Mutagenesis Advance Access originally published online on December 29, 2004
Mutagenesis 2005 20(1):29-37; doi:10.1093/mutage/gei003
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Mutagenesis vol. 20 no. 1 © UK Environmental Mutagen Society 2005; all rights reserved.
Evaluation of the potential genotoxicity of the phosphate binder lanthanum carbonate
Biosciences, Shire Pharmaceutical Development Ltd, Chineham, Basingstoke, UK and 1Covance Laboratories Ltd, Harrogate, North Yorkshire, UK
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Abstract |
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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 µg/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 µg/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.
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Introduction |
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Lanthanum carbonate (Fosrenol®) is a new non-aluminium, non-calcium phosphate-binding agent developed for the treatment of hyperphosphataemia associated with end-stage renal disease. The emerging non-clinical and clinical profile indicates good efficacy and tolerability in long-term studies (Damment et al., 2003
; D'Haese et al., 2003; Behets et al., 2004).
Although the oral bioavailability of lanthanum is extremely low, 
0.0007% in animals (Damment and Gill, 2003), limited retention in bone, liver and the gastrointestinal tract occurs with prolonged use (Damment and Gill, 2003; D'Haese et al., 2003). It is important, therefore, to ensure that any lanthanum retained in tissues does not present a latent genotoxic or carcinogenic hazard to patients.
Damment and colleagues found no adverse consequences of tissue-deposited lanthanum during stringent toxicological testing of lanthanum carbonate, which included evaluation for potential carcinogenicity in lifetime rodent studies (Damment et al., 2003). The published literature on the genotoxicity of this class of chemical contains contradictory results for different lanthanum salts and other rare earth metals. Two ‘rec assays’ in Bacillus subtilis on lanthanum trichloride and lanthanum trinitrate were negative (Kanematsu et al., 1980; Environmental Mutagen Information Centre, 1981), as were more standard bacterial reversion assays (Ames tests) on lanthanum trichloride using either Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 (Zeiger et al., 1992) or Escherichia coli WP2 uvrA (Seo and Lee, 1993), indicating that lanthanum, in various salt forms, is not a bacterial mutagen. Lanthanum nitrate was reported to cause increases in micronucleus frequency, single-strand DNA breaks and unscheduled DNA synthesis (UDS) in cultured human lymphocytes, however, these effects were seen only at cytotoxic concentrations and, hence, are unlikely to be relevant (Yongxing et al., 2000). In vivo lanthanum chloride has been reported to induce chromosome damage in the bone marrow of mice after chronic administration (Das et al., 1988), but only limited experimental details were provided. Conversely, lanthanum carbonate itself has been reported to possess antimutagenic properties both in vitro (in UDS and chromosome aberration assays) and in vivo in the mouse micronucleus test (Zhang and Zhang, 1997).
It has been reported that lanthanides are able to bind to DNA, RNA and nucleotides in vitro (Evans, 1990). It is possible that the chemical can gain entry into cells as small, colloidal particles via phagocytosis or pinocytosis and may also enter dead, dying or damaged cells (Squier and Rooney, 1976). Intracellular deposits have been observed following extended incubation of cells with lanthanum ions in vitro, but were ascribed to a time-dependent loss of cellular integrity. The prevailing evidence from extensive electron microscopy studies indicates that lanthanides are unable to penetrate the plasmalemmae of healthy, intact cells (Evans, 1990) and, therefore, are unable to interact directly with DNA in vivo.
To confirm this presumption, to clarify contradictory results from published genotoxicity studies and to provide robust evaluation of safety prior to medicinal use a thorough evaluation of the genotoxic potential of lanthanum carbonate was carried out. The battery of genotoxicity tests performed exceeded the current recommendations of the International Conference on Harmonization (1995a,b) for new medicinal products.
Lanthanum carbonate was subjected to a range of in vitro tests and an oral in vivo genotoxicity assay (mouse bone marrow micronucleus test). To enhance the robustness of the evaluation, two in vivo genotoxicity assays were also performed on lanthanum as the more soluble chloride salt, using the i.v. route of administration either as a single dose (rat bone marrow micronucleus test) or after dosing for 28 days (in vitro/in vivo rat liver UDS assay). As lanthanum is poorly absorbed from the gastrointestinal tract, use of the i.v. route resulted in much higher systemic exposure levels than could be achieved in the oral study, thereby enhancing the sensitivity of the assays to identify potential genotoxic effects. Use of a multiple dose regimen in the UDS assay maximized deposition of lanthanum in the target tissue for this assay, the liver, further enhancing the likelihood of detecting any inherent genotoxic properties of the drug.
