Results of a comparative study on the Salmonella pre-incubation and plate incorporation assays using test samples from the IPCS collaborative study.

Three complex mixtures (air particles, diesel particles and a coal tar fraction) and two pure compounds (benzo[a]pyrene and 1-nitropyrene) were tested in both the pre-incubation and the plate incorporation assay employing Salmonella typhimurium TA98 and TA100. Each experiment was conducted independently 2 or 4 times in duplicate in the presence and absence of metabolic activation. The mutagenic activities were calculated by least squares linear regression from the slope of the linear portion of each dose-response curve. Although slightly higher mutagenic activity was observed in the pre-incubation assay for the two pure compounds and with the plate incorporation assay for the diesel particulate sample, the overall data from both assays gave similar values and good correlations in TA100 and TA98. The results indicate that the pre-incubation assay could be used for these samples instead of the plate incorporation assay.     

Mutagenicity of potentially carcinogenic mycotoxins produced byFusarium moniliforme

The mutagenic behaviour of two potentially carcinogenic mycotoxins produced byFusarium moniliforme was investigated in theSalmonella mutagenicity test using tester strains TA97a, TA98, TA100, and TA102. The mutagenic response obtained with fusarin C (1, 5, and 10μg/plate) against tester strains TA98 and TA100 in the presence of microsomal activation confirmed previous observations on the mutagenic behaviour of this mutagen while that obtained against TA97a is reported for the first time. No dose-response relationship could be detected for the concentration levels (0.2, 0.5, 1, 5, 10 mg/plate) tested for FB₁, FB₂, and FB₃ against any of the tester strains used in either the plate incorporation and / or the pre-incubation tests. A cytotoxic effect was obtained at concentration levels of 5 and 10mg/plate in the absence of the microsomal activation mixture. From the studies it became evident thatF moniliforme produces two compounds, a mutagenic compound, fusarin C which has been shown to lack carcinogenic activity in rats and the non-mutagenic fumonisin B mycotoxins of which FB₁ is known to be responsible for the hepatocarcinogenicity of the fungus in rats.     

Results of the IPCS collaborative study on complex mixtures

The International Programme on Chemical Safety (IPCS) sponsored a collaborative study to examine the intra- and inter-laboratory variation associated with the preparation and bioassay of complex chemical mixtures. The mixtures selected were National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs). 20 laboratories worldwide participated in the collaborative trial. The participating laboratories extracted the organic portion of two particulate samples — an air-particulate sample and a diesel-particulate sample - and bioassayed the extracts. The laboratories simultaneously bioassayed a NIST-prepared extract of coal tar and two control compounds (benzo[a]pyrene, and 1-nitropyrene). The bioassay method used was the Salmonella/mammalian microsome plate-incorporation test using strains TA98 and TA100. Study design also allowed for a comparison of sonication and Soxhlet extraction techniques. The mean extractable masses for the air particles and diesel particles were approximately 5% and 1.75%, respectively. The particulate samples were mutagenic in both strains with and without activation in all 20 laboratories. For TA100 the with and without activation slope values for the air particulate were 162 and 137 revertants per mg particles, respectively. For TA98 the respective diesel slope values were 268 and 269. The mutagenicity slope values for the diesel particles ranged from 3090 (TA98, + S9) to 6697 (TA100, + S9) revertants per mg particles. The coal tar solution was negative for both strains when exogenous activation was not used but was mutagenic in both strains with exogenous activation. The benzo[a]pyrene and 1-nitropyrene were used as positive controls and gave results consistent with the literature. This paper provides a complete summary of the data collected during the collaborative study. Companion papers provide further analysis and interpretation of the results.     

Characterization of organic extracts from standard reference materials 1649, 'urban dust/organics,' and 1650, 'diesel particulate matter', using a microsuspension assay. A WHO/IPCS/CSCM study.

