Mutagenicity of carbon tetrachloride and chloroform in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, using a gas exposure method

The volatile solvents carbon tetrachloride and chloroform are carcinogens that are often reported as nonmutagenic in bacterial mutagenicity assays. In this study, we evaluated the mutagenicity of these compounds in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, with and without S9 mix, using a gas exposure method. Tests were also conducted with a glutathione-supplemented S9 mix. Carbon tetrachloride was mutagenic in TA98 without S9 mix, and in WP2/pKM101 and WP2uvrA/pKM101 with and without S9 mix; carbon tetrachloride was not mutagenic in TA100, TA1535 or TA1537. Chloroform was mutagenic in WP2/pKM101, but only in the presence of glutathione-supplemented S9 mix. Chloroform was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 with or without S9 mix, and was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 in the presence of glutathione-supplemented S9 mix. The data indicate that carbon tetrachloride and chloroform are bacterial mutagens when adequate exposure conditions are employed and suggest that a genotoxic mode of action could contribute to the carcinogenicity of these compounds.     

Propyldazine is mutagenic in Salmonella typhimurium and Escherichia coli: distinct specificity for strains TA1537 and TA97

The antihypertensive drug propyldazine (Atensil) was demonstrated to be mutagenic with auxotrophic mutants of Salmonella typhimurium and Escherichia coli. Addition of liver S9 mix (postmitochondrial supernatant fraction supplemented with an NADPH-generating system) had little, if any, effect on the mutagenicity. The mutagenicity showed an unusual pattern of strain specificity. Increased frequencies of reversion were observed with all strains whose auxotrophy was caused by frame-shift mutations: the number of revertant colonies per plate from S. typhimurium TA98, TA1538, TA97, and TA1537 was increased up to 5-, 9-, 43-, and 160-fold, respectively, above background. Among the strains that became auxotrophic by substitution mutations, S typhimurium TA102, E. coli WP2, and E coli WP2 uvrA yielded positive results (twofold above background). S. typhimurium TA1535 and TA100 were not reverted by propyldazine. It should be noted that propyldazine, due to its low toxicity and good solubility, could be tested up to very high doses. Hence, although quite impressive mutagenic effects occurred, the mutagenic potency was moderate even in the most responsive strains, TA1537 and TA97 (about 0.3 and 1.0 revertants per nmole, respectively). With the limitation that the strain specificities were different, the mutagenic potency of propyldazine was in the same order of magnitude as that of hydralazine and dihydralazine, two related antihypertensive drugs which were already known to be mutagenic. In our hands, both compounds were mutagenic in S typhimurium TA1535, TA100, TA1537, and TA98. These results differ from data in the literature in that we found clear but weak effects even with strains for which others have reported negative results.     

Mutagenic activity and mutational specificity of antiprotozoal drugs with and without nitrite treatment

We examined the mutagenic activities of six antiprotozoal drugs (three diaminopyrimidine compounds [pyrimethamine, diaveridine, and trimethoprim] and three 8-aminoquinoline derivatives [primaquine, pentaquine, and pamaquine]) in Escherichia coli WP2uvrA/pKM101 and Salmonella typhimurium TA100 and TA98 with and without nitrite treatment. The diaminopyrimidine compounds showed no mutagenic activity under any condition in any strain. The 8-aminoquinoline derivatives after nitrite treatment at 5-20 mM for 5 min at pH 3, on the contrary, showed clear mutagenicity in TA100 and WP2uvrA/pKM101 in the presence and absence of S9 mix. We concluded that 8-aminoquinoline derivatives became mutagenic following nitrite treatment. In the Lac(+) reversion assay with E. coli WP3101P-WP3106P, these nitrite-treated compounds induced G:C --> A:T transitions and G:C --> T:A transversions in the absence of S9 mix. On the other hand, A:T --> T:A transversions were induced only in the presence of S9 mix, suggesting a different kind of products may be responsible for the mutagenicity.     

