9; the inhibitory factor was contained in the microsomal fraction. Mouse or rat liver microsomes did not inhibit the DMN demethylase activity of hamster liver S-9. The microsomal inhibitor from rat or mouse liver was stable at 60 but was inactivated at 70 degrees. DMN demethylase from both rat and mouse liver was inactivated at 60 degrees. Although the DMN demethylase activity of hamster liver S-9 was contained in the microsomal fraction, DMN mutagenesis under conditions of the assay required the presence of both microsomal and cytosolic (S-105) fractions; the cytosols from hamsters, mice, and rats were all effective. The cytosolic factor required for DMN mutagenesis was sensitive to trypsin and was not dialyzable. The presence of an inhibitor of DMN activation in rat and mouse microsomes may account for, or contribute to, the failure of liver S-9 preparations from these species to activate DMN to a mutagen under standard conditions of the Ames test. The requirement for the cytosolic fraction may indicate that DMN demethylase is not sufficient for the activation of DMN to a mutagen under the conditions used in these studies.

Sequence analysis of mutations arising during prolonged starvation of Salmonella typhimurium.

We have examined the effects of prolonged histidine deprivation on the reversion of Salmonella typhimurium histidine auxotrophs containing either hisG46, a missense mutation (CTC----CCC), or hisG428, an ochre mutation (CAA----TAA). Both of these mutants can revert to His+ via intragenic and extragenic mechanisms. Whereas the hisG46 mutant site consists of G/C base pairs, extragenic suppression of hisG46 requires mutation at an A/T site. Conversely, the hisG428 site itself contains only A/T base pairs, and extragenic suppression of hisG428 occurs principally at G/C sites. Thus, by examining the mutational spectrum of hisG46 and hisG428 revertants that occurred in the presence and in the absence of histidine, it was possible to determine the effects of histidine starvation on mutations at G/C vs. A/T sites as well as on intragenic sites vs. extragenic suppressor sites. Using DNA-colony hybridization, we determined the DNA sequences of over 1300 hisG46 and hisG428 revertants. Histidine-independent revertants that arose during growth in liquid medium that contained histidine included both intragenic and extragenic suppressor mutations. The relative frequency of such extragenic suppressors was greatly reduced among the His+ revertants that were isolated after 5-10 days of histidine starvation on agar medium. Moreover, DNA sequence analysis revealed striking differences in the distribution of particular transversions at the hisG428 locus in revertants arising after prolonged histidine starvation as compared to those arising after growth in the presence of histidine.

Adaptive mutation and slow-growing revertants of an Escherichia coli lacZ amber mutant.

We have studied revertants, selected on lactose minimal agar medium, of the Escherichia coli lacZam strain that was first used by Cairns and his colleagues to demonstrate the phenomenon of "adaptive mutation." We have found, by performing appropriate reconstruction studies, that most of the late-arising Lac+ revertants of this lac amber strain (appearing as colonies in 3-5 days) are slow-growing ochre suppressor mutants that probably existed in the culture prior to plating and cannot, therefore, be classified as "adaptive." The appearance of a small number of fast-growing, late-arising Lac+ revertants may result from residual cell growth and turnover or from phenomena related to the fact that the lacZam mutation in strain SM195 is carried on an F' plasmid. Thus, the appearance of late-arising revertants in this lacZam system does not provide convincing evidence that selective conditions specifically increase the rate of occurrence of favorable mutations.

Evaluation of the TOPKAT system for predicting the carcinogenicity of chemicals.

The TOPKAT computer-based system for predicting chemical carcinogens was evaluated by determining its ability to predict the carcinogenicity of chemicals tested by the National Toxicology Program. TOPKAT was not effective in identifying potential rodent carcinogens and noncarcinogens in the data set analyzed. The chemicals in the TOPKAT database of known carcinogens and noncarcinogens that the software identifies as most "similar" to unknown chemicals are illustrated using six examples. These "similar" chemicals generally bear no apparent relationship to the chemical of interest with regard to metabolism or potential mechanism of carcinogenicity. Environ. Mol. Mutagen. 37:55-69, 2001 Published 2001 Wiley-Liss, Inc.

Reevaluation of the mutagenicity and carcinogenicity of chemicals previously identified as "false positives" in the Salmonella typhimurium mutagenicity assay.

