Systemic Toxicity of the Heavy Fraction of a Coal Coprocessing Product in Male Rats Following Subchronic Dermal Exposure

The systemic toxicity of a coal coprocessing product [heavygas oil II (HGOII)] following subchronic, dermal exposure inmale Sprague-Dawley rats was investigated. HGOII was appliedto the dorsal skin daily at doses of 8.7, 20.8, 50.0, or 120.0mg/ kg body weight (bw) for 13 weeks. Another group of ratstreated with a medium boiling coal liquefaction product (CLP)served as positive controls. Growth suppression and decreasedfood consumption were noted in the groups exposed to HGOII at20.8 mg/kg and higher, and to CLP starting at the third weekof treatment. Relative liver, kidney, and brain weights in the20.8 mg/kg HGOII group and up were higher than those of thecontrol. Increased spleen weight was observed in all HGOII-treatedgroups. CLP treatment also caused increased relative kidneyand brain weights. Serum cholesterol was elevated in the HGOII-treatedgroups starting at 8.7 mg/kg while increased uric acid and lactatedehydrogenase were observed at 20.8 mg/kg and up. Decreasederythrocyte, hemoglobin, and platelet counts were observed at20.8 mg/kg and higher. All HGOII-treated groups had elevatedreticulocytes. These biochemical and hematological changes werenot observed in the CLP-treated group. Mild to marked histologicalchanges were observed in the thyroid, thymus, liver, spleen,and bone marrow of HGOII groups. In contrast, morphologicalchanges were relatively mild in CLP-treated animals. Data fromthe present study demonstrated that the hematological endpointswere sensitive to the liquid fuels and that HGOII was more toxicthan CLP. Based on the growth rate and biochemical, hematological,and morphological changes, the no observable adverse effectlevel for HGOII is judged to be below 8.7 mg/kg bw in the rat.     

Mutagenicity evaluation of azaperone in the Salmonella/microsome test

Azaperone was evaluated for its mutagenic potential by the Salmonella/microsome test. No mutagenic activity towards six S. typhimurium strains could be evidenced with azaperone at doses up to 2,000 micrograms/plate, either without or with metabolic activation at usual test conditions. Higher concentrations of liver post-mitochondrial fraction from Aroclor 1254 (ARO)-pretreated rats did not reveal any increase in the number of revertants towards S. typhimurium strains TA1537, TA1538 and TA98. Moreover, a plate-incorporation test with liver post-mitochondrial fractions from mice pretreated with phenobarbital (PB) and a liquid preincubation test with liver post-mitochondrial fractions from rats pretreated with ARO also failed to reveal any mutagenic action of azaperone towards S. typhimurium strain TA98. Thus, none of the tests used provided any indication of azaperone having a mutagenic action.     

Mutagenicity Evaluation of Azaperone in the Salmonella/Microsome Test

ABSTRACT

Azaperone was evaluated for its mutagenic potential by the Salmonella/microsome test. No mutagenic activity towards six S. typhimurium strains could be evidenced with azaperone at doses up to 2,000 µg/plate, either without or with metabolic activation at usual test conditions. Higher concentrations of liver post-mitochondrial fraction from Aroclor 1254 (ARO)-pretreated rats did not reveal any increase in the number of revertants towards S. typhimurium strains TA1537, TA1538 and TA98. Moreover, a plate-incorporation test with liver post-mitochondrial fractions from mice pretreated with phenobarbital (PB) and a liquid preincubation test with liver post-mitochondrial fractions from rats pretreated with ARO also failed to reveal any mutagenic action of azaperone towards S. typhimurium strain TA98. Thus, none of the tests used provided any indication of azaperone having a mutagenic action.     

Mutagenicity testing of selected analgesics in Ames Salmonella strains

Acetaminophen (APAP), aspirin (ASA), phenacetin (PA) and ibuprofen (IB) were tested for mutagenic activity in the Ames Salmonella plate incorporation assay using strains TA98, TA100, TA1535, TA1537 and TA1538. These analgesics were tested in four separate tests: without metabolic activation, and in the presence of a rat, hamster or mouse liver post-mitochondrial supernatant (S-9, Aroclor 1254-induced). Treatment of all five strains of Salmonella with APAP, ASA or IB under all four metabolic conditions did not induce any appreciable increases in revertant colony counts, as compared to the negative controls. A dose-related increase in revertant colony counts, reaching levels twice the negative control values, were seen with PA at doses greater than or equal to 500 micrograms per plate. This response was only seen in strain TA100 in the presence of hamster S-9. Therefore, these findings constitute a positive result for PA in the Ames test. APAP, ASA and IB did not show any mutagenic potential under these conditions of testing. These findings are discussed along with previously published results concerning the genotoxicity of these analgesics.     

