Verne A. Ray

The Salmonella typhimurium/mammalian microsomal assay: A report of the U.S. Environmental Protection Agency Gene-Tox Program☆

The Salmonella assay has been in use for almost 15 years and can be defined as a routine test for mutagenicity and for predicting potential carcinogenicity. It detects the majority of animal carcinogens and consequently plays an important role in safety assessment. The test is also routinely used as the frontline screen for environmental samples (complex mixtures) isolated from air, water and food. This role will continue to remain an area of growth as or because sample volumes associated with these testing areas are generally very limited and more extensive testing is generally impossible. While this test, like all others, has some limitations, it is recommended that it be regularly included in all genetic testing batteries.

Chemical carcinogens a review and analysis of the literature of selected chemicals and the establishment of the Gene-Tox carcinogen data base. A report of the U.S. environmental protection agency Gene-Tox program

The literature on 506 selected chemicals has been evaluated for evidence that these chemicals induce tumors in experimental animals and this assessment comprises the Gene-Tox Carcinogen Data Base. Three major sources of information were used to create this evaluated data base: all 185 chemicals determined by the International Agency for Research on Cancer to have Sufficient evidence of carcinogenic activity in experimental animals, 28 selected chemicals bioassayed for carcinogenic activity by the National Toxicology Program/National Cancer Institute and found to induce tumors in mice and rats, and 293 selected chemicals which had been evaluated in genetic toxicology and related bioassays as determined from previous Gene-Tox reports. The literature data on the 239 chemicals were analyzed by the Gene-Tox Carcinogenesis Panel in an organized, rational and consistent manner. Criteria were established to assess individual studies employing single chemicals and 4 categories of response were developed: Positive, Negative, Inconclusive (Equivocal) and Inconclusive. After evaluating each of the individual studies on the 293 chemicals, the Panel placed each of the 506 chemicals in an overall classification category based on the strength of the evidence indicating the presence or absence of carcinogenic effects. An 8-category decision scheme was established using a modified version of the International Agency for Research on Cancer approach. This scheme included two categories of Positive (Sufficient and Limited), two categories of Negative (Sufficient and Limited), a category of Equivocal (the evidence of carcinogenicity from well-conducted and well-reported lifetime studies had uncertain significance and was neither clearly positive nor negative), and three categories of Inadequate (the evidence of carcinogenicity was insufficient to make a decision, however, the data suggested a positive or negative indication). Of the 506 chemicals in the Gene-Tox Carcinogen Data Base, 252 were evaluated as Sufficient Positive, 99 as Limited Positive, 40 as Sufficient Negative, 21 as Limited Negative, 1 as Equivocal, 13 as Inadequate with the data suggesting a positive indication, 32 as Inadequate with the data suggesting a negative indication, and 48 Inadequate with the data not suggesting any indication of activity. This data base was analyzed and examined according to chemical class, using a 29 chemical class scheme.(ABSTRACT TRUNCATED AT 400 WORDS)

Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells II. Test validation and interpretation

The L5178Y Mouse Lymphoma TK assay was studied extensively to determine if this mammalian cell assay for gene mutations at the thymidine kinase (TK) locus could provide valid, interpretable determinations of mutagenic potential, and whether this information is of value in the safety evaluation of chemicals. We first determined that test-derived TFTR mutants were phenotypically stable, possessing little or no thymidine kinase activity as measured by labeled thymidine uptake, but demonstrating 100% cross resistance to bromodeoxyuridine. Common solvent vehicles such as acetone, dimethylsulfoxide and ethanol were shown to produce little cytotoxicity and no mutagenic activity when present at 1% levels. Out of a total of 10 noncarcinogens tested, all were negative when results were analyzed by a 2-sample loge t test on control and treated mutant count means. Of the 13 putative animal carcinogens tested, 10 were positive, 2 were negative (auramine O and sodium phenobarbital), and 1 showed sporadic activity (hydrazine sulfate) in the TK assay on the basis of test-derived t statistics. 2 compounds, 1,2-epoxybutane and ICR 191, which have been described as Ames positive non-carcinogens, were also positive in the TK assay. Although this sampling of a total of 29 compounds is insufficient for precise estimations of expected false-positive or false-negative frequencies, these data indicate the TK assay can be expected to detect a majority of carcinogens as mutagens including some missed by more established point-mutation assays.

Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells: I. Application to genetic toxicological testing

We have systematically evaluated the mouse lymphoma TK+/- leads to TK-/- mutagenesis assay to determine if this somatic-cell test system would be a useful addition to the routine screening battery already used in our laboratory for the detection of chemical mutagens. During these investigations we observed that, with certain modifications of the basic assay, mutagenicity data could be obtained in as little as 9 days once the relative cytotoxic properties of the test substance were known. By improving the culturing conditions, we were able to reduce the serum requirements by as much as 50--75% without appreciably altering either cell viability or the recovery of chemically-induced mutants. Phenotypic stability of test-derived trifluorothymidine resistant (TFTR) mutants was confirmed by demonstrating cross-resistance to bromodeoxyuridine and concomitant sensitivity to methotrexate (THMG) in TFTR cells grown for 20 generations under non-selective conditions. While reduced growth rates resulting from temporary cell-division delay in treated cells is probably not a contributing factor to the observed mutation frequencies, only TFTR colonies which formed large distinct colonies in the presence of trifluorothymidine were clearly phenotypically stable mutants when spontaneous mutants were isolated and verified. When a non-mutagen, a weak mutagen, and a well-established mutagen were compared at equitoxic doses under these modified conditions, clear quantitative differences were seen in the respective mutation frequencies induced by these 3 types of agents. With these technical modifications, we feel this assay is both reliable and amenable to the screening of diverse chemical compounds for point-mutational activity in a mammalian cell.

An evaluation of the Escherichia coli WP2 and WP2uvrA reverse mutation assay

The methodology and status of the Escherichia coli WP2 reverse mutation system as it applies to chemical screening were reviewed using the available published literature. 163 documents were reviewed by the Working Group. These included abstracts, research articles, review articles and publicly available contract and grant final reports. From this group, 115 documents were rejected for critical evaluation by the Working Group. 48 documents were reviewed and the test results summarized. The general conclusion of the Working Group was the the E. coli WP2 reverse mutation system is a valuable tool for mutagenesis research, but that there is no evidence from a review of the literature that this assay will contribute significantly to the results obtainable from careful application of the Ames Salmonella assay. Another review of the role of this system in general screening may be warranted after more research and development with the plasmid-containing WP2 derivatives.

A comparative study on the genetic effects of hycanthone and oxamniquine

Oxamniquine (UK-4271; 6-hydroxymethyl-2-isopropylaminomethyl-7-nitro-1,2,3,4-tetrahydroquinoline) is a new schistosomicidal agent currently undergoing clinical investigation in South America. Essentially a complete cure rate against Brazilian Schistosoma mansoni has been seen in adults with a single im dose of 7.5 mg/kg or a single oral dose of 15 mg/kg. These regimens were tolerated without significant toxicity. To assess its mutagenic potential, oxamniquine was examined in a battery of genetic tests designed to detect mutations at the gene and chromosome levels. For comparative purposes, hycanthone, a schistosomicide with extensively studied mutagenic properties, was evaluated in a similar series of tests. Point mutations were measured in a series of histidineless auxotrophs of Salmonella typhimurium in direct plate and host-mediated assays. Gross chromosomal aberrations were assessed in human leucocyte cultures and in bone marrow preparations from drug-treated mice. Effects on germ cells were tested in the dominant-lethal assay. Hycanthone showed significant mutagenic activity in the direct bacterial tests and the in vivo and in vitro cytogenetic assays. No response was detected in the host-mediated or dominant-lethal assays. On the other hand, oxamniquine produced no drug-related mutagenic effects in the cytogenetic, host-mediated, or dominant-lethal tests at doses up to 150 mg/kg administered parenterally. Oxamniquine produced a weak response in the frameshift mutant, TA1538, of Salmonella typhimurium in direct plate tests with and without liver microsomal enzymes. However, this response was achieved only by using a concentration of compound which was several orders of magnitude higher than that required to produce a similar response to hycanthone.

