Induction of prophage lambda by chlorinated pesticides

Chlorinated organics represent an important class of environmental carcinogens. However, only a small percentage of the carcinogens of this chemical class are genotoxic in prokaryotic bioassays such as the Salmonella assay. In an effort to identify a short-term assay sensitive to chlorinated carcinogens, we have tested a group of chlorinated pesticides, most of which are carcinogenic in rodents, in a prophage-induction assay developed by Rossman et al. (1984). The Microscreen phage-induction assay is a rapid, inexpensive, miniaturized system that uses the induction of prophage lambda in Escherichia coli as an indicator of genetic damage. It has been used successfully to screen complex environmental samples for genotoxicants and has detected carcinogenic metals that are refractory in the Salmonella assay. The pesticides tested were malathion, monuron, p,p'-DDT, mirex, lindane, nitrofen, chlordane, toxaphene, captan, and dichlorvos. All but the first 4 induced prophage. The remaining pesticides were ranked as follows according to induction potency in the presence of S9: captan greater than dichlorvos greater than toxaphene greater than lindane greater than nitrofen greater than chlordane. Rankings were similar in the absence of S9. Of these 6 pesticides, only nitrofen required S9 to induce prophage. Comparisons with mutagenesis data in Salmonella indicated that the Microscreen assay detected as genotoxic each of the pesticides that were mutagenic in Salmonella; moreover, it detected 2 additional carcinogens (chlordane and lindane) that were not mutagenic in the Salmonella assay. The possible use of the Microscreen phage-induction assay to detect chlorinated organics is discussed.     

Evaluation of the GreenScreen GADD45alpha-GFP indicator assay with non-proprietary and proprietary compounds

The GreenScreen GADD45alpha indicator assay has been assessed for its concordance with in vitro genotoxicity and rodent carcinogenicity bioassay data. To test robustness, sensitivity, and specificity of the assay, 91 compounds with known genotoxicity results were screened in a blinded manner. Fifty seven of the compounds were classified as in vitro genotoxic whereas 34 were non-genotoxic. Out of the 91 compounds, 50 had been tested in 2-year carcinogenicity assays, with 33 identified to be rodent carcinogens and 17 non-carcinogens. Gadd45alpha assay sensitivity and specificity for genotoxicity was 30% and 97%, respectively (17/57 and 33/34), whereas its sensitivity and specificity for rodent carcinogenicity was 30% and 88%, respectively (10/33 and 15/17). Gadd45alpha assay genotoxicity results from this validation study exhibited a high concordance with previously published results as well as for compound test results generated at two different sites (91%, 19/21), indicating that the assay is both robust and reproducible. In conclusion, results from this blinded and independent validation study indicate that the GreenScreen GADD45 indicator assay is reproducible and reliable with low sensitivity and high specificity for identifying genotoxic and carcinogenic compounds.     

Bacterial mutagenicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenylacrylates

The mutagenic potential of three alkyl 2-cyanoacrylate adhesives, three commercial alkyl 2-cyanoacrylate adhesives and three methyl 2-cyano-3-phenylacrylates, was assessed using the Salmonella/microsome mutagenicity assay. Compounds were tested with and without Aroclor 1254-induced rat-liver homogenate (S9 mix). The methyl 2-cyanoacrylate adhesives were mutagenic in the standard plate test with S. typhimurium strain TA100 with and without S9 activation. Methyl 2-cyano-3-(2-bromophenyl)acrylate revealed a direct mutagenic action to S. typhimurium strain TA1535. The compounds most toxic towards the bacterium S. typhimurium, were the methyl 2-cyanoacrylate adhesives (greater than 500 micrograms/plate). All alkyl 2-cyanoacrylate adhesives were tested in a modified spot test for volatile compounds with tester strain TA100. Mutagenic and toxic effects were observed with the three methyl 2-cyanoacrylate adhesives. It can be concluded from the results that the bacterial toxicity and mutagenicity of methyl 2-cyanoacrylate adhesives may be due to the methyl 2-cyanoacrylate monomer.     