An accurate determination of the correlation between the carcinogenicity and the mutagenicity of chemicals has been hampered by the lack of a well-documented list of noncarcinogens. To overcome this problem, Shelby and Stasiewicz (Environ Mutagen 6:871-878, 1984) published a list of 70 chemicals that showed no evidence of carcinogenicity in the National Cancer Institute (NCI) or National Toxicology Program (NTP) rodent carcinogenesis bioassays. More recently, Tennant et al. (Science 236:933-941, 1987) published a list of chemicals, including 29 noncarcinogens, that had been adequately tested for carcinogenicity by the NTP. Of the chemicals listed by Shelby and Stasiewicz or by Tennant and co-workers as noncarcinogenic, the NTP has evaluated 25 as mutagenic in Salmonella typhimurium; 48 of the noncarcinogens were evaluated as nonmutagenic. Thus, of the 73 noncarcinogens that have been evaluated as either positive or negative for mutagenicity, 34% (25/73) were "false positives" (mutagenic noncarcinogens) in the S. typhimurium assay. We re-evaluated the same mutagenicity and carcinogenicity data to determine whether the frequency of "false positives" is really as high as it appears to be. Our reevaluation of the mutagenicity data used more stringent criteria for calling a compound mutagenic than those used by the NTP, resulting in a substantial reduction in the frequency of "false positives" in the S. typhimurium mutagenicity assay. However, application of these same stringent criteria also substantially reduced the frequency of "true positives" (mutagenic carcinogens). Thus, it is concluded that modification of the evaluation criteria for the mutagenicity test can increase the specificity of the assay for the detection of carcinogens, but only at the cost of a corresponding reduction in sensitivity. We also performed a separate reevaluation of the NCI/NTP carcinogenicity data for the 25 S. typhimurium "false positives," assuming that the NTP evaluations of the mutagenicity data were correct. These reevaluations were based on the methodologies and findings of Griesemer and Cueto (In Montesano R, Bartsch H, Tomatis L (eds): Molecular and Cellular Aspects of Carcinogen Screening Tests.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenicity of nitro compounds in Salmonella typhimurium in the presence of flavin mononucleotide in a preincubation assay.

A series of nitro compounds (18 aromatic and one aliphatic) was evaluated using a modification of the standard Salmonella typhimurium mutagenicity assay. A preincubation protocol was used with flavin mononucleotide (FMN) incorporated into the assay mixture to facilitate nitro reduction. Several aromatic nitro compounds (m-nitroaniline, p-nitroaniline, 2,6-dinitrotoluene, 2,4-dinitrotoluene,2,3-dinitrotoluene,1,8-dinitronaphthalene), which were negative or only weakly mutagenic when tested in the standard plate incorporation assay, showed FMN-dependent mutagenic responses with this procedure. For some nitro compounds, the addition of FMN was not needed for the detection of mutagenicity in the modified protocol. Not all nitro compounds were positive using the preincubation procedure with FMN. The lack of mutagenicity, however, does not appear to be the result of the inability of the modified method to reduce nitro compounds, since it was found that reduction does occur under the assay conditions for the two nonmutagens evaluated for nitro reduction (nitrobenzene and p-nitrophenol). It is suggested that the modified protocol may be useful for evaluating the mutagenicity of many nitro compounds.

Evolution of social concerns and environmental policies for chemical mutagens.

The founding of the Environmental Mutagen Society 20 years ago coincided with the beginning of general social concern about exposure to chemical mutagens. Initially, this concern focused on the potential of chemicals to induce heritable genetic damage in humans. Within a few years, however, mutagenicity tests came to be regarded primarily as short-term tests for carcinogenicity. Serious doubts have recently been cast upon the relationship between mutagenicity and carcinogenicity, and, as a result the real utility of mutagenicity tests is being questioned. Justification for the continued use of these tests will require 1) more detailed mechanistic knowledge concerning the role of genetic changes in the development of cancer and 2) an improved ability to relate the results of mutagenicity tests to the potential for inducing heritable genetic effects in humans.

Anomalous mutagenicity profile of cyclohexanone oxime in bacteria: cell survival in background lawns.