Mutagenicity associated with replicate organic extracts from standard reference materials 1649 'urban dust/organics' (air particles), and 1650, 'diesel particulate matter' (diesel particles), was determined using a Salmonella microsuspension assay. The results indicate that the mutagenicity of samples such as these can readily be determined using the microsuspension assay with only 5% of the mass required for the standard plate incorporation assay. In general, 80% of the variation in mutagenic activity was due to the bioassay procedure and 20% to the extraction process. Extracts from both samples had primarily direct-acting mutagenicity as there were no significant differences in responses with and without metabolic activation (S9). The TA98-S9 mean air particles mutagenic activities (C.V., %) based on mass of extractable organics or particles were 4.4 (4.7%) and 0.29 (3.6%) revertants/micrograms, respectively, and for the diesel particles were 66 (44%) and 12 (29%) revertants/microgram, respectively. More of the observed direct-acting mutagenicity in the diesel particles extracts was due to nitro-substituted compounds because there were significant reductions in activity with TA98NR (45% of TA98 -S9) and TA98-1,8-DNP6 (21% of TA98 -S9). In the air particles extracts, the TA98NR activities were not significantly different from TA98 -S9 but the TA98-1,8-DNP6 levels were.     

Salmonella mutagenicity of three complex mixtures assayed with the microsuspension technique. A WHO/IPCS/CSCM study.

In a collaborative study on complex mixtures, three complex mixtures and two pure compounds were assayed with the Salmonella microsuspension technique. The two pure compounds were benzo[a]pyrene (BaP) and 1-nitropyrene (1-NP). The three complex mixtures were standard reference materials (SRMs) from the U.S. National Institute of Standards and Technology, SRM 1649, SRM 1650 and SRM 1597. The two samples SRM 1649, an urban dust particulate matter, and SRM 1650, a diesel particulate matter, were sonicated with dichloromethane. Sample SRM 1597 was an extract of a coal tar sample with a complex mixture of polycyclic aromatic hydrocarbons. The microsuspension assay was performed with Salmonella strains TA98 and TA100 according to Kado et al. (1983) with minor modifications (L?froth et al., 1988). The results showed that the microsuspension technique is a more sensitive assay than the plate incorporation method. Depending on sample, strain and metabolic condition the mutagenic responses were 3-37 times higher in the microsuspension assay than in the conventional plate incorporation assay. The microsuspension method is thus useful for environmental samples which are often available in only small amounts.     

Characterization of organic extracts from standard reference materials 1649, ‘urban dust/organics,’ and 1650, ‘diesel particulate matter’, using a microsuspension assay. A WHO/IPCS/CSCM study

Mutagenicity associated with replicate organic extracts from standard reference materials 1649 ‘urban dust/organics’ (air particles), and 1650, ‘diesel particulate matter’ (diesel particles), was determined using a Salmonella microsuspension assay. The results indicate that the mutagenicity of samples such as these can readily be determined using the microsuspension assay with only 5% of the mass required for the standard plate incorporation asssay.In general, 80% of the variation in mutagenic activity was due to the bioassay procedure and 20% to the extraction process. Extracts from both samples had primarily direct-acting mutagenicity as there were no significant differences in responses with and without metabolic activation (S9). The TA98 - S9 mean air particles mutagenic activities (C.V., %) based on mass of extractable organics or particles were 4.4 (4.7%) and 0.29 (3.6%) revertants/μg, respectively, and for the diesel particles were 66 (44%) and 12 (29%) revertants/μg, respectively. More of the observed direct-acting mutagenicity in the diesel particles extracts was due to nitro-substituted compounds because there were significant reductions in activity with TA98NR (45% of TA98 -S9) and TA98-1,8-DNP6 (21% of TA98 -S9). In the air particles extracts, the TA98NR activities were not significantly different from TA98 - S9 but the TA98-1,8-DNP6 levels were.     

Mutagenic activities of ten imidazole derivatives in <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

Ten imidazole derivatives were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 both in the absence and presence of metabolic activation by the microsomal fraction S9 mix. In a general manner, derivatives tested exhibited a greater mutagenic activity in the TA100 strain comparing to the responses in TA 98. In the standard plate incorporation assay, 8 of these substances (80%) were found to be mutagenic for at least one of the two strains in the presence or absence of metabolic activation. Two compounds showed positive results in TA98 and 6 compounds were also mutagenic in TA100 without S9. In the presence of S9 mix, all of the 10 substances were non-mutagenic in TA98, whereas 4 compounds were positive in TA100. The results suggested the mutagenic potentials of the imidazole derivatives particularly inducing the reversion of base-pair substitutions. According to the structure-activity relationships phenyl groups in position 2 with different substituents can confer the mutagenic activity of the tested compounds. Methyl groups in different positions of these phenyl substituents can cause different types of mutations. This mutagenic effect is observed more clearly when the phenyl group is inhibited with a nitro group.     