Study on the mutagenicity of nifurtimox and eight derivatives with the L-arabinose resistance test of Salmonella typhimurium

The mutagenicity of nifurtimox (nfx) and 8 nfx analogues has been investigated with the L-arabinose forward-mutation assay of Salmonella typhimurium. The nfx analogues tested were obtained by replacing the 3-methyl-4-yl-tetrahydro-1,4-thiazine-1,1-dioxide group of the parent compound with the following other groups: indazol-1-yl (1); pyrazol-1-yl (2); benzimidazol-1-yl (3); 1,2,4-triazol-4-yl (4); 1-methyl-3-methylthio-1,2,4-triazol-4-yl-5-thione (5); 3,5-bis(methylthio)-1,2,4-triazol-4-yl (6); 1-adamantyl (7); 4,6-diphenylpyridin-1-yl-2-one (8). The mutagenic activity of each chemical was determined by the standard plate-incorporation test, in the presence or absence of the S9 activation mixture. The 9 compounds were mutagenic and exhibited linear dose-mutagenic response relationships. They were direct-acting mutagens and showed a nearly 1000-fold range in mutagenic potency from chemical 1 to nfx. In most cases, the addition of S9 mixture to the test plates decreased the mutagenicity of compounds. This effect was particularly noticeable in the case of chemicals 1-3, 5 and 7 where a more than 70% decrease in mutagenic activity was observed in the presence of the S9 mixture. The mutagenic potency of compounds in the Ara test showed a negative linear correlation with previously reported antitrypanosomal activity. Thus, chemicals 6 and 8 with in vitro activities against Trypanosoma cruzi clearly superior to that of nfx showed 2 of the lowest mutagenic potencies in the Ara test and these were only somewhat higher than the mutagenicity of the reference drug.     

Evaluation of the Allium anaphase-telophase test in relation to genotoxicity screening of industrial wastewater

The Allium anaphase-telophase test was evaluated to find out if it could be recommended in the screening of wastewater for genotoxicity. Five mutagenic or carcinogenic chemicals usually found in wastewater were tested in the Allium anaphase-telophase test. Sodium dichromate (25 μM), benzene (100 μM), dichloromethane (175 μM) and 1,1,1-trichloromethane (175 μM) increased the frequency of chromosome aberrations in the root cells, whereas formaldehyde (1 mM) was found to be non-mutagenic in this test system. Other studies where chemicals were tested in the Allium test were reviewed. For 15 chemicals the results were compared with results from the Ames test, the Microscreen assay, and carcinogenicity tests in rodents. The sensitivity of the Allium test was calculated to be 82%. In conclusion the Allium test is recommended for the screening of wastewater because it has a high sensitivity, is cheap, rapid, easy to handle, and because it can be used on wastewater without pretreatment of the sample.     

Genotoxicity and embryotoxicity of urban air particulate matter collected during winter and summer period in two different districts of the Czech Republic

This study is the in vitro part of a long-term program to investigate the impact of air pollution on the health of a population in a polluted region of Northern Bohemia. In order to assess the possible health risks associated with a complex mixture of hundreds of organic compounds adsorbed to air particles, we used a biomarker-directed fractionation procedure to evaluate biological activities of different chemical compound classes. The extractable organic compounds from the air particles collected in both the polluted and the control districts during the summers and winters of 1993-1994 were investigated. The principal aim of this study was to compare the DNA binding activities of those compound classes using an in vitro acellular assay coupled with 32P-postlabeling and an embryotoxicity assay using Chick Embryotoxicity Screening Test (CHEST). In both assays, the highest activity was due to the neutral fractions from which the aromatic subfractions containing mainly polycyclic aromatic hydrocarbons (PAHs) and their methyl-derivates were the most active for both localities and seasons. A good correlation between the levels of DNA adduct formation using S9 metabolic activation and the ED50 for all different complex mixtures of organic compounds was observed (r=0.773, p<0.001). DNA adduct maps and high performance liquid chromatography (HPLC) profiles were similar for samples from both districts and seasons. The major DNA adducts resulting from the crude extracts were identical to those derived from aromatic fractions. The DNA adducts tentatively identified constituted about 50% of the total adducts formed by the crude extracts following S9-metabolic activation. Our results confirmed the similarities of the major ubiquitous emission sources of organic compounds in both districts. This is the first report in which the biological activities of complex mixtures in short-term assays with remarkably different endpoints such as DNA adduct formation and embryotoxicity have been compared.     

Mutagenicity tests on anthelmintics: microsomal activation of viprynium embonate to a mutagen.

