Earle R. Nestmann

Mutagenicity of constituents identified in pulp and paper mill effluents using the Salmonella/mammalian-microsome assay

About 300 compounds have been reported in the literature as constituents of pulp-mill effluent. Previously, in our screening program, 10 resin acids identified in effluent were examined for potential mutagenicity in the Salmonella/mammalian-microsome assay. Neoabietic acid was the only resin acid which was found to be mutagenic. Now, a program to screen for mutagenicity of 48 additional compounds, belonging to chemical classes of chlorinated aliphatic and aromatic hydrocarbons, phenols, aldehydes, quinones, and carboxylic acids, has been completed. Only 2 of these compounds, tetrachloropropene and pentachloropropene, were found to be mutagenic, showing dose-related increases in His+ reversion mutations, in the standard Salmonella test. Metabolic activation with a preparation of Aroclor 1254-induced liver homogenate (S9) greatly reduced the mutagenic responses of these 2 compounds. Modifications of the Salmonella test for volatile mutagens enabled the detection of the mutagenicity of 3 additional chlorinated aliphatic hydrocarbons dichloromethane, dichloroethane and trichloroethane.

Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity

Since its development by Dr. Bruce Ames and his coworkers, the Salmonella typhimurium/mammalian microsome mutagenicity assay has been used widely throughout the world. Many authors have suggested various modifications and made recommendations in regards to this assay. Although the recommendations of a panel of experts was published in 1979 by de Serres and Shelby, a committee of members of the Environmental Mutagen Society (EMS) initiated this effort in response to the encouragement by the American Society of Testing and Materials (Committee E47.09.01) and because of new developments within the field of microbial mutagenesis testing. Its purpose is to provide a guide for people who perform or evaluate microbial mutagenesis tests, but it is not intended for these recommendations to replace or diminish the usefulness of presently available protocols and procedures.

Detection of the mutagenic activity of lead chromate using a battery of microbial tests.

The potential mutagenicity of the carcinogen lead chromate was tested by the following battery of microbial tests: the Escherichia coli PolA+/PolA- survival test; the Salmonella/microsome His+ reversion assay; the E. coli Trp+ reversion test as a plate assay; the E. coli Gal+ forward mutation test; and the Saccharomyces cerevisiae assay for mitotic recombination. Lead chromate is mutagenic in Salmonella and in Saccharomyces and is thus identified as a microbial mutagen by this battery. Metabolic activation by rat liver homogenate (S9) is not required for the mutagenic activity of lead chromate. The most statistically significant, positive result is found with a supplementary assay, the E. coli fluctuation test. To determine whether the lead ion and/or the chromate ion were responsible for the mutagenicity observed, lead chloride and chromium trioxide (chromic acid) were also tested. In E. coli fluctuation test, the ranges of maximal mutagenicity for chromium trioxide and lead chromate overlap at the concentration 10(-5)M, whereas lead chloride shows no mutagenicity and little lethality at concentrations up to 10(-3)M. Thus, it appears that the chromate ion is responsible for the mutagenicity of lead chromate.

Mutagenicity of constituents of pulp and paper mill effluent in growing cells of Saccharomyces cerevisiae

42 compounds identified in pulp mill effluent were screened for genetic activity in growing yeast cells using strains D7 and XV185-14C without S9. In addition to 3 of these compounds that had been shown previously to be mutagenic in Salmonella. 5 more were mutagenic in strain XV185-14C. All 8 induced 2-5-fold increases in reversion frequencies over background at the trp5 locus--5 of these induced 1.5-3-fold increases at the hom3 locus and 1 induced a doubling at his1. None of the compounds induced absolute increases of gene convertants per plate in strain D7, although 3 showed increased convertant frequencies as survival decreased.

Determination of mutagenic potential and organic contaminants of Great Lakes drinking water

Abstract Extracts of organic compounds were obtained, using XAD-2 macroreticular resin, from drinking water supplies in 12 Great Lakes municipalities. The extracts were tested for mutagenic potential using the Salmonella/mammalian-microsome assay and analysed for organochlorine pesticides, polyaromatic hydrocarbons, organophosphorous pesticides and trialkyl-arylphosphates. Grab samples of drinking water were also analysed for volatile organic compounds. Dose-related increases in mutagenicity were found in extracts from 11 of the drinking water supplies.

Genotoxic activity of pulp mill effluent in Salmonella and Saccharomyces cerevisiae assays.

