Evaluation of the potential genotoxicity of the phosphate binder lanthanum carbonate.

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.

The single laser flow cytometric micronucleus test: a time course study using colchicine and urethane in rat and mouse peripheral blood and acetaldehyde in rat peripheral blood.

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.

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application.

Alternatives to Aroclor 1254-induced S9 in in vitro genotoxicity assays.

A working party was set up by the UK Environmental Mutagen Society to consider alternatives to Aroclor 1254 (Aroclor)-induced S9 in in vitro genotoxicity assays, with the aims of considering whether a replacement for Aroclor in its role in general screening assays could be readily identified. The working party concluded that there was sufficient support in the literature to justify the use of an appropriate phenobarbital/beta-naphthoflavone regime as an acceptable alternative to Aroclor.

The differential clastogenicity of Solvent Yellow 14 and FD & C Yellow No. 6 in vivo in the rodent micronucleus test (observations on species and tissue specificity).

Solvent Yellow 14 is carcinogenic in rats, inducing neoplastic nodules of the liver, but is non-carcinogenic in mice. The present paper shows that Solvent Yellow 14 induces micronuclei in the bone marrow of rats after a single oral dose of 250 mg/kg and above. In mice, however, there was no increased incidence of micronuclei after single oral doses of up to 2000 mg/kg Solvent Yellow 14, thus reflecting the species specific carcinogenic effect of the compound. The structurally related azo dye FD & C Yellow No. 6 is noncarcinogenic to rats and mice and gave a negative result in both rat and mouse bone marrow micronucleus tests after a single oral dose of up to 2000 mg/kg. The rat bone marrow micronucleus test is therefore capable of discrimination between the carcinogenic and the non-carcinogenic azo dye. A negative result was obtained for Solvent Yellow 14 in an in vivo liver unscheduled DNA synthesis assay after oral doses up to 1000 mg/kg. This result demonstrates the inability of the two in vivo assays used to predict target organ specificity seen in the cancer bioassay.

Investigations into the induction of aneuploidy and polyploidy in mammalian cells by the anti-tussive agent noscapine hydrochloride.

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.

Effects of aniline hydrochloride in the mouse bone marrow micronucleus test after oral administration.

Mice are non-responsive to aniline-induced carcinogenicity. However, the present paper shows that aniline hydrochloride induces micronuclei in the bone marrow of male CRH mice 24 h after a single oral dose of 1000 mg/kg (in terms of free base) though not at the lower doses of 400 and 500 mg/kg. No micronucleus inductions was found 48 h after oral dosing. A positive response was also seen 24 h after a single i.p. dose of 380 mg/kg aniline. The high oral dose of aniline needed for micronucleus induction, the presence of micronuclei with abnormal morphology and the large inter-animal variability found within this group may possibly indicate a more complex mechanism of micronucleus induction than simple clastogenicity.

Uptake of tritiated thymidine by cells of the rat gastric mucosa after exposure to loxtidine or omeprazole.

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)

Micronucleus induction by azobenzene and 1,2-dibromo-3-chloropropane in the rat: evaluation of a triple-dose protocol.

A triple-dose protocol was evaluated in the rat bone-marrow micronucleus test using azobenzene and 1,2-dibromo-3-chloropropane (DBCP) as test compounds. Dosing with 250 mg/kg azobenzene over 3 consecutive days led to an accumulation of micronucleated polychromatic erythrocytes. The magnitude of the effect was the same as that obtained after a single dose of 750 mg/kg in a previous study. In the single-dose study the effect was only detected at the 48 h sampling time. With the triple-dose protocol the effect was apparent 24 h after the last dose--thus eliminating the need for more than one sampling time. In the case of DBCP, the positive effect observed after a single dose diminished with increasing number of doses for the low dose but plateaued for the high dose, accompanied by a marked cytotoxic effect. It is therefore concluded, that for one of the compounds, azobenzene, the triple-dose protocol provided an advantage whereas for the other, DBCP, it failed to do so and led to results which are rather difficult to interpret. Furthermore, the number of animals necessary to select an appropriate dose level appears to be higher than in the single-dose protocol.

Comparison of Salmonella typhimurium TA102 with Escherichia coli WP2 tester strains.

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)

Induction of mutations in TK6 human lymphoblastoid cells by ethyl methanesulphonate, benzo[a]pyrene and benzidine.

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.

A demonstration of the in vitro bacterial mutagenicity of procarbazine, using the microtitre fluctuation test and large concentrations of S9 fraction.

The anti-neoplastic agent procarbazine is genetically active in a variety of short term mutagenicity tests, and it also possesses carcinogenic and teratogenic potential. This compound has consistently yielded false negative results in in vitro microbial mutagenicity tests in the presence and absence of mammalian metabolic activation. In this study, procarbazine was not mutagenic in standard and preincubation Ames tests using large S9 concentrations and bacterial test strains devoid of the rfa mutation. The microtitre fluctuation test is a sensitive technique for the detection of bacterial mutagens. Using this assay, procarbazine proved to be a bacterial mutagen after in vitro metabolic activation at concentrations as low as 200 micrograms/ml. Activity was dependent upon liver S9-concentrations which were higher than those usually attained within agar assays, and was only observed when such fractions were derived from aroclor-treated or phenobarbitone plus beta-naphthoflavone treated rat livers. The excision-repair proficient strain E. coli WP2 was equally, if not more, sensitive than the repair deficient strain E. coli WP2 uvrA. Furthermore, the differential mutagenic effects obtained using S. typhimurium strains TA1535, TA1530 and his G46 indicated that the absence of an rfa mutation was essential for procarbazine mutagenesis. Procarbazine was also mutagenic for E. coli D494 in a forward mutation fluctuation assay measuring resistance to ampicillin. In this assay lower concentrations of S9-fraction were sufficient, reflecting the increased sensitivity of the test strain towards the mutagenic metabolites of the drug. In conclusion, the results suggest that rat liver S9-fraction contains only low levels of enzymes capable of activating procarbazine to a mutagen. This may be a result of rapid breakdown during storage, or due to intrinsically low levels in rat liver extracts. Inducing agents appear to increase these levels. The low concentrations of mutagenic species formed in vitro, may only be detectable using a highly sensitive assay such as the microtitre fluctuation test.