Eight anthelmintic preparations readily available in Australia were tested for mutagenicity in the Salmonella typhimurium test system. A slightly modified version of the procedure recommended by Ames et al. [2] was adopted, in that the test samples were placed in "wells" cut out of the agar of a plate previously seeded with the appropriate tester strain. Addition of a mixture of rat liver microsomal enzymes and appropriate co-factors ("S-9 mix") to one of the two wells on a single plate allowed a possible requirement for metabolic activation to be recognised. Using this procedure, viprynium embonate was found to be non-mutagenic. It was however, activated by the rat liver microsome preparation to a mutagen capable of causing both base-pair substitution (detected with strain TA100) and frameshift (detected with strain TA98) mutations. The other seven compounds tested all gave negative results in this system.     

Mutagenicity of 43 structurally related heterocyclic compounds and its relationship to their carcinogenicity.

43 heteropolycyclic compounds belonging to a homologous series were investigated for mutagenicity. The results are compared with carcinogenicity data obtained with the same batches of compounds under conditions identical for all of them. Mutagenicity was tested in the Ames test with Salmonella typhimurium strains TA1535, TA1537 and TA100 in the presence and absence of liver 10 000 g supernatant from rats treated with Aroclor 1254. Carcinogenicity was tested by injection of the compounds into subcutaneous tissue of XVIInc/Z mice. 18 test compounds showed carcinogenic activity, some strongly, others only weakly. Of these, 17 were detected as mutagens: one weak carcinogen did not revert the Salmonella strains. No quantitative correlation was observed between the extents of the mutagenic and the carcinogenic effects. Of the 25 substances that did not produce tumours, 13 showed mutagenicity (12 in the presence, 2 in the absence, of the liver homogenate). The mutagenic effects of these compounds were quantitatively similar to those of the compounds that produced tumours. The most sensitive strain of Salmonella typhimurium was TA100. It detected all 30 mutagens. TA98 was mutated by 25 compounds, TA1537 by 16 compounds. No mutagenic effects were seen with TA1535. Possible reasons for the high percentage of apparently "false positives" in the Ames test and the lack of a quantitative correlation between the potency of the mutagenic and carcinogenic effects are discussed. It is suggested that the complexity of the metabolism of these heterocyclic compounds may lead to critical differences in metabolism in mouse subcutaneous tissue in vivo and in liver homogenates from rats treated with Aroclor. Therefore the present study will be extended to life-long oral and intrahepatic carcinogenicity tests leading to a higher proportion of metabolism in the liver.     

Mutagenicity studies on coffee. The influence of different factors on the mutagenic activity in the Salmonella/mammalian microsome assay

Recently, mutagenic activity on several strains of Salmonella typhimurium has been found in many heat-processed foodstuffs. The previously reported direct-acting mutagenic activity of coffee in Salmonella typhimurium TA100 (Ames assay) was confirmed in our study. In addition to TA100, a mutagenic effect of coffee was also found by using the newly developed strain TA102. The mutagenic activity was abolished by the addition of rat-liver homogenate. 10% S9 mix completely eliminated the mutagenic activity of 30 mg of coffee per plate. The addition of reduced glutathione to active S9 further decreased the mutagenic activity and also reduced the mutagenicity together with inactivated S9. The compound or compounds responsible for this inactivation are heat-labile and seem to be located in the cytosol fraction of the S9. Part of the mutagenicity of coffee was also lost spontaneously upon incubation at temperatures between 0 degrees and 50 degrees C. The loss of activity was dependent on temperature, being more pronounced at 50 degrees C compared to 0 degrees C (at 50 degrees C approximately 50% of the mutagenic activity was lost after 6 h). As anaerobic conditions prevented this loss of mutagenicity almost totally, oxidative processes are probably responsible for the inactivation. The stability of the mutagen was not influenced by incubation at low pH values (pH 1-3), with or without the addition of pepsinogen. The mutagenic properties of methylglyoxal, which to some extent could be responsible for the mutagenic activity of coffee, were compared with those of coffee. Methylglyoxal was strongly mutagenic towards Salmonella typhimurium TA100 and TA102. Its mutagenic activity was partially inactivated by the addition of 10% S9. Glyoxalase I and II together with reduced glutathione abolished the mutagenic activity of methylglyoxal but reduced the mutagenicity of coffee by only 80%. Since these enzymes occur in mammalian cells, the mutagenic compound(s) of coffee could also be degraded in vivo. This conclusion is supported by the fact that a long-term carcinogenicity study with rats was negative. These results clearly demonstrate that the effects observed in vitro do not necessarily also occur in vivo, but that in vitro experiments may contribute to the understanding of fundamental mechanisms of chemical carcinogenesis.     

