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Lanthanum was evaluated for potential genotoxicity using a range of in vitro assays (as the carbonate) in the presence and absence of post-mitochondrial fraction (S9) and in vivo in three independent tests for mutagenicity and clastogenicity (as the carbonate and chloride). The drug was devoid of mutagenic activity in bacterial assays (maximum concentration 5000 microg/plate) using a range of test strains (Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102 and Escherichia coli WP2 uvrA and WP2 uvrA pkm101). No effects were seen in the hgprt gene mutation assay in Chinese hamster ovary cells in the presence of S9. In the absence of S9, sporadic increases in revertant numbers were not dose-related or reproducible in subsequent experiments and hence were concluded to be chance events. In an in vitro chromosome aberration assay using Chinese hamster ovary cells, chromosome damage in the presence and absence of S9 (concentration 200-5000 microg/ml) was attributed to overt cell toxicity. To confirm this, a comprehensive in vivo evaluation of the drug was performed. Negative results were obtained in two independent rodent micronucleus tests. In the first mice were given oral doses (of carbonate) up to 2000 mg/kg, in the second rats were given a single i.v. bolus injection (of chloride) up to 0.1 mg/kg. Negative results were also obtained in a rat liver unscheduled DNA synthesis assay after treatment for 28 days with i.v. bolus injections (of chloride) up to 0.1 mg/kg/day. In these in vivo studies lanthanum plasma concentrations were >3000 times higher than the steady-state peak plasma concentration observed in dialysis patients given therapeutic doses of lanthanum carbonate. It can be concluded that lanthanum is not genotoxic and that lanthanum carbonate is unlikely to present a latent hazard in therapeutic use.

A single laser flow cytometric procedure to quantify micronucleus frequency in rat and mouse peripheral blood was evaluated. Reticulocytes express the transferrin receptor (also known as the CD71-defined antigen). When combined with a DNA stain, antibodies against this antigen can be used to differentially label and quantify micronucleated reticulocytes. The object of this study was to evaluate the method for rat and mouse peripheral blood using flow cytometry and compare the results obtained between two laboratories (GlaxoWellcome and Litron Laboratories). The compounds selected were the rodent carcinogens colchicine, urethane and acetaldehyde. Colchicine gives a positive response in the rat bone marrow micronucleus assay and an inconclusive result in the rat peripheral blood micronucleus assay. The latter two are both established rat carcinogens readily detected in both the bone marrow and peripheral blood micronucleus assays. In these experiments both rat and mice were treated with either colchicine or urethane and rats alone treated with acetaldehyde. After a single treatment, repeat sampling of peripheral blood was made at 0, 24, 48 and 72 h. Replicate blood samples were obtained and fixed for flow cytometric analysis at both facilities. The micronucleated reticulocyte frequency of each blood sample was determined by analysing 20 000 total reticulocytes per blood sample. The data suggest that the single laser flow cytometric procedure resulted in consistent reticulocyte and micronucleated reticulocyte frequencies between laboratories. Furthermore, these flow cytometric data compare favourably with previously published data.

Sanfetrinem cilexetil, a hexetil ester of a Trinem antibiotic, does not induce micronuclei in rat bone marrow cells or induce DNA repair synthesis in rat hepatocytes following oral dosing. However, in vitro chromosome damage and mutations are induced in mammalian cells lacking carboxylesterase activity (human lymphocytes and mouse lymphoma L5178Y cells). In cells possessing carboxylesterase activity (CHL cells), chromosome damage induced by Sanfetrinem cilexetil is not observed. Similarly, if induced rat liver preparations or non-induced preparations from rat or human intestinal cells are present during exposure, genotoxic activity is lost, even in those cells lacking carboxylesterase enzymes. Thus the lack of demonstrable genotoxicity in vivo, in the assays used, is likely to be due to hydrolysis of the parent molecule by non-specific carboxylesterases present within the intestinal epithelium. In turn this data indicates that a genotoxic hazard to humans under therapeutic conditions is unlikely.