An XAD-2 resin concentrate of chlorination-stage pulp mill effluent was found to induce mutations in Salmonella typhimurium strains TA1535, TA100 and TA98 but not in strains TA1537 or TA1538. The presence of either S9 mix, S9 mix without cofactors, or heat-inactivated S9 mix, reduced the mutagenic effects. Dose-related increases in gene conversion, mitotic recombination and aberrant colony formation in Saccharomyces cerevisiae strain D7 also were found.

Mutagenic activity of 3 industrial chemicals in a battery of in vitro and in vivo tests

3 chemicals were selected for mutagenicity testing from a priority list, based on production volume and available mutagenicity data. Propargyl alcohol (PA), 2-nitroaniline (2NA), and 5-methyl-1H-benzo-triazole (MBT) were selected for testing using the approach recommended in the Health Protection Branch Genotoxicity Guidelines. The battery of tests included the Salmonella/mammalian microsome mutation assay, the in vitro chromosomal aberration assay, and the bone-marrow micronucleus assay. The results indicate that 2 of the 3 chemicals, PA and 2NA, were clastogenic in vitro. Both PA and 2NA induced chromosomal aberrations in CHO cells in vitro with and without metabolic activation, while none induced reverse mutations detectable with the Salmonella/mammalian microsome assay. Because PA and 2NA were found to be in vitro clastogens, they also were tested in the mouse bone-marrow micronucleus assay. 2NA induced a small increase in micronuclei in males but not females. PA did not induce an increase in micronuclei.

Suitability of the modified fluctuation assay for evaluating the mutagenicity of unconcentrated drinking water

Filter-sterilized, unconcentrated tap water induced mutagenic responses (p less than 0.01) in Salmonella strain TA100 in fluctuation assays, usually with dose-related increases in positive tubes. Additional experiments were performed to study possible artifacts that could lead to falsely positive results. Determinations of bacterial survival revealed that cell populations in the tubes containing tap water were larger than in the controls. Since spontaneous mutation is a function of cell generation, the increased numbers of bacteria appeared to be responsible for the higher numbers of mutants observed. Therefore, the positive responses must be regarded as artifactual. This study suggests that survival determination should be a routine part of this method, and care should be exercised in the interpretation of positive results.

Genetic activity in Saccharomyces cerevisiae of compounds found in effluents of pulp and paper mills

20 compounds identified in pulp mill effluents were screened for genetic activity in growing cells using Saccharomyces cerevisiae strains D7 and XV185-14C without and with S9. Nine compounds were positive in one or the other yeast strain (7 in D7; 2 in XV185-14C). One additional compound showed weak effects and two others showed elevated frequencies/survivor without absolute increases of mutants. The presence of S9 enabled detection of one positive and two weak effects, it enhanced the genetic activity of one compound in each strain, and it reduced the mutagenic effects of 4 others in strain D7. 7 of the 20 chemicals tested have been shown previously to be mutagenic in the Salmonella/mammalian-microsome assay. Of the 7 bacterial mutagens, 6 were positive and 1 had a weak effect in yeast.

Mutagenic activity of diallate and triallate determined by a battery of in vitro mammalian and microbial tests

Diallate and Triallate are carbamate herbicides used mainly for the pre-emergence control of wild oats in various crops. The genetic activity of these compounds was studied using a battery of microbial and mammalian in vitro tests. In the Salmonella/mammalian-microsome assay, Diallate and Triallate show dose-related increases without metabolic activation in strains TA1535, TA100 and TA98, indicating that these compounds cause both frameshift and base-substitution mutations. Mutagenicity of both herbicides was enhanced greatly by incubation with Aroclor 1254 induced rat-liver S9. Genetic activity in mammalian cells was determined using a number of in vitro tests with Chinese hamster ovary (CHO) cells combined with metabolic activation as described above. Both Diallate and Triallate caused dose-related decreases in colony-forming ability, with concomitant dose-related increases in the frequencies of cells with chromosome damage and in the number of sister-chromatid exchanges. However, only Diallate caused a reduction in DNA molecular weight as determined by alkaline sucrose gradient (ASG) sedimentation. DNA damage was negligible even at concentrations of Triallate that reduced colony-forming ability to zero. This suggests that the lesions in DNA detected by the ASG technique are not necessarily related to those that produce chromosomal damage. These data, taken together, strongly implicate both Diallate and Triallate as capable of causing mutations in mammals. However the risk to man in terms of inherited disease or cancer remains to be established by appropriate in vivo methodology.