Mutagenic activity in synthetic pyrethroids in Salmonella typhimurium

Four pyrethroids, allethrin, resmethrin, permethrin and fen-valerate,were tested for mutagenicity in bacterial reversion assay systemswith seven strains (TA1535, TA100, TA1538, TA98, TA1537, TA97and TA104) of Salmonella typhimurium. Our results show thatthree pyrethroids, namely resmethrin, permethrin and fenvalerate,were not found to be mutagenic in S. typhimurium in the presenceor absence of a rat liver activation system. Allethrin was foundto be mutagenic with TA100, TA104 and TA97 strains and requiredmetabolic activation (S9 mix) in order to show its activity,mainly with TA100 and TA104 strains.     

Photomutagenicity of thiabendazole,a postharvest fungicide,in bacterial assays

We investigated the photomutagenicity of thiabendazole (TBZ), a postharvest fungicide commonly used on imported citrus fruits. Using UVA light (320-400 nm), we irradiated bacterial cultures with or without TBZ in a 24-well multiplate. UVA-irradiation without TBZ was not mutagenic to the tester strains, nor was unirradiated TBZ. TBZ was strongly photomutagenic in Escherichia coli WP2uvrA and WP2uvrA/pKM101 strains, weakly photomutagenic in Salmonella typhimurium TA100 and TA98, and not photomutagenic in S. typhimurium TA1535 and TA1538. The photomutagenicity of TBZ was more evident in WP2uvrA/pKM101, which carries the trpE65 ochre mutation (TAA), than in TA100, which carries the hisG46 missense mutation (CCC). In E. coli WP3101-WP3106 and the corresponding pKM101-containing strains, photoactivated TBZ induced predominantly G:C-->A:T transitions and A:T-->T:A transversions. In the plasmid-containing strains only, TBZ induced a moderate number of A:T-->G:C transitions and a few A:T-->C:G and G:C-->T:A transversions. The observation that UVA-irradiated TBZ mutated both G:C and A:T basepairs may explain why WP2uvrA/pKM101 was more sensitive to its mutagenicity than TA100. TBZ that was irradiated before it was added to the WP2uvrA/pKM101 cells was not photomutagenic, which suggests that the photomutagenic products of TBZ were unstable or rapidly reacted with other molecules before being incorporated into cells.     

Formaldehyde is a bacterial mutagen in a range of Salmonella and Escherichia indicator strains

Formaldehyde was examined for bacterial mutagenicity using Escherichiacoli WP2(pKM101) and WP2uvrA(pKM101), and Salmonella typhimuriumTA1535, TA1537, TA1538, TA98, TA100 and TA102, in the absenceof any exogenous source of metabolic activation. Using pre-incubationexposure, clear mutagenicity was seen for TA98, TA100 and TA102,and both E.coli strains. In standard plate-incorporation assays,consistent mutagenicity was seen only for TA100 and WP2uvrA(pKM101).No evidence of mutagenicity was seen for TA1535, TA1537 or TA1538using either method of exposure. These data confirm the enhancedability of the pre-incubation method to detect the mutagenicityof formaldehyde both quantitatively, as expressed by numbersof revertant colonies, and qualitatively, in terms of the rangeof indicator strains reverted. The relatively greater sensitivityof the pre-incubation assay is probably due to better containmentof a volatile agent and/or lack of interaction with agar duringthe initial period of exposure. The findings are consistentwith the suggestion that formaldehyde induces lesions in bacteriawhich are, at least to some extent, excision-repairable, andindicate that the presence of the R-factor plasmid may be requiredfor the expression of its mutagenicity in excision repair-deficientSalmonella.     