The number and structural diversity of new compounds that are currently being introduced into the environment underlies the need to provide more sensitive toxicology tests. Ideally, these should involve tests that reduce dependence upon animal experimentation; over the past few years a step towards achieving this goal has arisen from the ability to construct transgenic animals. By the inclusion of a suitable marker gene or genetic predisposition to a given disease state, several potentially valuable new tests have become available. Here we assess the potential of these systems for use by the toxicologist and consider the future of this technology to the industry.

A collaborative trial was carried out to determine the intra/interlaboratory variability of Salmonella typhimurium strains TA102 and TA97a with regard to spontaneous revertant frequency and in response to four model mutagens (cumene hydroperoxide and bleomycin for strain TA102, and 4-nitrophenylenediamine and 4-aminoantipyrine for strain TA97a). A secondary objective of the trial was to monitor the stability of the strains after storage for up to 8 months and identify any technical problems associated with their use. Thirteen different laboratories participated in the trial, all receiving identical stock cultures of the bacterial strains and samples from the same batch of mutagenic compound. A standard protocol was followed and two independent experiments were carried out within 1 month of receipt of the strains/compounds (phase I), and again after a period of 6-8 months (phase II). Comparative studies with the standard strain TA100 after treatment with 4-nitrophenylenediamine were carried out as part of phase II. Overall, both strains gave acceptably consistent results in different laboratories and are considered useful for screening purposes when used under standardized conditions. One major source of interlaboratory variability identified for TA102 appears to be the sensitivity of different types of automatic colony counter for detecting the micro-colony revertants that this strain produces.

Noscapine, a non-narcotic, centrally-acting anti-tussive drug induces polyploidy in Chinese hamster CHL cells; further studies were carried out to investigate whether similar effects could be induced in other rodent cells (Chinese hamster V79) and in human lymphocytes. In both cases, large increases in the frequency of polyploid cells were induced at test concentrations ranging from 15 to 120 micrograms/ml after 24 and 48 h continuous treatment in the absence of S9 mix. In addition, spindle damage was observed in V79 cells and human skin fibroblasts after 24 h treatment with test concentrations of 30 and 60 micrograms/ml. Furthermore, after treatment of human skin fibroblasts there was a marked increase in the proportion of cells containing chromosomes which had become dislocated from the spindle. Treatment of the mouse/human hybrid cell line R3-5 induced a significant increase in the number of 6-thioguanine resistant colonies and it was confirmed cytogenetically that these colonies had arisen due to loss of human chromosome 2. From these experiments it can be concluded that noscapine induces polyploidy in both rodent and human somatic cells, and that this could arise through a direct effect upon spindle structure and/or function. The aneugenic properties of noscapine are less certain and further work is required in this area. Exposure to the drug through its therapeutic use (15mg up to four times daily) could exceed, at least locally within the gastrointestinal (GI) tract, the concentration range shown to be active in these in vitro studies. An immediate topical hazard might exist within the buccal cavity and GI tract, but further confirmation of these in vitro results are required using suitable in vivo systems before definite conclusions can be made regarding any potential hazard associated with the administration of this drug.