Microbial mutagenicity of 3- and 4-ring polycyclic aromatic sulfur heterocycles

The stable isomers of 3- and 4-ring polycyclic aromatic sulfur heterocycles were tested for mutagenicity in the Ames standard plate incorporation test and a liquid pre-incubation modification of the Ames test. Of the 4 three-ring compounds tested, only naphtho[1,2-b]thiophene was mutagenic. Of the four-ring compounds, 7 of 13 were mutagenic in the standard Ames or pre-incubation Ames test. The highest activity for the 4-ring compounds was observed for phenanthrol[3,4-b]thiophene, a compound of approximately the same mutagenic potency in the Ames test as benzo[a]pyrene. The other active 4-ring compounds were of considerable less mutagenic potency than phenanthrol[3,4-b]thiophene. Mutagenicity for two of the 4-ring aromatic thiophenes could only be detected in the liquid pre-incubation Ames test. Salmonella typhimurium TA100 was the most sensitive strain to mutagenesis by these compounds, followed by TA98. All mutagenesis was indirect, requiring metabolic activation.     

Mutagenicity assessment of Salacia chinensis by bacterial reverse mutation assay using histidine dependent Salmonella typhimurium tester strains

Background and objectiveGenotoxicity analysis is one of the most important non-clinical environmental safety investigations required for pharmaceutical and agrochemical product registration. Any medicinal product must undergo a risk evaluation to determine its mutagenicity and carcinogenicity.Materials and methodsThe Ames test is a commonly used in vitro test for determining a test chemical's mutagenic activity. Histidine-dependent Salmonella typhimurium strains with a defective gene that causes the bacteria to synthesis the necessary amino acid histidine for life were tested for mutagenic potential. In order to reveal pro-mutagens and mutagens, the mutagenic potential of both plate integration and pre-incubation techniques was examined in the presence and absence of metabolizing system. Salacia chinensis has been widely used in ayurveda to treat various ailments. However, the information of mutagenicity of Salacia chinensis is scarce as per available literature.ResultsThe mutagenicity of a Salacia chinensis root extract was investigated utilizing the Ames assay with plate incorporation and pre-incubation protocols using the appropriate Salmonella typhimurium tester strains: TA98, TA100, TA1537, TA1535, and TA102 in the presence and absence of S9. The concentrations used were 0.3123, 0.625, 1.25, 2.5 and 5 mg/plate. The extract of Salacia chinensis root did not show any mutagenic effect in any of the Salmonella typhimurium strains at the concentrations tested in the absence or presence of metabolic activation.ConclusionThe root of Salacia chinensis was hence confirmed to be non-mutagenic and at least according to the results of this genotoxicity evaluation can be regarded as being safe for human use.     

Results of the IPCS collaborative study on complex mixtures.

The International Programme on Chemical Safety (IPCS) sponsored a collaborative study to examine the intra- and inter-laboratory variation associated with the preparation and bioassay of complex chemical mixtures. The mixtures selected were National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs). 20 laboratories worldwide participated in the collaborative trial. The participating laboratories extracted the organic portion of two particulate samples--an air-particulate sample and a diesel-particulate sample--and bioassayed the extracts. The laboratories simultaneously bioassayed a NIST-prepared extract of coal tar and two control compounds (benzo[a]pyrene, and 1-nitropyrene). The bioassay method used was the Salmonella/mammalian microsome plate-incorporation test using strains TA98 and TA100. Study design also allowed for a comparison of sonication and Soxhlet extraction techniques. The mean extractable masses for the air particles and diesel particles were approximately 5% and 17.5%, respectively. The particulate samples were mutagenic in both strains with and without activation in all 20 laboratories. For TA100 the with and without activation slope values for the air particulate were 162 and 137 revertants per mg particles, respectively. For TA98 the respective diesel slope values were 268 and 269. The mutagenicity slope values for the diesel particles ranged from 3090 (TA98, +S9) to 6697 (TA100, +S9) revertants per mg particles. The coal tar solution was negative for both strains when exogenous activation was not used but was mutagenic in both strains with exogenous activation. The benzo[a]pyrene and 1-nitropyrene were used as positive controls and gave results consistent with the literature. This paper provides a complete summary of the data collected during the collaborative study. Companion papers provide further analysis and interpretation of the results.     