Enzyme-mediated mutagenicity in Salmonella typhimurium of contaminants of synthetic indigo products

The mutagenic potential of two natural and seven synthetic, commercial indigo dye products was investigated. The natural products showed no mutagenicity in Salmonella typhimurium stains TA98 and TA100. In the presence of rat-liver homogenate from Aroclor 1254 pretreated rats all of the synthetic products were mutagenic towards strain TA98 but not towards strain TA100. The mutagenic effect produced was highly dependent on the amount of rat-liver homogenate added. Because of its high mutagenic potential, one product was further investigated. In the presence of rat-liver homogenate this product was weakly mutagenic towards strain TA1537 and strongly mutagenic towards strain TA1538. No mutagenicity was observed in strain TA1535. Experiments with purified synthetic indigo and natural indigo revealed that the mutagenic activity of the synthetic commercial products can be ascribed to one or more contaminants.     

Mutagenicity evaluation of phthalic acid esters and metabolites in Salmonella typhimurium cultures

The mutagenic potential of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEPH), as well as metabolites of DEHP--i.e., mono-2-ethylhexyl phthalate (MEHP), 2-ethylhexanol (2-EH), and phthalic acid (PA)--were tested in Salmonella typhimurium cultures using the Ames test procedure. The compounds were tested on strains TA98, TA100, TA1535, TA1537, TA1538, and TA2637 for base-pair substitution or frameshift-type mutations. Spot tests yielded negative responses for all compounds with the strains tested. Each compound was tested for a dose-effect relationship in the TA98, TA100, TA1535, and TA1538 systems. DEP and DBP exhibited a mildly positive response in both TA100 and TA1535 cultures, and DMP showed a similar response in TA1535. Normalization of the data for cytotoxicity of DMP suggests TA100 has a mildly positive effect. The higher doses of these compounds exhibited some cytotoxic effects. The mutagenic effects were apparently abolished by the addition of S9 fraction in TA100 and TA1535 cultures, while no effect, other than cytotoxicity, was observed in the TA98 and TA1538 systems. DEHP, MEHP, 2-EH, and PA exhibited no mutagenicity in any of the strains of Salmonella typhimurium tested, with or without S9 metabolic activation. MEHP and 2-EH, however, exhibited a moderate cytotoxic effect in most cultures.     

Strong mutagenic effects of diesel engine emissions using vegetable oil as fuel

Diesel engine emissions (DEE) are classified as probably carcinogenic to humans. In recent years every effort was made to reduce DEE and their content of carcinogenic and mutagenic polycyclic aromatic compounds. Since 1995 we observed an appreciable reduction of mutagenicity of DEE driven by reformulated or newly designed fuels in several studies. Recently, the use of rapeseed oil as fuel for diesel engines is rapidly growing among German transportation businesses and agriculture due to economic reasons. We compared the mutagenic effects of DEE from two different batches of rapeseed oil (RSO) with rapeseed methyl ester (RME, biodiesel), natural gas derived synthetic fuel (gas-to-liquid, GTL), and a reference diesel fuel (DF). The test engine was a heavy-duty truck diesel running the European Stationary Cycle. Particulate matter from the exhaust was sampled onto PTFE-coated glass fibre filters and extracted with dichloromethane in a soxhlet apparatus. The gas phase constituents were sampled as condensates. The mutagenicity of the particle extracts and the condensates was tested using the Salmonella typhimurium/mammalian microsome assay with tester strains TA98 and TA100. Compared to DF the two RSO qualities significantly increased the mutagenic effects of the particle extracts by factors of 9.7 up to 59 in tester strain TA98 and of 5.4 up to 22.3 in tester strain TA100, respectively. The condensates of the RSO fuels caused an up to factor 13.5 stronger mutagenicity than the reference fuel. RME extracts had a moderate but significant higher mutagenic response in assays of TA98 with metabolic activation and TA100 without metabolic activation. GTL samples did not differ significantly from DF. In conclusion, the strong increase of mutagenicity using RSO as diesel fuel compared to the reference DF and other fuels causes deep concern on future usage of this biologic resource as a replacement of established diesel fuels.     