The basis for the observed mutagenicity of cyclohexanone oxime in the presence of hamster liver S9 in Salmonella typhimurium strain TA1535, but not in TA100, was explored. While the chemical had no effect on the appearance of the background lawn in either strain, it did cause a reduction in mutant colony counts in strain TA100, raising the possibility of selective toxicity to this strain. Viability of the two strains was determined directly by titering the cells in background lawns over a 3 day period. In order to do this, cells embedded in top agar overlays were released by extruding agar plugs through small holes in the bottoms of centrifuge tubes, followed by vigorous vortexing. Viable cell counts in background lawns of strain TA100, but not strain TA1535, were greatly reduced in the presence of cyclohexanone oxime. Most of the loss of viable TA100 cells occurred on days 2 and 3 following plating, after the cells had exhausted the histidine in the medium and stopped growing. Therefore, the observed loss of background lawn viable cells is unlikely to be the cause of the non-mutagenicity of cyclohexanone in strain TA100. Analysis of reversion spectra showed that cyclohexanone oxime-induced C-->T transitions in the second position of the CCC triplet at the his mutation site in strain TA1535, but had no significant effect on any transition or transversion in strain TA100.

Chemicals mutagenic in Salmonella typhimurium strain TA1535 but not in TA100.

The standard Salmonella mutagenicity test uses two strains of Salmonella typhimurium (TA1535 and TA100) containing the same base pair substitution mutation (hisG46). These strains differ only in that strain TA100 contains the plasmid pKM101, whose mucAB gene products enhance SOS mutagenesis. This makes strain TA100, in general, the more sensitive of the two for mutagen detection, raising the question as to whether or not to include strain TA1535 in the core battery of strains in routine testing. Out of 659 chemicals judged as mutagens in the S. typhimurium assay when subjected to the National Toxicology Program's screening protocol, 36 (5%) were evaluated as positive in strain TA1535 but not in strain TA100. Of these, 23 were judged as negative and 13 as equivocal in strain TA100, and 5 were positive or equivocal in at least one other strain (TA97 or TA98). In general, the data on these chemicals indicate that the absolute increases in revertants per plate induced in strain TA1535 were too small to have been judged as positive if similar increases occurred in strain TA100, which has a much higher spontaneous background. For three chemicals (acetaldehyde oxime, 6-mercaptopurine, and 1,3-butadiene) the absolute increases in revertants in strain TA1535 greatly exceeded those in strain TA100. Evaluation of the reproducibility of these findings and of the mechanisms and relevance of unique TA1535 positives should be useful when decisions are made as to whether this strain should be kept as a part of the core battery of strains in the S. typhimurium assay.

Effects of pH on weak and positive control mutagens in the Ames Salmonella plate assay.

The effects of pH on the mutagenic activity of several chemicals were evaluated in the standard Ames Salmonella typhimurium plate-incorporation assay. The pH of the base agar was varied between 6.0 and 8.0. The positive control compounds routinely used in this laboratory, 2-aminoanthracene, 4-nitro-o-phenylenediamine, sodium azide and nitrofurantoin, showed increasing mutagenic activity as the pH was decreased to 6.0. However, the activity of two weakly mutagenic cosmetic ingredients, 2,2',4,4'-tetrahydroxybenzophenone and trans-4-phenyl-3-buten-2-one, was completely eliminated at pH levels near 6.0. It is concluded that plates poured with agar with pH levels below 7.0 can result in strong responses for the positive control chemicals but give negative results for some mutagens.

Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S9.

A protocol for assessing the mutagenic activity of azo dyes derived from mutagenic or potentially mutagenic aromatic amines was evaluated, using 4 model compounds. This protocol is based upon one developed in Sugimura's laboratory with modifications, including the use of flavin mononucleotide (FMN) rather than riboflavin to reduce the azo compounds to free amines, and hamster liver S9 rather then rat liver S9 for metabolic activation. The protocol developed differs from the standard Ames Salmonella plate incorporation assay in 5 ways: (1) uninduced hamster liver S9 rather than Aroclor 1254-induced rat liver S9 is used; (2) 150 microliters of S9 is used rather than the maximum of 50 microliter of S9 used in the standard assay; (3) FMN is added to the cofactor mix; (4) the cofactor mix is modified to include exogenous glucose 6-phosphate dehydrogenase, NADH, and 4 times the standard amount of glucose 6-phosphate; and (5) a 30-min "pre-incubation" step is used before addition of top agar. We found that each of these 5 changes is necessary for optimal mutagenic activity of azo dyes derived from the mutagenic aromatic amines benzidine, o-tolidine or o-dianisidine. The use of hamster liver S9 rather than rat liver S9 was also required for optimal mutagenic activity of benzidine itself. Rat liver S9 inhibited the ability of hamster S9 to activate benzidine to a mutagen. The presence in rat liver S9 of an inhibitor of the metabolic activation of benzidine may account for the failure of benzidine and a benzidine dye (Congo red) to be strongly mutagenic when tested with this type of S9.