Compatibility of organic solvents with the Microscreen prophage-induction assay: solvent--mutagen interactions

The following solvents did not induce prophage lambda in the Escherichia coli WP2s(lambda) Microscreen assay: acetone, benzene, chloroform, ethanol, n-hexane, isopropanol, methanol, toluene, and a mixture of the three isomers of xylene. Dimethyl sulfoxide was genotoxic in the presence and absence of S9, and methylene chloride was weakly genotoxic in the presence of S9. The genotoxic potencies of 2-aminoanthracene and 2-nitrofluorene were reduced when dissolved in DMSO or methanol compared to their potencies when dissolved in acetone.     

Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NCI/NTP

A survey has been conducted of 222 chemicals evaluated for carcinogenicity in mice and rats by the United States NCI/NTP. The structure of each chemical has been assessed for potential electrophilic (DNA-reactive) sites, its mutagenicity to Salmonella recorded, and the level of its carcinogenicity to rodents tabulated. Correlations among these 3 parameters were then sought. A strong association exists among chemical structure (S/A), mutagenicity to Salmonella (Salm.) and the extent and sites of rodent tumorigenicity among the 222 compounds. Thus, a approximately 90% correlation exists between S/A and Salm. across the 115 carcinogens, the 24 equivocal carcinogens and the 83 non-carcinogens. This indicates the Salmonella assay to be a sensitive method of detecting intrinsic genotoxicity in a chemical. Concordance between S/A and Salm. have therefore been employed as an index of genotoxicity, and use of this index reveals two groups of carcinogens within the database, genotoxic and putatively non-genotoxic. These two broad groups are characterized by different overall carcinogenicity profiles. Thus, 16 tissues were subject to carcinogenesis only by genotoxins, chief among which were the stomach, Zymbal's glands, lung, subcutaneous tissue and circulatory system. Conclusions of carcinogenicity in these 16 tissues comprised 31% of the individual chemical/tissue reports of carcinogenicity. In contrast, both genotoxins and non-genotoxins were active in the remaining 13 tissues, chief among which was the mouse liver which accounted for 24% of all chemical/tissue reports of carcinogenicity. Further, the group of 70 carcinogens reported to be active in both species and/or in 2 or more tissues contained a higher proportion of Salmonella mutagens (70%) than observed for the group of 45 single-species/single-tissue carcinogens (39%). 30% of the 83 non-carcinogens were mutagenic to Salmonella. This confirms earlier observations that a significant proportion of in vitro genotoxins are non-carcinogenic, probably due to their non-absorption or preferential detoxification in vivo. Also, only 30% of the mouse liver-specific carcinogens were mutagenic to Salmonella. This is consistent with tumors being induced in this tissue (and to a lesser extent in other tissues of the mouse and rat) by mechanisms not dependent upon direct interaction of the test chemical with DNA. Detection of 103 of the 115 carcinogens could be achieved by use of only male rats and female mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of the formaldehyde releasing preservatives dimethylol urea and diazolidinyl urea in several short-term genotoxicity tests