Ames mutagenicity tests on purified 3-nitropropionic acid

3- Nitropropionic acid is a toxic compound produced by several moulds involved in food fermentation or spoilage. An impure commercial sample of this compound was previously reported as being mutagenic to Salmonella typhimurium strains TA1535 and TA100. In the present study, a sample from the same lot of 3- nitropropionic acid was mutagenic in strain TA100 without metabolic activation, but this activity was diminished after recrystallization. This sample was not mutagenic in strain TA98, before or after recrystallization. A new, purer commercial sample was non-mutagenic in strains TA98, TA100 and TA1538, with or without metabolic activation. Therefore the mutagenicity reported to be due to 3- nitropropionic acid was considered to be due to the impurity(ies).     

Mutagen formation in the reaction of maillard browning products, 2-acetylpyrrole and its analogues, with nitrite

Three 2-substituted pyrroles (2-acetylpyrrole, pyrrole-2-carboxaldehyde and pyrrole-2-carboxylic acid), which are products of the Maillard browning reaction, were reacted with nitrite in buffer solution (pH 3) at 50°C for 24 hr. The reaction mixtures were extracted with methylene chloride and the extracts were tested for mutagenicity using Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104, with and without S-9 metabolic activation. The methylene chloride extract of the 2-acetylpyrrole-nitrite reaction mixture showed strong mutagenicity to all the tester strains, both in the presence and absence of S-9 mix. The reaction product of pyrrole-2-carboxaldehyde with nitrite only gave a weak mutagenic response with strain TA100, while the pyrrole-2-carboxylic acid-nitrite reaction product did not produce a mutagenic response in any of the tester strains. Two mutagenically active fractions, separated by thin-layer chromatography, were found in the reaction of 2-acetylpyrrole with nitrite. The formation of mutagenic products in the latter reaction was found to vary with reaction pH, time and temperature, with nitrite level and with 2-acetylpyrrole concentration.     

An Ab initio study of the relationship between nitroarene mutagenicity and electron affinity

Electron affinities, approximated by lowest unoccupied molecular orbital (LUMO) energies, were determined for an extensive group of nitrated polycyclic aromatic hydrocarbons by ab initio methods at the STO-3G level. Significant correlations were demonstrated between nitroarene LUMO energy and the corresponding mutagenic activity in Salmonella typhimurium strains TA98, TA100, TA1537, and TA1538. An analogous correlation using Hückel calculations was substantially poorer. A correlation between nitro group rotation and LUMO energy was related to pi-conjugation about the C--N bond. Analysis of aryl substituent effects on nitrenium ion stability implicated additional nitro substitution in certain systems to be destabilizing. The results suggest a means for predicting nitroarene mutagenic activity and for assessing the role of metabolic intermediates.     

Mutagenicity evaluation of KB-944, a new calcium antagonist, in the Ames bacterial system

Evaluation of the mutagenic activity of diethyl 4-(benzothiazol-2-yl) benzylphosphonate (KB-944) was performed using bacteria. The method consisted of mutagens or KB-944 with and without metabolic activation, and two bacteria; Salmonella typhimurium 5 test strains, TA 1535, TA 100, TA 1537, TA 1538 and TA 98, and Escherichia coli WP 2 uvr A. Results indicated that KB-944 had no mutagenic activity against S. typhimurium and E. coli.     

The antimutagenic activity of Lavandula angustifolia (lavender) essential oil in the bacterial reverse mutation assay.

Essential oils from Melaleuca alternifolia (tea-tree oil) and Lavandula angustifolia (lavender oil) are commonly used to treat minor health problems. Tea-tree oil possesses broad-spectrum antimicrobial activity, and is increasingly used for skin problems. Lavender oil, traditionally used as an antiseptic agent, is now predominantly used as a relaxant, carminative, and sedative in aromatherapy. Despite their growing use no data are available on their mutagenic potential. In this study, after determining the chemical composition of tea-tree oil and lavender oil, by gas-chromatography and mass spectrometry, we investigated their mutagenic and antimutagenic activities by the bacterial reverse mutation assay in Salmonella typhimurium TA98 and TA100 strains and in Escherichia coli WP2 uvrA strain, with and without an extrinsic metabolic activation system. Neither essential oil had mutagenic activity on the two tested Salmonella strains or on E. coli, with or without the metabolic activation system. Conversely, lavender oil exerted strong antimutagenic activity, reducing mutant colonies in the TA98 strain exposed to the direct mutagen 2-nitrofluorene. Antimutagenicity was concentration-dependent: the maximal concentration (0.80 mg/plate) reduced the number of histidine-independent revertant colonies by 66.4%. Lavender oil (0.80 mg/plate) also showed moderate antimutagenicity against the TA98 strain exposed to the direct mutagen 1-nitropyrene. Its antimutagenic property makes lavender oil a promising candidate for new applications in human healthcare.     