Bacterial genotoxicity of nitrosated famotidine

Famotidine, a histamine H2-receptor antagonist, was devoid ofmutagenic activity in seven his^- Salmonella typhimurium strains(TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102) and wasequitoxic in repair-proficient (WP2) and repair-deficient (WP2uvr,WP67, CM561, CM571, WP100 and CM871) Escherichia coli strains,both in the presence and in the absence of S9 mix containingliver S9 fractions from Aroclor-treated rats. However, aftera short pre-incubation step with nitrite in an acidic environment,the drug increased, by a direct mechanism, the number of his⁺revertants in Salmonella strains TA100, TA102 and TA97 (a decreaseof mutagenicity being conversely observed in TA1535) and oftrp+ revertants in E. coli strains WPluvrA and WP67. Moreover,it enhanced the induction of non-reparable DNA damage in E.coli strains simultaneously lacking the uvrA-dependent excisionrepair and the lexA post-replication repair pathways. The mutagenicityof acidified nitrite-famotidine mixtures was related to dosesof both precursors, with a maximum production of mutagenic derivativesin a slight molar excess of nitrite. The optimal pH of the nitrosationreaction (2.0) was intermediate between the one required forcimetidine (1.5) and ranitidine (2.5). Potency of famotidineas a precursor of mutagenic derivatives was considerably lowerthan the one of the other two H₂ blockers. The nitrosation productsof all three drugs mainly induced base-pair substitutions inSalmonella DNA, to a greater extent at sites containing G-Cbase pairs (strain TA100) in the case of famotidine and cimetidine,and at sites containing AT base pairs (TA102) in the case ofranitidine. Although these experimental findings may suggestpossible toxicological consequences in ulcer patients receivinganti-secretory drugs, various considerations tend to minimizetheir practical in vivo relevance, especially for risk-benefitevaluations. Additionally, as in the case of cimetidine andranitidine, formation of mutagenic nitrosated famotidine wasefficiently prevented by equimolar ascorbic acid.     

Mutagenicity spectra in Salmonella typhimurium strains of glutathione, L-cysteine and active oxygen species

Glutathione and L-cysteine, in the presence of rat kidney post-mitochondrialsupernatant (S9) fraction, and various forms of active oxygenwere investigated for mutagenicity in seven his^– strainsof Salmonella typhimurium. Glutathione and L-cysteine showedqualitatively and quantitatively virtually identical mutagenicactivities. The number of mutants induced in strain TA97 was3–4 times higher than in TA100, the strain in which themutagenicity was originally detected. Mutagenic effects werealso observed in strains TA92, TA102 and TA104, but not in TA1535and TA1537. Hydrogen peroxide, superoxide and glucose/glucoseoxidase in the presence and absence of kidney S9 fraction showedpronounced mutagenic effects in strains TA104 and TA102. Additionally,weak mutagenic effects were observed in TA100, while the remainingstrains, including TA97, were not responsive. These mutagenicityspectra suggest that the mutagenic species formed from glutathioneand L-cysteine are similar, if not identical, and are differentfrom hydrogen peroxide, superoxide and other oxygen speciesderived from them. Further support for this notion was givenwhen it was observed that catalase did not affect the mutagenicityof glutathione and that superoxide dismutase showed a significanteffect only when used in milligram quantities. This study showsthat mutagenicity spectra may be useful in the elucidation ofactivation pathways. Furthermore, it is interesting to notethat all the compounds and preparations showing a positive responsein the Ames test in the present study occur endogenously inorganisms: glutathione, L-cysteine, hydrogen peroxide, superoxide,glucose, glucose oxidase and kidney S9 fraction (which was mutagenicin several strains).     

The SIMULTEST: a new approach to screening chemicals with the Salmonella reversion assay

A new Salmonella mutagenicity test method is under development to test a chemical with more than one strain simultaneously (the "SIMULTEST"), that is, different Salmonella typhimurium tester strains are used in combination on the same plate. Strains are combined in two sets: strains with plasmid pKM101 (TA97, TA98, TA100, and TA102) and strains without the plasmid (TA1535, TA1537, and TA1538). The SIMULTEST combinations successfully detect the mutagenic activity of five mutagens in different chemical classes. This approach may be useful in reducing the workload associated with mutagenicity testing with Salmonella.     