The H2-antagonist loxtidine and the H+/K(+)-ATPase inhibitor omeprazole inhibit gastric acid secretion and both have been associated with the appearance of gastric tumours in rat cancer studies. Loxtidine is not genotoxic in a range of in vitro and in vivo assays. As false negative results can occur if the organotropic nature of the drug is not considered, both drugs were evaluated using an assay which estimates the uptake of tritiated thymidine by cells of the gastric mucosa (the target tissue) in comparison with the positive control, N-methyl-N-nitro-nitrosoguanidine (MNNG), which others have shown to induce genetic damage in the stomach mucosa of rats. Such uptake may be, in part, indicative of unscheduled DNA synthesis (UDS) resultant from genotoxic damage. Serum gastrin levels were also determined at various times after either loxtidine or omeprazole treatment. Increased uptake of tritiated thymidine was only obtained after omeprazole or MNNG treatment, when this was estimated scintillometrically. The nature of the formulation of omeprazole was critical. The uptake of tritiated thymidine was greatest when omeprazole was administered in vehicle which had been buffered to pH 9. These effects were unlikely to be due to the trophic effects of gastrin since serum gastrin levels were similar after either loxtidine or omeprazole treatment. Autoradiographic analysis of stomach sections was also carried out and revealed a 2- to 3-fold increase in the number of labelled cells within the fundic mucosa as compared to the control values after treatment with MNNG or Losec (enteric coated granules of omeprazole).(ABSTRACT TRUNCATED AT 250 WORDS)

In 1982, Levin et al. published a paper describing a new Salmonella typhimurium strain, TA102, for detecting mutagenic agents that react preferentially with AT base pairs. This strain has an AT base pair at the critical mutation site within the hisG gene, which is located on a multicopy plasmid, pAQ1; the chromosomal copy of the hisG gene has been deleted. It also has an intact excision repair system, thus facilitating the detection of cross-linking agents, and carries the mutator plasmid, pKM101. Although TA102 has been shown to be reverted by certain mutagenic agents that are not detected in the usual battery of strains (TA1535, TA1537, TA1538, TA98 and TA100), there has been a general reluctance within the field to include TA102 as one of the standard screening strains. This may in part result from the difficulties which have been experienced in many laboratories in maintaining the strain, and in obtaining reproducible spontaneous and induced revertant counts. At Glaxo we routinely include certain Escherichia coli strains in our microbial test battery, and were aware that some of the genetic features offered by TA102 were already being covered by these strains. For example, E.coli WP2 (pKM101) has an AT base pair at the critical mutation site within the trpE gene, is excision proficient (and thus will detect cross-linking agents) and carries the pKM101 plasmid to enhance error-prone repair. From the published literature it was apparent that a number of the 'TA102 specific' mutagens could be detected in E.coli e.g. neocarzinostatin, UV and 8-MOP plus UV.(ABSTRACT TRUNCATED AT 250 WORDS)

The experiments described were carried out as part of the UKEMS Third Collaborative Trial. The method used was essentially that described by Thilly et al., based on a cloning assay using microtitre test plates. The induction of mutations at both the thymidine kinase (tk) locus and the hypoxanthine guanine phosphoribosyl transferase (hprt) locus was determined following exposure to ethyl methanesulphonate (EMS), benzidine (BZD) and benzo[a]pyrene (B[a]P). EMS was tested in the absence of a metabolic activation system (S9 mix), BZD was tested in the presence and absence of S9 mix and B[a]P was tested only in the presence of S9 mix. Incubation with EMS or B[a]P caused the induction of mutations at both the tk and hprt loci. The effects of benzidine were not reproducible, although some individual data points at the tk locus were found to be significant.

Loxtidine, a potent, non-competitive histamine H2-receptor antagonist was evaluated for genotoxic potential using a range of short-term mutagenicity assays. Unequivocally negative results were obtained in a Salmonella/plate incorporation assay and a liquid pre-incubation assay (using S. typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98), a fluctuation assay [using Escherichia coli strains WP2, WP2 uvrA (R46) and 343/113 lys60 (R46)], a gene conversion assay (using Saccharomyces cerevisiae JD1) and a human peripheral lymphocyte cytogenetic assay. All of these in vitro tests were carried out in the presence and absence of rat liver S9 mix. In addition, the major metabolites of loxtidine in the rat were also negative in the same range of microbial mutagenicity assays. Loxtidine was inactive in the mouse micronucleus test after oral administration. The potential nitrosatability of loxtidine was investigated using an expanded version of the WHO Nitrosation Assay Procedure, and detectable quantities of mutagenic nitroso-species were not formed. The subsequent appearance of carcinoid tumours within the gastric fundus of rodents treated orally with loxtidine for most of their natural lifespan, led to additional assays being carried out on this compound to determine whether the tumorigenic effects were due to alternative mutagenic mechanisms. Negative results were obtained in an in vitro unscheduled DNA synthesis assay using primary rat hepatocytes, and an assay for spindle damaging agents using Muntjac skin fibroblasts. It can be concluded from these results that loxtidine is unlikely to be a genotoxic carcinogen. The increase in carcinoid tumour incidence observed in rats and mice after loxtidine treatment was probably related to the prolonged achlorhydria produced by this potent unsurmountable histamine H2-receptor antagonist.