Absence of mutagenic activity of benorylate,paracetamol and aspirin in the Salmonella//mammalian microsome test

Benorylate and its two major hydroyssis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Mutagenicity of aryl propylene and butylene oxides with salmonella

10 aryl propylene oxides and 6 aryl butylene oxides were synthesized. Dose-mutagenicity relationships were studied for these compounds and for 1,2-epoxybutane, using both the preincubation and plate incorporation Ames tests with Salmonella typhimurium strains TA100 and TA1535. Structure-mutagenicity relationships were further examined by concurrent testing at single doses with the plate incorporation assay in strain TA100. In both series of compounds, mutagenicity showed very correlation to chemical reactivity, molar volume and partition values. However, all compounds were mutagenic in at least one system with the propylene oxides being more mutagenic than the corresponding butylene oxide derivatives. The naphthyl derivatives in each series were the most mutagenic.     

Detection of mutagenic activity in automobile exhaust

Using the Ames Salmonella-microsome system, we detected mutagenic activity in the exhaust from two kinds of 4-cycle gasoline engines of unregulated and regulated cars, and from diesel engines, as well as in the particulates from air collected in tunnels. The mutagenicity of particulates from a car equipped with a catalyst (regulated car), as compared with that from an unregulated car, was reduced very much (down to 500 from 4500 revertants/plate/m3 in tester strain TA98). However, the mutagenicity of the ether-soluble acid and neutral fractions from the condensed water of emissions from a regulated car was still high (down to 2880 from 10 900 revertants/plate/m3 in tester strain TA100). The mutagenic activity of emission exhaust from old diesel car engines was very high; the particulates showed 9140 and 19 600 revertants/plate/m3 from strain TA98 incubated with an activating rat-liver S9 fraction. A small diesel engine of the type used for the generation of electric power or in farm machinery also produced exhaust with highly mutagenic particulates. The mutagenic activity of a methanol extract of particulate air pollutants collected in a highway tunnel showed 39 revertants/plate/m3 toward strain TA98 and 87 toward strain TA100. The ether-soluble neutral fraction yielded 86 revertants/plate/m3 from strain TA98 and 100 from strain TA100. This fraction also contained carcinogenic compounds, including benzo[a]pyrene, benzo[e]pyrene, benz[a]anthracene, benzo[ghi]perylene and chrysene. Very high mutagenic activity was detected, especially in the particulate air pollutants collected at night, in another tunnel on a superhighway: 60-88 revertants/plate/m3 from strain TA100 for the sample collected by day, but 121-238, by night. Night traffic includes many more diesel-powered vehicles compared with gasoline-powered automobiles.     

Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.     

Mutagenic action of methyl 2-cyanoacrylate vapor

Alkyl 2-cyanoacrylate adhesives were tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1538. Both a normal spot test and a spot test specially designed to test volatile compounds were used. The adhesives were also tested in the plate incorporation assay. These investigations showed that methyl 2-cyanoacrylate adhesives are mutagenic in strain TA100. The spot test for volatile compounds showed that it is the vapors from the methyl 2-cyanoacrylate monomer that are responsible for the mutagenic effect. One can conclude that working with methyl 2-cyanoacrylate adhesives entails exposure to vapors with a mutagenic effect and may therefore pose a carcinogenic hazard. Because the adhesives are used in industry, their mutagenic effect has a special importance in work environment.     

Detection of oxidative mutagens in an urban air-particulate extract: a preliminary study.

The Ames assays strains TA98 and TA100 have been useful in characterizing complex mixtures from organic solvent extracts of particles from diesel-powered vehicles, ambient air, and other sources. In this paper we report preliminary experiments using TA102, a bacterial strain that detects compounds that can oxidize DNA, to characterize the mutagenicity of an ambient air sample collected in Ann Arbor, MI. Four sets of ambient air filters were collected in duplicate over a period of several days. The mutagenicities of methylene chloride extracts of these filters were compared using strains TA98, TA100 and TA102. The concentration-mutagenicity data for TA98 and TA100 were linear over the concentration range 0-200 micrograms extract/plate. The mutagenicity of the extracts using TA102 was much lower than the other two strains and was non-linear over the concentration range tested. These results suggest that it would be difficult to use TA102 to identify the oxidative mutagens present in an ambient air particulate extract.     