Mutagenicity assay in Salmonella for thirteen 2-substituted-1 H-phenanthro (9,10-d) imidazoles

Some 2-substituted-1 H-phenanthro [9,10-d] imidazole compounds synthesized as a predrugs were tested in mutagenicity assays in Salmonella strains TA97, TA98, and TA100 using a plate incorporation assay both with and without S9 mix. The 10 substances were mutagenic in TA97 and five of them were mutagenic only with metabolic activation, whereas one of them did not require the addition of S9. The eight substances were mutagenic in TA98 only with S9. For TA100, seven substances showed positive results both with and without S9, however another four required S9, whereas only one of them did not required metabolic activation. In summary, all of 13 substances derived from phenanthro [9,10-d] imidazole were found to be mutagenic for at least one or two of the three strains and their mutagenicity are discussed.     

Differential activation of promutagens by alloenzymes of human sulfotransferase 1A2 expressed in Salmonella typhimurium

Various enzymatically formed sulfuric acid esters are chemically reactive and mutagenic. This metabolic activation pathway is not detected in standard in-vitro mutagenicity test systems. We describe the construction of Salmonella typhimurium TA1538-derived strains expressing alloenzymes *1, *2, *3, *5, *6 of human sulfotransferase 1A2 (SULT1A2). The reference compounds, 1-hydroxymethylpyrene (1-HMP), N-hydroxy-2-acetylaminofluorene (OH-AAF) and 2-hydroxylamino-5-phenylpyridine (OH-APP), were activated to mutagens in these strains. Their activity differed 7- to 16-fold between strains expressing various alloenzymes. It was strongest and weakest in the strains expressing the common alloenzymes, *1 and *2, respectively. The SULT1A2 protein expression levels, and the V(max) and K(m) values with the reference substrate 4-nitrophenol, varied 2.5-, 4-, and 110-fold, respectively, in cytosolic preparations from strains TA1538-SULT1A2*1 and *2. Strains with varying protein levels were constructed via insertion of silent mutations in the 5'-part of the cDNA. TA1538-SULT1A2*1Z and TA1538-SULT1A2*2Y showed equal expression levels of alloenzymes *1 and *2, respectively, which were 3 times above those of TA1538-SULT1A2*1. The mutagenicity of OH-AAF and OH-APP was unchanged in strain TA1538-SULT1A2*1Z versus *1, and moderately increased in TA1538-SULT1A2*2Y versus *2. The influence of the protein level was stronger with 1-HMP. Nevertheless, mutagenic activity of 1-HMP was still 11 times higher in TA1538-SULT1A2*1Z than in TA1538-SULT1A2*2Y. Thus, differences in the properties between alloenzymes can lead to differences in the activation of promutagens. The model compounds were also tested in strains expressing the other ten human SULTs identified. Whereas OH-AAF and OH-APP showed the highest mutagenic activities in strains expressing SULT1A2, 1-HMP was more potent in strains expressing other SULT forms. With the limitation that little is known about the tissue distribution and regulation of SULT1A2, the findings suggest that its polymorphism may affect the individual susceptibility towards procarcinogens, in particular certain aromatic amines and amides.     

Mutagenicity studies on dihydroergocristine

Dihydroergocristine (DHEC) is an ergot derivative used for the therapy of patients with cerebrovascular insufficiency. It was tested for mutagenicity by means of four tests. In the mutagenicity in vitro assay on Salmonella typhimurium, DHEC was checked at 10,000 micrograms/plate on TA98 and TA1538 strains and at 3000 micrograms/plate on TA1535, TA1537 and TA100 strains with and without metabolic activation. In a quantitative in vitro test for mutagenicity in V79 Chinese hamster cells, DHEC was studied at concentrations between 30 and 0.3 microgram/ml with and without metabolic activation. DHEC was tested for its ability to induce chromosomal damage in human lymphocyte cultures utilizing the concentrations of 10, 3 and 1 microgram/ml. In the in vivo mouse (Swiss strain) micronucleus assay, DHEC was orally administered at two dosages (50% and 16% of LD50) following the schedule of the test. Dihydroergocristine is a drug free of mutagenic activity on the basis of all the results obtained from the above in vitro and in vivo tests.     