Further studies on the detection of mutagenic and genotoxic activity in human faeces: aerobic and anaerobic fluctuation tests with S. typhimurium and E. coli, and the SOS Chromotest

The product of six complete bowel movements were collected from one man (consuming a normal western diet) over a period of 5 months. Aqueous faecal extracts were prepared (0.75 g wet wt./ml water) under aerobic and anaerobic conditions. Graded doses were assayed aerobically and anaerobically for mutagenicity in replicate microtitre fluctuation tests employing histidine reversion in Salmonella typhimurium TA100 and tryptophan reversion in Escherichia coli WP2uvrA(pKM101), and in aerobic SOS Chromotests, using E. coli PQ37. The histidine and tryptophan content of each extract was determined by (i) pre-column derivatisation with o-phthaldialdehyde followed by reverse-phase h.p.l.c. and (ii) by aerobic and anaerobic bioassays employing appropriate auxotrophic strains of S. typhimurium and E. coli. All six faecal samples contained enough histidine and tryptophan or their precursors to compromise the interpretation of fluctuation tests. The h.p.l.c. technique, though quantitatively detecting free tryptophan added to faecal extracts, did not account for growth-enhancing effects of faecal extracts detected by bioassay using bacteria with a requirement for tryptophan. However, h.p.l.c. detected histidine more efficiently than bioassays. A combination of reconstruction experiments and bioassays enabled construction of 'growth windows' within which, in fluctuation tests, dose-related increases in numbers of positive wells could be ascribed to mutation rather than to feeding of auxotrophic bacteria. Under aerobic conditions of extraction and assay, five out of six samples were mutagenic to S. typhimurium TA100, and all six were mutagenic to E. coli WP2uvrA(pKM101). None of the samples extracted and assayed anaerobically was mutagenic to S. typhimurium, but all six were positive in E. coli WP2uvrA(pKM101). There was an increase both in faecal histidine and tryptophan and in mutagenicity during the period of collection, the association between faecal tryptophan and faecal mutagenicity being statistically significant. All the samples were negative in the SOS Chromotests. We conclude that this donor regularly excretes faeces which contain at least two classes of water-soluble directly-acting mutagens which are not detected in SOS Chromotest at doses five times those giving positive effects in fluctuation tests.     

The effectiveness of Salmonella strains TA100, TA102 and TA104 for detecting mutagenicity of some aldehydes and peroxides

Several aldehydes and peroxides were tested for mutagenicityusing Salmonella typhimurium tester strains TA97a, TA100, TA102and TA104, in the presence and absence of Aroclor-induced liverS9 mix from F344 rats and B6C3F₁ mice, in either preincubationor vapour phase rotocols. Some chemicals were tested in additionalSalmonella strains. Benzaldehyde, butyraldehyde, benzoyl peroxide,4-chlorobenzaldehyde, isobutyraldehyde, propionaldehyde andveratraldehyde were non-mutagenic Acetaldehyde and dicumyl peroxidegave inconsistent results and furfural gave equivocal responsesin TA100 and TA104. Cumene hydroperoxide, formaldehyde and glutaraldehydewere mutagenic in TA100, TA102 and TA104. trans-Cinnamaldehydeexhibited a weak mutagenic response in TA100 with mouse liverS9 only. 2,4,5-Trimethoxybenzaldehyde was mutagenic only instrain TA1538 with rat liver S9. With the exception of butanoneperoxide, which was mutagenic only in TA104, all chemicals mutagenicin strains TA102 and/or TA104 were also mutagenic in TA100.The data do not, therefore, support the preferential use ofstrains TA102 and TA104 for screening aldehydes and peroxidesfor mutagenicity. For a number of these chemicals the advantagesof using TA102 or TA104 was in the increased responses comparedwith those obtained with TA100. Two of the four peroxides weremutagenic and one of these was mutagenic only with TA104. Thissuggests that strains TA102 and TA104 be used if peroxides arenot mutagemc in TA100 or TA97. ⁴Present addresses: ⁴British American Tobacco Ltd, SouthamptonSO15 8TL, UK ⁵FRAME, Nottingham NG1 4EE, UK ³To whom correspondence should be addressed. Tel: +1 919 541 4482; Fax: +1 919 541 2242; Email: zeiger{at}niehs.nih.gov      