The two formaldehyde (FA)-releasers dimethylol urea (DMU) and diazolidinyl urea (DZU) are widely used as preservatives or additives. They were tested for genotoxicity in three short-term test systems, i.e. in the Salmonella typhimurium mutagenicity assay, in the in vitro micronucleus test with V79 Chinese hamster cells and in the in vitro tubulin assembly assay using isolated tubulin from pig brains. The polymerization products obtained in the tubulin assembly assay were examined additionally by electron microscopy.In the S. typhimurium mutagenicity assay with the pre-incubation assay both FA-releasers tested show a clear and concentration-dependent increase in the number of revertants in strains TA98, TA100 and TA102 with and without metabolic activation (rat liver S9 mix). In all cases, a biologically relevant increase in the number of revertants was achieved within the concentration range tested (DZU: 0.04-1.8 micromol per plate, DMU: 0.21-8.33 micromol per plate). FA was tested at 0.06-2.5 micromol per plate and lead to similar effects.Both compounds induce the formation of micronuclei (concentration range tested: DZU: 2.5-50 micromol/l, DMU: 3.3-333 micromol/l). However, DMU shows a comparatively weaker effect exclusively in the absence of the metabolizing enzymes. By contrast, DZU yields a distinct increase of the micronucleus rate in the absence and in the presence of S9. In addition, DZU predominantly causes an increase of large micronuclei, which suggests that this compound has a marked aneugenic potential. Cytotoxic effects accompany the clastogenic effects of both DMU and DZU.The examination of DMU and DZU in view of a possible aneugenic potential in the tubulin assembly assay yielded the following results: DMU at concentrations up to 10 mmol/l did not influence the formation of microtubuli, whereas DZU inhibited this process completely at 3 mmol/l. FA at 6 mmol/l completely inhibited the tubulin assembly. These results could clearly be confirmed by electron microscopy examination. The different potential of the two compounds with respect to the inhibition of tubulin formation is apparently due to a significant difference in the degree of FA release.According to these results, both compounds have to be considered as genotoxic in vitro. On account of these data and because of the widespread use of these two compounds in various products used in daily life, a reevaluation of the risk associated with these compounds seems to be necessary.     

Mutagenic activity of 2-chloro-4-nitroaniline and 5-chlorosalicylic acid in Salmonella typhimurium: two possible metabolites of niclosamide

Niclosamide is an anti-helminthic drug susceptible to being metabolized into a bacterial mutagen by the action of enzymes present in the S9 activation mixture. Additional results from genotoxic studies in rodents and humans suggest that the drug is absorbed from the gastrointestinal tract, and mutagenic metabolites are excreted both in the free form and as conjugated glucuronides. As in the case of other secondary amides, phase I metabolism of niclosamide may result in a hydrolytic cleavage of the amide bond, giving rise to 5-chlorosalicylic acid and 2-chloro-4-nitroaniline as the main metabolites. In this study, the mutagenicity of these compounds was tested using the Salmonella typhimurium assay. Bacterial mutagenicity tests with these 2 compounds reveal a non-mutagenic response with 5-chlorosalicylic acid and a mutagenic one with 2-chloro-4-nitroaniline. However, the mutagenic potency observed with this compound is lower than that of niclosamide. The role of nitroreduction in the activation of niclosamide and 2-chloro-4-nitroaniline was also investigated with the help of S. typhimurium strains TA98NR, YG1020, YG1021 and YG1024. The results show a pattern of response which is qualitatively similar for both compounds and this indicates that its mutagenicity depends on both nitroreduction and transacetylation.     

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.     

Studies on the antimutagenesis of Phyllanthus orbicularis: mechanisms involved against aromatic amines.

Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties. This plant extract is being studied for treatment of viral diseases in animals and humans. Antimutagenic activities of this plant aqueous extract have been investigated as an additional and possible valuable property. Antimutagenesis was assayed against the mutagenic activity of m-phenylenediamine (m-PDA), 2-aminofluorene (2-AF), 1-aminopyrene (1-AP), 2-aminoanthracene (2-AA) and 9-aminophenantrene (9-AP) in Salmonella typhimurium (S. typhimurium) YG1024, in different co-treatment approaches. This plant extract produced a significant decrease of the mutagenesis mediated by these aromatic amines (AA) in the following order: m-PDA>2-AA>2-AF>9-AP>1-AP. Interactions with S9 enzymes and transformation of promutagenic amines and their mutagenic metabolites by chemical reactions to non-mutagenic compounds are proposed as possible mechanisms of antimutagenesis. Mutagenesis mediated by m-PDA was almost completely abolished when S9 mixture was co-incubated with the plant extract during 40 min, previous to the addition of the m-PDA and bacterial cells to the assay. Similar results were found with 2-AA and 1-AP, but the reduction of the mutation rate was not so dramatic. In contrast, the most significant antimutagenic effect against 2-AF and 9-AP was seen when these chemicals were co-incubated with the plant extract, before addition of the S9 mixture and bacterial cells to the assay. Therefore, inhibition or competition for S9 enzymes seems to be the main antimutagenic mechanism of this plant extract against m-PDA, 2-AA and 1-AP, whilst a chemical modification of 2-AF and 9-AP into non-promutagenic derivatives is likely to be the main mechanism of antimutagenesis against both compounds.     