Co-mutagenicity of glyco- and tauro-deoxycholic acids in the Ames test

Mutagenicity and co-mutagenicity of glyco- and tauro-deoxycholic acids (GDCA and TDCA), which are abundant in human bile, were examined by the Ames test. The two chemicals were not mutagenic for themselves to Salmonella typhimurium TA98 and TA100, with and without S9 mix. They enhanced, however, the mutagenic activities of the pro-mutagens, 2-aminoanthracene (2AA) and benzo[a]pyrene (BaP), for both TA98 and TA100 with S9 mix. They were more co-mutagenic for the pro-mutagens on TA98 than on TA100. On TA98, the mutagenic activities of 2AA with GDCA (5 μmol/plate) and with TDCA (5 μmol/plate) were 9.7-fold and 11.8-fold as high as that of the corresponding control (2AA only), respectively. BaP with GDCA (2.5 μmol/plate) and with TDCA (2.5 μmol/plate) showed 2.8-fold and 3.0-fold increases over the corresponding control level (BaP only), respectively. It is hence concluded that GDCA and TDCA may enhance the activity of some mutagens existing in bile.     

Chemical purity and mutagenicity: Case study of a drug in development

During a routine Ames assay of a potential antipsychotic drug candidate, the compound appeared to be a frameshift mutagen in Salmonella typhimurium strains TA98 and TA1538. Additional testing indicated the mutagenic activity was due to one or more contaminants incurred during synthesis. While the compound was initially shown to be > 98% pure by high-performance liquid chromatography, the presence of small amounts (0.01 – 0.1%) of a highly mutagenic impurity produced positive mutagenicity results. The need to assess for chemical purity before discontinuing development of drug candidates found positive in the Ames assay is discussed.     

Evaluation of the mutagenic potential of endosulfan using the Salmonella/mammalian microsome assay

The mutagenic potential of endosulfan, a polychlorinated insecticide, was assessed using the highly sensitive Salmonella tester strains TA97(a), TA98, TA100 and TA102. It exhibited a toxic effect at dose levels of 50 micrograms/plate and higher. Plate incorporation studies did not show mutagenic response with any of the tester strains used. A modification of the assay using a preincubation procedure showed mutagenic activity with and without metabolic activation with TA97(a) only. Increased toxicity was observed after addition of phenobarbital-induced S9 mix.     

Bacterial mutagenicity of two cyclopentafused isomers of benzpyrene

Two novel cyclopentafused polycyclic aromatic hydrocarbons, naphtho(1,2,3-mno)acephenanthrylene (cyclopenta benzo[e]pyrene) and naphtho(2,1,8-hij)acephenanthrylene (cyclopenta(ij)benzo[a]pyrene) were evaluated for mutagenic activity in the Ames Salmonella typhimurium plate incorporation assay. Both compounds required S9 metabolic activation, and showed optimal activity at low S9 concentrations (below 0.6 mg/plate). Both compounds induced frameshift and base-pair substitution mutations, being active in strains TA98, TA100, TA1537, TA1538 and TA104, but not in strain TA1535. Cyclopenta(ij)benzo[a]pyrene was more active than cyclopentabenzo[e]pyrene, and both were more potent than their parent ring systems, benzo[a]pyrene and benzo[e]pyrene, respectively. Cyclopenta(ij)benzo[a]pyrene was more active in strain TA104 than in TA100 or TA98 (250-470, 340 and 80-100 rev/nmole) as was benzo[a]pyrene (120, 70 and 40 rev/nmole respectively); cyclopentabenzo[e]pyrene was more active in TA100 than TA104 or TA98 (70 versus 50 and 40 rev/nmole), and benzo[e]pyrene showed a similar pattern (4, 3.5 and 0.6 rev/nmole). The relative potencies of the four compounds are in accord with predictions based on perturbational molecular orbital calculations. The peak of activity at low S9 concentrations is consistent with epoxidation at the cyclopentafused ring being the major route of metabolic activation for both these cyclopentafused compounds.