Bacterial mutagenicity assays: Vehicle and positive control results from the standard Ames assay,the 6‐ and 24‐well miniaturized plate incorporation assays and the Ames II™ assay

Bacterial mutation assays are conducted routinely as part of the safety assessment of new chemicals. The OECD Test Guideline (TG) 471 describes the conduct of the standard agar plate Ames assay, required for regulatory submissions. Higher throughput non‐OECD 471 TG assays, such as the miniaturized plate incorporation and Ames II? assays, can be used for prescreening purposes. We have compiled historical vehicle and positive control data generated using these methods. The historical database is comprised from experiments spanning 9 years and includes >1000 experiments from the standard Ames assay using the plate incorporation and pre‐incubation methods (TA98, TA100, TA1535, TA1537, and WP2 uvrA), >50 experiments from the 6‐well (TA98, TA100, TA1535, TA97a, and WP2 uvrA) and >100 experiments from the 24‐well (TA98, TA100, TA102, TA1535, TA1537, and TA97a) plate incorporation assays, and >1000 experiments from the Ames II? assay (TA98 and TAMix). Although miniaturization to a 24‐well format made the measurement of control revertant colonies in TA1537 and TA1535 more difficult; this can be overcome by using an alternative strain with a higher spontaneous reversion rate (i.e., using TA97a instead of TA1537) or by increasing the number of replicate wells to 12 (for TA1535). All three miniaturized methods, including the Ames II? assay, were responsive to known mutagens and the responses were reproducible over years of use. These data demonstrate the excellent reproducibility of the standard and miniaturized bacterial mutation assays using positive control chemicals. Environ. Mol. Mutagen. 57:483–496, 2016. © 2016 Wiley Periodicals, Inc.     

Mutagenic evaluation of trichlorfon using different assay methods with Salmonella typhimurium

Evaluation of the mutagenicity of trichlorfon pesticide was carried out with strains TA1535, TA100, TA97, TA98 and TA104 of Salmonella typhimurium by means of several assay methods: (i) spot test; (ii) standard plate incorporation test; (iii) plate incorporation test with preincubation; (iv) fluctuation test and (v) fluctuation test with preincubation, with and without post-mitochondrial liver fraction (S9) from Wistar rats pretreated with phenobarbital and 5,6-benzoflavone as a metabolic activation system. Trichlorfon induced base-pair substitution mutations, and its mutagenic activity was decreased by the addition of S9 mix. The fluctuation test and fluctuation test with preincubation were the most sensitive assay methods for detecting the mutagenicity of trichlorfon.     

DNA damage and mutagenicity induced by endosulfan and its metabolites

Endosulfan is a widely used broad-spectrum organochlorine pesticide, which acts as a contact and stomach poison. Nontarget species, such as cattle, fish, birds, and even humans, are also affected. Studies on the genotoxicity and mutagenicity of endosulfan have been inconsistent and nothing is known about the genotoxicity of its metabolites. In the present study, endosulfan (as a commercial isomeric mixture and as the alpha- and beta-isomers), and metabolites of endosulfan (the sulfate, lactone, ether, hydroxyether, and diol derivatives) were assayed for their ability to induce DNA damage in Chinese hamster ovary (CHO) cells and human lymphocytes using the Comet assay and were assayed for their mutagenicity using the Salmonella reversion assay (Ames test with TA98, TA97a, TA102, TA104, and TA100, with and without S9 activation). The compounds produced statistically significant (P < 0.01), concentration-dependent (0.25-10 microM) increases in DNA damage in both CHO cells and human lymphocytes. Endosulfan lactone caused the most DNA damage in CHO cells, while the isomeric mixture of endosulfan produced the greatest response in lymphocytes. The test compounds also were mutagenic in Salmonella strains at concentrations of 1-20 mug/plate (P < 0.05), with TA98 being the most sensitive strain and the diol and hydroxyether metabolites producing the highest responses. The results indicate that exposure to sublethal doses of endosulfan and its metabolites induces DNA damage and mutation. The contribution of the metabolites to the genotoxicity of the parent compound in Salmonella and mammalian cells, however, is unclear, and the pathways leading to bacterial mutation and mammalian cell DNA damage appear to differ.     