Mutagenicity of 22 N-nitrosamides and related compounds for Salmonella typhimurium TA1535.

Twenty-two N-nitrosamides and related compounds, including 14 nitrosoureas, 5 nitrosocarbamates, and one nitrosocyanamide, were tested at various concentrations for mutagenic activity towards Salmonella typhimurium TA1535 without the use of microsomes. The ether-water partition coefficient, solubility in water, and half-life in aqueous solution were also measured. Twenty compounds were mutagenic, with "standard mutagenic concentrations" (i.e. those producing 100 mutants/dish) of 0.0024--6500 micron. Standard mutagenic concentration was negatively correlated with the partition coefficient. Three compounds (ethyl 2-acetoxyethylnitrosocarbamate, nitrosocarbaryl, and methylnitrosobenzamide) were more active than the classic mutagen methylnitrosonitroguanidine. Nitrosocarbamates were at least 50 times more mutagenic than the corresponding nitrosoureas. Nitrosodihydrouracil and propylene-nitrosourea were more active than related compounds. Ethylnitrosocyanamide was 730 times more mutagenic than ethylnitrosourea. Fifteen of the test compounds (of which 14 were mutagenic) had previously been assayed in rats for carcinogenicity, all with positive results.     

Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium.

To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual alpha and beta subunits. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.     

Genotoxicity and carcinogenicity studies of antihypertensive agents

This survey is a compendium of genotoxicity and carcinogenicity information of antihypertensive drugs. Data from 164 marketed drugs were collected. Of the 164 drugs, 65 (39.6%) had no retrievable genotoxicity or carcinogenicity data; this group was comprised largely of drugs marketed in a limited number of countries. The remaining 99 (60.4%) had at least one genotoxicity or carcinogenicity test result. Of these 99, 48 (48.5%) had at least one positive finding: 32 tested positive in at least one genotoxicity assay, 26 in at least one carcinogenicity assay, and 10 gave a positive result in both at least one genotoxicity assay and at least one carcinogenicity assay. In terms of correlation between results of the various genotoxicity assays and absence of carcinogenic activity in both mice and rats 2 of 44 non-carcinogenic drugs tested positive in the in vitro bacterial mutagenesis assay, 2 of 9 tested positive in the mouse lymphoma assay, none of 14 tested positive for gene mutation at the hprt locus, 5 of 25 tested positive in in vitro cytogenetic assays, none of 31 in in vivo cytogenetic assays, and none of 14 in inducing DNA damage and/or repair in in vitro and/or in vivo assays. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, 75 drugs had both genotoxicity and carcinogenicity data; of these 37 (49.3%) were neither genotoxic nor carcinogenic, 14 (18.7%) were non-carcinogens which tested positive in at least one genotoxicity assay, 14 (18.7%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, and 10 (13.3%) were both genotoxic and carcinogenic. Only 42 of the 164 marketed antihypertensives (25.6%) had all data required by the guidelines for testing of pharmaceuticals.     

Mutagenesis by 4-nitrobenzofurazans and furoxans.

15 nitrobenzofurazans and 10 nitrobenzofuroxans synthesized primarily for testing as potential anti-rheumatic drugs were also tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100. The method used involved placing each compound in a "well" cut out of a plate of selective medium previously seeded with the appropriate tester strain, and then adding a rat liver microsome/cofactor mixture to one of the two wells on each plate. This method is considerably cheaper and more convenient than the conventional agar overlay technique, but in the present series of experiments failed to detect 4 compounds which could be detected by the overlay technique. Using a combination of the two techniques, 21 of the 25 compounds tested were found to be mutagenic. All 10 benzofuroxans and 9 of the 15 benzofurazans were detected as direct-acting mutagens with at least one of the two tester strains. Two of the benzofurazans gave positive results only in the presence of rat liver microsomes, and hence are pro-mutagens. One of the 4 benzofurazans which gave negative results for mutagenicity in these tests was found to be an efficient inhibitor of a neutral protease activity found in rheumatic synovial fluid, and may therefore have some potential as an anti-rheumatic drug.     

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.