Bacterial mutagenicity testing of 49 food ingredients gives very few positive results.

49 substances permitted for use in food in the United States was tested for mutagenicity in the Ames Salmonella typhimurium assay and in Escherichia coli strain WP2. Four of these substances caused increases in revertant counts in S. typhimurium. Two of these four (papain and pepsin) were found to contain histidine, and therefore the results of the tests on these two substances could not be taken as demonstrating mutagenicity. The other two substances causing increases in revertant counts (hydrogen peroxide and potassium nitrite) were mutagenic. The results on one chemical, beta-carotene, were evaluated as inconclusive or questionable. The remaining 44 substances were nonmutagenic in the test systems used. It is concluded that, for those generally physiologically innocuous chemicals tested, there are very few 'false positives' in the bacterial test systems used.

Isolation and characterization of an isogenic set of Salmonella typhimurium strains analogous to the "Ames" tester strains.

The standard Ames tester strains of Salmonella typhimurium contain a number of genetic differences at loci other than his. The fact that these strains contain independently isolated uvrB-bio-gal deletions and rfa mutations implies that these are likely to vary from strain to strain. Since the strains were isolated from different parental stocks of S. typhimurium LT-2, they differ in their ability to metabolize arabinose. Other, unknown differences may exist because the isolation of some of the strains involved ultraviolet and chemical mutagenesis. We have isolated a set of isogenic S. typhimurium strains that contain the relevant genetic markers of the standard Ames tester strains. These strains all contain the same uvrB-bio-gal deletion and the same rfa mutation; they differ only in the nature of their his mutations and in the presence or absence of the plasmid pKM101. We have assessed the responsiveness of these strains to a number of mutagens and conclude that their mutagenic specificities are the same as those of the corresponding Ames strains: TA98, TA100, TA1535, TA1537 and TA1538. Therefore, the specificity of the standard Ames strains with respect to these mutagens is a result solely of the differences in the nature of their his mutations and the effects of pKM101.

Evaluation of azo food dyes for mutagenicity and inhibition of mutagenicity by methods using Salmonella typhimurium.

The mutagenicity of 4 azo dyes (FD&C Yellow No. 5, FD&C Yellow No. 6, FD&C Red No. 40 and amaranth) that are widely used to color food has been evaluated. 4 different methods were used: (1) the standard Ames plate-incorporation assay performed directly on the dyes in the absence of S9 and in the presence of rat- or hamster-liver S9; (2) application of the standard plate assay to ether extracts of aqueous solutions of the dyes; (3) a variant of the standard assay, using hamster liver S9, preincubation, flavin mononucleotide (FMN) and other modifications designed to facilitate azo reduction; and (4) reduction of the dyes with sodium dithionite, followed by ether extraction and the standard plate assay. Assays that include chemical reduction (methods 3 and 4) were included because azo compounds ingested orally are reduced in the intestine with the release of free aromatic amines. No mutagenic activity was seen for any of the azo dyes tested by using the standard Ames plate assay (method 1). Ether extracts of some samples of FD&C Yellow No. 6, FD&C Red No. 40 and amaranth were active (method 2), but only at high doses, generally 250 mg-equivalents or more per plate. These results indicate the presence of low levels of ether-extractable mutagenic impurities. The FMN preincubation assay (method 3) gave negative results for all dye samples tested. Most batches of FD&C Red No. 40 tested had mutagenic activity that was detectable when the ether extract of less than 1 mg of dithionite-reduced dye was plated in the presence of S9 (method 4). This finding implies that an impurity in these samples of FD&C Red No. 40 can be reduced to yield an ether-extractable mutagen. Dithionite-reduced samples of FD&C Yellow No. 6 and amaranth showed ether-extractable mutagenic activity only at much higher doses than those at which activity was seen with most dithionite-reduced samples of FD&C Red No. 40 (method 4). FD&C Yellow No. 5 showed no mutagenic activity with this method. Mutagenic activity was not detected when FD&C Red No. 40 was tested by using the azo reduction preincubation assay with FMN (method 3).(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenicity of benzidine and benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay.