SOS induction in Escherichia coli and Salmonella mutagenicity: a comparison using 330 compounds

To examine the concordance of two microbial genotoxicity short-termassays, 330 experimental results for the SOS chromotest usingtester strain Escherichia coli PQ37 were compared with the resultsof the Salmonella/mammalian microsome mutagenicity assay withSalmonella typhimurium TA97, TA98, TA100, TA102, TA104, TA1535,TA1537 and/or TA1538. With respect to qualitative features,the concordance between SOS chromotest and Salmonella mutagenicitytest results was 86.4% (sensitivity, 78.6%; specificity, 100%;     

Comparison of the sensitivities of Salmonella typhimurium strains TA102 and TA2638A to 16 mutagens

The qualitative and quantitative sensitivity of the genetically related, histidine-auxtrophic Salmonella typhimurium strains TA102 and TA2638a to 16 compounds was examined. The compounds were mainly cross-linking and oxidising mutagens, the effects of which were known to be detected by strain TA102 preferentially or by a combination of Escherichia coli WP2 (pkM101) and uvrA/pkM101. The morphology and number of spontaneous revertants was also compared. Fourteen of the 16 compounds caused reversion in both strains. Bleomycin and streptomycin induced reversion in strain TA102 but not TA2638a. The greater sensitivity of TA102 to these compounds may be associated with the extrachromosomal location of the target genes. The overall quantitative sensitivity of the two strains was similar for the other compounds. The number of compounds that caused reversions at lower doses or produced greater proportional increases were the same in TA102 as in TA2638a. The spontaneous number of revertants, without and with metabolic activation, respectively, was 98 and 130 for TA2638a and 322 and 465 for TA102. Strain TA2638a formed larger, more uniform colonies than TA102. The present results together with those of previous studies indicate a high degree of concordance between the sensitivity of strains TA102 and TA2638 for the detection of mutagens. The uniform colony size and lower spontaneous reversion frequency seen with strain TA2638a compared with TA102 would make it more reliable and convenient for routine testing. It is concluded that strain TA2638a should be considered as an alternative to TA102 and included, as well as the two E.coli strains, in the set of bacterial strains used in the standard test battery for mutagenicity testing.     

Comparative mutagenic effects of structurally similar flavonoids quercetin and taxifolin on tester strains Salmonella typhimurium TA102 and Escherichia coli WP‐2 uvrA

Quercetin (QT) and Taxifolin (TF) are structurally similar plant polyphenols. Both have been reported to have therapeutic potential as anti‐cancer drugs and antioxidants. Mutagenic effects of QT and TF were evaluated using Salmonella typhimurium TA102 and Escherichia coli WP‐2 uvrA tester strains. Either in the presence or absence of S9 mix, QT was mutagenic to TA102 and WP2 uvrA. However, the mutagenicity of QT was significantly enhanced in the presence of S9 mix. Likewise, in the presence of Iron (Fe2+) and NADPH generating system (NGS) and absence of S9 mix, QT induced significantly high mutations in both TA102 and WP‐2 uvrA. Mutagenicity of QT decreased in both strains in the presence of Iron (Fe2+) or NGS alone. TF was not mutagenic in the presence or absence of S9 mix in both TA102 and WP‐2 uvrA 2, regardless of the presence of iron or NGS. Incorporation of antioxidants (ascorbate, superoxide dismutase (SOD), catalase (CAT)) and/or iron chelators (desferroxamine (DF) and ethylenediamine‐tetraacetate (EDTA)) in the test systems markedly decreased QT‐induced mutations in both tester strains. These results suggest that QT but not TF, could induce mutations in the presence or absence of rat liver S9 or Iron (Fe2+) and NGS in both tester strains by redox cycling and Fenton reactions to produce oxygen free radicals. Our results indicate that a minor structural variation between the two plant polyphenols could elicit a marked difference in their genotoxicities. These results provide a basis for further study into the potential use of QT in combination with iron supplements. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Mutagenic screening of marker grenade dyes by the Salmonella reversion assay, L5178Y/TK+/- mouse lymphoma assay, and in vivo sister chromatid exchange analysis in mice