1,2-Dimethylhydrazine is confirmed as active in the mouse bone-marrow micronucleus assay when administered as an aqueous solution via oral gavage to three different strains of mice. It is also shown to be inactive in a corresponding rat assay under similar conditions of test. The observations were independently repeated in two laboratories. In contrast, 1,2-dibromo-3-chloropropane showed the reverse profile, being active in the rat bone-marrow assay but inactive in the mouse; the latter observations were also made in two laboratories. The carcinogen procarbazine was active in both species. These findings are discussed within the context of the present GeneTox revision of the standard test protocol of the rodent bone-marrow micronucleus assay.

This review discusses features of the standard protocols currently used in bacterial mutation assays which can have a critical effect upon the test outcome. Such features if not strictly controlled may affect the results qualitatively as well as quantitatively. These include the dose-intervals of the test compound used; the number of bacterial cells exposed to the test compound; the phase of growth such cells are in during exposure; and the length of time that the cells are exposed prior to the addition of soft agar (in the Salmonella/plate incorporation assay). The following possible modulating effects will also be discussed: the nature of the solvent used; the nature and quantity of the exogenous metabolizing system (usually liver S9-fraction) and the quantity of amino-acid (e.g. histidine) within the test system being that either deliberately added or present unavoidably within the test sample (e.g. biological fluids). If bacterial mutagenicity assays are to realize their full potential for the detection of genotoxic carcinogens, the use of rigid protocols should be discouraged. Where possible, consideration of the compound's structure should lead to the employment of the optimal protocol for the detection of genotoxic carcinogens within that chemical class. The relative speed and low cost of bacterial assays should be exploited in this way to avoid the generation of false negative results during the primary screening of novel compounds.

The in vitro and in vivo clastogenic potential of three beta-lactam antibiotics, ampicillin, carbenicillin and penicillin VK, was investigated using cultured human lymphocytes and the rat micronucleus test. Neither ampicillin nor carbenicillin induced significant increases in chromosome damage in vitro up to test concentrations of 10 mg/ml. These results contrast with other published studies on these compounds. Both drugs were also inactive in vivo in the rat micronucleus test, using single- or double-dosing regimens (ampicillin 5 g/kg orally; carbenicillin 500 mg/kg i.m., either dosed once 30 h before marrow preparation, or dosed twice 48 and 24 h before marrow preparation). In vitro, penicillin VK induced a dose-related increase in chromosome and chromatid gaps and breaks, down to concentrations of 1.25 mg/ml. It is likely that the increase in aberration frequency was partly the result of exposing the cells to increased K+ ion concentration, as similar results were obtained when potassium chloride was evaluated over the same molar concentration range. However, the occurrence of 'ion-mediated' clastogenic effects as reported by other workers, does not fully account for the positive effects obtained with this compound, as clastogenic effects were also observed with penicillin V in this test system at similar test concentrations. It is known that exposure of mammalian cells to extremely high concentrations of beta-lactams can affect DNA polymerase alpha activity. An inhibitory effect upon DNA polymerase alpha resulting in a breakdown in the structural integrity of the chromosomes, is suggested as an additional mechanism of action for penicillin VK.(ABSTRACT TRUNCATED AT 250 WORDS)