Mutagenic activity of some textile dyes in different test systems

The Salmonella/microsome mammalian test, the micronucleus and the dominant lethal tests on mice were used to study mutagenic effects of three dyes widely used in the textile industry. Direct Black 19:1, Direct Red 81 and Acid Blue 62 increased the frequency of micronucleated polychromatic erythrocytes in bone marrow of mice. Out of all dyes tested, only Direct Black 19:1 appeared to be an indirect mutagen inducing reverse mutations in two strains of Salmonella typhimurium TA 1538 and TA 98. None of them produced dominant lethal mutations in germ cells of male mice.     

Mutagenic activity in roadside soils

Mutagenicity of soils sampled at median strips, roadsides and a park neighboring arterial roads in Kurume City was determined by Ames test. Organic extracts of soils were mutagenic in strains TA98, TA100, YG1041 and YG1042 with and without S9mix. No sample showed mutagenic responses in strains YG3003 or YG7108. Extracts from soils of median strips and beside intersections showed higher mutagenicity and concentrations of PAHs and heavy metals than others, and mutagenic activity of soils correlated significantly with concentrations of PAHs and heavy metals. However, PAHs accounted for less than 12% of total mutagenicity in strains TA98 and TA100 of soil extracts. These extracts showed much higher mutagenicity in YG strains than in TA strains. The results indicate that these soils may be polluted with nitroarenes and aromatic amines.     

Methemoglobinogenic potential of primaquine and its mutagenicity in the Ames test

Single doses of primaquine did not produce methemoglobinemia in beagle bitches. Repeated daily administration for 12 days produced a gradually rising level of methemoglobin over that time period, unaccompanied by depletion of erythrocytic reduced glutathione. Primaquine was mutagenic in the Ames test in Salmonella typhimurium strain TA 1537, with or without S9, using a liquid preincubation assay. Primaquine was non-mutagenic in this assay to strains TA 1535, TA 1538, TA 98 and TA 100, regardless of the presence or absence of S9. In the standard overpour Ames test, the drug was non-mutagenic in all 5 Salmonella strains, both with and without S9 metabolic activation.     

A teratological assessment of coal liquefaction products in the rat

Teratogenicity of coal liquefaction products (CLP) was assessed in the pregnant rat. Three product streams of CLP (medium, hydrotreated medium and heavy fractions) were each administered dermally on Sprague-Dawley rats at doses of 125, 250 or 500 mg kg-1 day-1 from Day 6 through to Day 15 of gestation. Depressed maternal weight gain and reduced number of fetuses resulting from an increased resorption rate, decreased fetal weight and retarded ossification were observed in the group treated with the heavy fraction at a dose of 500 mg kg-1 day-1. The heavy fraction at 500 mg kg-1 day-1 also caused anaemia and increased liver and spleen weights in dams. The dams exposed to the highest dose of three CLP fractions had mild and adaptative hepatic changes consisting of increased cytoplasmic eosinophilia and nuclear anisokaryosis. No treatment-related histological changes were observed in fetuses. None of the fractions demonstrated any teratological effects.     

Bacterial metabolism of 2,6-dinitrotoluene with Salmonella typhimurium and mutagenicity of the metabolites of 2,6-dinitrotoluene and related compounds.

1. Metabolites produced by the incubation of 2,6-dinitrotoluene (2,6-DNT) with Salmonella typhimurium strains TA 98, TA 98/1,8-DNP6 and TA 98NR were examined. Mutagenicities of bacterial products and related compounds were also examined in the Ames assay using TA 98 and TA 100. 2. 2,6-DNT was converted to 2-nitroso-6-nitrotoluene, 2-hydroxylamino-6-nitrotoluene and 2-amino-6-nitrotoluene, with concurrent spontaneous formation of 2,2'-dimethyl-3,3'-dinitroazoxybenzene, in the incubation with TA 98 and TA 98/1,8-DNP6. Capacity of TA 98NR to reduce 2,6-DNT was much lower than that of TA 98 and TA 98/1,8-DNP6. 3. Bacterial products, including 2,2'-dimethyl-3,3'-dinitroazoxybenzene, showed no mutagenic activity in the Ames assay. 4. Results indicate that the lack of mutagenic activity of 2,6-DNT is not due to low reductive metabolism of 2,6-DNT by bacteria, but due to the lack of mutagenic activity of the bacterial reductive products of 2,6-DNT.