We have evaluated the mutagenic activity of a series of diazo compounds derived from benzidine and its congeners o-tolidine, o-dianisidine and 3,3'-dichlorobenzidine as well as several monoazo compounds. The test system used was a modification of the standard Ames Salmonella assay in which FMN, hamster liver S9 and a preincubation step are used to facilitate azo reduction and detection of the resulting mutagenic aromatic amines. All of the benzidine and o-tolidine dyes tested were clearly mutagenic. The o-dianisidine dyes except for Direct Blue 218 were also mutagenic. Direct Blue 218 is a copper complex of the mutagenic o-dianisidine dye Direct Blue 15. Pigment Yellow 12, which is derived from 3,3'-dichlorobenzidine, could not be detected as mutagenic, presumably because of its lack of solubility in the test reaction mixture. Of the monoazo dyes tested, methyl orange was clearly mutagenic, while C.I. Acid Red 26 and Acid Dye (C.I. 16155; often referred to as Ponceau 3R) had marginal to weak mutagenic activity. Several commercial dye samples had greater mutagenic activity with the modified test protocol than did equimolar quantities of their mutagenic aromatic amine reduction products. Investigation of this phenomenon for Direct Black 38 and trypan blue showed that it was due to the presence of mutagenic impurities in these samples. The modified method used appears to be suitable for testing the mutagenicity of azo dyes, and it may also be useful for monitoring the presence of mutagenic or potentially carcinogenic impurities in otherwise nonmutagenic azo dyes.

Evaluation, using Salmonella typhimurium, of the mutagenicity of seven chemicals found in cosmetics.

Six chemicals used as ingredients in cosmetics were evaluated for mutagenic activity in Salmonella typhimurium. Two of these ingredients, trans-4-phenyl-3-butene-2-one and 2,2',4,4'-tetrahydroxybenzophenone, were mutagenic in the presence of rat liver S-9 towards strains TA100 and TA1537 respectively. An impurity found in some cosmetic products, N-nitrosodiethanolamine, was mutagenic to S. typhimurium strains TA1535 and TA100 in the presence of hamster-liver S-9 but not rat-liver S-9.

Determination of combined benzidine in FD&C Yellow No. 6 (Sunset Yellow FCF).

Samples from 67 manufactured lots of FD&C Yellow No. 6 (Sunset Yellow FCF; Colour Index No. 15985) were analysed for combined benzidine. These samples were selected from those submitted to the US Food and Drug Administration for certification between October 1991 and December 1992 by 13 dye distributors. Dithionite was used to reduce any combined benzidine present in the form of azo or disazo dyes to free benzidine. This reduction was followed by extraction, diazotization and coupling with 2-naphthol-3,6-disulfonic acid disodium salt (R salt). The total benzidine was quantified as benzidine-R salt disazo dye by HPLC with detection at 540 nm and a quantification limit of 10 ng benzidine/g FD&C Yellow No. 6. Of the 67 samples analysed, 34 were found to contain more than 10 ng combined benzidine/g. Of these, 30 samples were from one manufacturing company, including three of its subsidaries. The level of combined benzidine found ranged from 11 to 104 ng/g, except for one sample containing 941 ng/g.

Determination of combined benzidine in FD & C yellow no. 5 (tartrazine), using a highly sensitive analytical method.

53 samples of FD & C Yellow No. 5 (tartrazine; Colour Index No. 19140) were examined for combined benzidine. These samples, which represent separate lots from 12 dye distributors, were submitted to the US FDA for certification between 28 February 1990 and 27 June 1991. A method was developed to reduce the dye matrix with dithionite so that combined benzidine present in the form of azo or disazo dyes would be converted to free benzidine. Reduction was followed by extraction, diazotization and coupling with pyrazolone T, and the total benzidine present was quantitated as benzidine-pyrazolone T disazo dye (BZPT) by HPLC with detection at 500 nm. The limit of quantitation for benzidine in FD & C Yellow No. 5 by this method is 5 ng/g. 25 samples of FD & C Yellow No. 5 were found to contain 7-83 ng/g of combined benzidine that was released by dithionite reduction. 23 of these samples were from the same manufacturer. The identify of the BZPT from two FD & C Yellow No. 5 samples was confirmed by spectral analysis using HPLC with a photodiode array detector.