Two dyes (C.I. Solvent Yellow No. 33 and a mixture of C.I. Solvent Yellow No. 33 and C.I. Solvent Green No. 3) were tested for mutagenicity in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay, and also for sister chromatid exchange (SCE) induction in vivo in C57B1/6J mice. In addition, a greater than 99.9% pure sample of the yellow dye [2-(2'-quinolyl)-1,3-indandione] was tested with and without exogenous activation in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay. Neither C.I. Solvent Yellow No. 33 nor the C.I. Solvent Yellow No. 33 and Solvent Green No. 3 mixture was positive for inducing SCEs in vivo. All three dyes were tested in the standard plate incorporation test in seven Salmonella strains TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538. The dyes were negative with and without exogenous activation in TA98, TA1535, and TA1538. One test with TA1537 was positive with the greater than 99.9% purified yellow dye. All three dyes gave weakly positive results (less than a twofold increase) with S-9 in TA100 and were clearly positive in TA102 and TA104 both with and without S-9. They also induced mutation at the thymidine kinase locus in mouse lymphoma cells, produced both large- and small-colony trifluorothymidine-resistant mutants, and were clastogenic. The purified yellow dye was further tested for SCE induction in mouse lymphoma cells and was determined to give a slightly positive response in the presence of S-9.     

Comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine

Comparative mutagenic and genotoxic effects of three antimalarialdrugs, chloroquine, primaquine and amodiaquine, were assessedin the Ames mutagenicity assay (in strains TA97a, TA100, TA102and TA104) and in vivo sister chromatid exchange (SCE) and chromosomeaberration (CA) assays in bone marrow cells of mice. These arethe most commonly used antimalarial drugs available at presentthroughout the world. The results of the bacterial mutagenicityassays showed a very weak mutagenic effect of all three drugsin Salmonella strains TA97a and TA100 both with and withoutS9 mix and in TA104 only with S9 mix. The results of the invivo SCE and CA assays indicate that these three drugs are genotoxicin bone marrow cells of mice. ³To whom correspodence should be addressed. Tel: +91 33 473 3491; Fax: +91 33 473 5197; Email: iichbio{at}giascl01.vsnl.net.in     

Evaluation of the potential genotoxicity of the phosphate binder lanthanum carbonate

Lanthanum was evaluated for potential genotoxicity using a range of in vitro assays (as the carbonate) in the presence and absence of post-mitochondrial fraction (S9) and in vivo in three independent tests for mutagenicity and clastogenicity (as the carbonate and chloride). The drug was devoid of mutagenic activity in bacterial assays (maximum concentration 5000 microg/plate) using a range of test strains (Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102 and Escherichia coli WP2 uvrA and WP2 uvrA pkm101). No effects were seen in the hgprt gene mutation assay in Chinese hamster ovary cells in the presence of S9. In the absence of S9, sporadic increases in revertant numbers were not dose-related or reproducible in subsequent experiments and hence were concluded to be chance events. In an in vitro chromosome aberration assay using Chinese hamster ovary cells, chromosome damage in the presence and absence of S9 (concentration 200-5000 microg/ml) was attributed to overt cell toxicity. To confirm this, a comprehensive in vivo evaluation of the drug was performed. Negative results were obtained in two independent rodent micronucleus tests. In the first mice were given oral doses (of carbonate) up to 2000 mg/kg, in the second rats were given a single i.v. bolus injection (of chloride) up to 0.1 mg/kg. Negative results were also obtained in a rat liver unscheduled DNA synthesis assay after treatment for 28 days with i.v. bolus injections (of chloride) up to 0.1 mg/kg/day. In these in vivo studies lanthanum plasma concentrations were >3000 times higher than the steady-state peak plasma concentration observed in dialysis patients given therapeutic doses of lanthanum carbonate. It can be concluded that lanthanum is not genotoxic and that lanthanum carbonate is unlikely to present a latent hazard in therapeutic use.