Mutagenicity studies with 6-mercaptopurine: I. Cytogenetic activity in vivo

Abstract Cytogenetic studies have been performed to determine the effect of 6-mercaptopurine (6-MP) on mammalian chromosomes in vivo . An elevated level of chromosome breakage is present in mouse bone marrow cells within 12 h after oral or parenteral dosing with 200 mg/kg. Damage is most severe at 24 h after oral dosing and at 72 h following intraperitoneal injection. A dose-response curve was constructed using oral doses of 10 to 200 mg/kg. The no-effect dose level lies within the 10–25 mg/kg range when administered as a single dose. In subacute studies, positive results have been obtained with as little as 2.5 mg/kg/day administered orally for 5 days.

Carbenicillin Indanyl Sodium, an Orally Active Derivative of Carbenicillin

Carbenicillin indanyl sodium, an orally active derivative of carbenicillin, is active against a broad spectrum of bacterial species. Although the ester has in vitro antimicrobial activity per se when evaluated in Brain Heart Infusion broth, the in vivo antibacterial activity seen in mice and rats reflects primarily the efficient hydrolysis of the ester to carbenicillin. With an acute systemic infection in mice as a test system, orally administered carbenicillin indanyl sodium protected mice against lethal infections produced by Escherichia coli, Salmonella choleraesuis, Pasteurella multocida, Proteus vulgaris, Staphylococcus aureus, and Streptococcus pyogenes. The dose that protected 50% of the animals against each of these infections was comparable to that of parenteral carbenicillin. Against experimental urinary-tract disease in rats produced by E. coli, P. vulgaris, and Pseudomonas aeruginosa, it was again observed that carbenicillin indanyl sodium provided activity comparable to that of parenterally administered carbenicillin.

Comparative studies of ethyl methanesulfonate-induced mutations with host-mediated, dominant lethal, and cytogenetic assays☆

Abstract Recent reports have advocated the use of a battery of tests for evaluating the mutagenic potential of chemicals. The present study was undertaken to compare three such procedures: the host-mediated, the dominant lethal, and the in vivo cytogenetic assays, using the mutagen ethyl methanesulfonate (EMS). The relative sensitivities of the three assay procedures were determined by establishing dose-response curves and no-effect dose levels. Dominant lethal effects were not evident until a dose of 150 mg/kg was used. Cytogenetic studies indicated significant breakage of somatic cell chromosomes did not occur until a dose of 115 mg/kg was utilized. Ethyl methanesulfonate is most effective on these cells in the late S or early G 2 phases of the cell cycle. The host-mediated assay was the most sensitive of the three procedures with a statistically significant response detectable at 35 mg/kg. This difference in sensitivities may reflect a difference in repair mechanisms between microbial and mammalian cells.

Preclinical toxicology studies with azithromycin: genetic toxicology evaluation

Azithromycin was subjected to a series of three in vitro and one in vivo genetic toxicology assays for the detection of drug-associated gene or chromosomal effects. In the Ames Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100, the presence of azithromycin was not associated with any increase in the number of his- revertants. Urine from mice dosed with up to 200 mg/kg of azithromycin also had no effect on the number of revertants in these same strains suggesting the absence of mutagenic excretory products following oral exposure. When tested up to the cytotoxic level of 240 micrograms/ml, azithromycin caused no increase in the mutant frequency at the thymidine kinase locus of L5178Y/TK cells. Both the mammalian and microbial gene mutation assays included the presence of rat-liver postmitochondrial (S9) fraction for the detection of mutagenic biotransformation products. Mitogen-stimulated human lymphocytes cultured in the presence of 2.5-7.5 micrograms/ml azithromycin for 24 h or 30.0-40.0 micrograms/ml azithromycin for 3 h in the presence of rat S9 had chromosomal aberration frequencies that were no different than negative control cells even though slight to moderate mitotic suppression was associated with these concentrations. In vivo assessment of this compound was completed in male and female mice with a single oral dose of 200 mg/kg followed by sacrifice at 6, 24 or 48 h later and metaphase analysis of bone marrow for chromosomal aberrations. No statistically significant elevations of chromosomally aberrant cells were found. We conclude that azithromycin does not cause gene mutations in microbial or mammalian cells, or chromosomal aberrations in cultured human lymphocytes or in mouse bone marrow in vivo.