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Materials and methods |
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Chemicals
Lanthanum carbonate (purity 100.5% w/w) and lanthanum chloride (purity 98.7% w/w) were supplied by Pharmaceutical Development Department, Shire Pharmaceutical Development Ltd and Fisher Scientific (Leicestershire, UK), respectively. For the in vitro assays stock solutions were prepared in sterile distilled water just prior to use. For the in vivo assays lanthanum carbonate was formulated in 0.5% w/v carboxymethylcellulose for oral administration, and lanthanum chloride was formulated as a 10% w/v aqueous stock solution in 0.9% w/v sodium chloride (physiological saline) for i.v. administration. The stock was diluted to deliver the required dose in a volume of 5 ml/kg. Sodium azide, mitomycin C, ethyl methane sulphonate and cyclophosphamide were supplied by Sigma Chemicals; 9-aminoacridine, 2-nitrofluorene, benzo[a]pyrene, cumene hydroperoxide, 2-acetylaminofluorene, 4-nitroquinoline-N-oxide and 2-aminoanthracene were supplied by Aldrich Chemical Co. Ltd.
Animals
The mice were supplied by Charles River (UK) Ltd (Margate, Kent). Male and female CD1 mice, aged 8 weeks, were used in the oral micronucleus test. The rats were also supplied by Charles River (UK) Ltd. In the i.v. micronucleus test male Sprague–Dawley Crl:CD® (SD) IGS BR rats were used, aged 7 weeks and weighing 236–296 g. In the liver UDS assay male Han Wistar Crl:WI (Glx/BRL/Han) IGS BR rats were used, aged 7–8 weeks and weighing 189–227 g.
Animal welfare practices and experimental details complied with UK national laws and guidelines, in particular the Animals (Scientific Procedures) Act, 1986. All protocols were reviewed and approved by a local ethics review committee.
Preparation of in vitro metabolizing system (S9 mix)
All of the in vitro assays were carried out in the presence and absence of S9 mix. The post-mitochondrial fraction (S9) was derived from the livers of male Fischer 344 rats that had been pretreated with a combination of sodium phenobarbitone (administered i.p. at 40 mg/kg) and ß-naphthoflavone (administered i.p. at 100 mg/kg) for 3 consecutive days prior to killing. The S9 fraction was prepared essentially as described by Maron and Ames (1983) and was used in conjunction with a NADPH-generating system.
Reverse mutation tests in bacterial cells (Ames test)
This procedure was carried out essentially as described by Ames et al. (1975) and Maron and Ames (1983) using S.typhimurium strains TA1535, TA1537, TA1538, TA98, TA100 and TA102 and E.coli strains WP2 uvrA and WP2 uvrA (pkm101). This strain selection more than complies with the most recently published recommendations (Gatehouse et al., 1994; International Conference on Harmonization, 1997). Treatments were performed in the presence and absence of metabolic activation in two independent experiments. Three replicate plates per dose of lanthanum carbonate were used in each part of the study and five doses were evaluated within each experiment. The highest concentration tested in each case was 5000 µg/plate, which is the maximum required by regulatory guidelines. Treatment plates were incubated at 37°C for 72 h before scoring for histidine-independent colonies using an automatic colony counter. Statistical analysis was carried out using Dunnett's statistic (Dunnett and Goldsmith, 1981). The positive control data were not included in the analysis.
Gene mutation assay at the hgprt locus in Chinese hamster ovary cells
Lanthanum carbonate was evaluated for its ability to induce hgprt gene mutations in Chinese hamster ovary (CHO) cells essentially as described by OECD Guideline 476 (OECD, 1997). This gene locus enables the detection of point mutations and intragenic deletions in mammalian cells. Two independent experiments were performed and four concentrations of drug were evaluated on each occasion up to a maximum concentration of 5000 µg/ml. Treatments were for 3 h in the presence and absence of metabolic activation by rat liver S9 fraction. Duplicate cultures were used for each treatment. Mutations at the hgprt locus were assessed by plating cells in medium containing 6-thioguanine. After plating, cells were incubated in a humidified atmosphere (at 37°C and 5% CO2) for up to 7 days. Colonies were counted after fixation and staining and the mutation frequency per survivor was determined. The significance of any changes in mutation frequency compared with the control was determined using the statistical methods described in Statistical Evaluation of Mutagenicity Test Data (Kirkland, 1989).
In vitro cytogenetics assay using CHO cells
To evaluate the potential of lanthanum carbonate to damage chromosomes, an in vitro cytogenetics assay was performed in CHO cells according to the recommendations of the United Kingdom Environmental Mutagen Society (UKEMS) Guidelines Committee (Scott et al., 1983). Three independent experiments were carried out in the absence of rat liver S9 fraction and two experiments in the presence of S9 fraction. In each case treatment was for 24 h up to a maximum concentration of 550 µg/ml in the absence of S9 and for 3 h up to a maximum concentration of 5000 µg/ml in the presence of S9. Duplicate cultures were used for each treatment. Cells were harvested 24 h (1.5 cell cycles) or 48 h after treatment initiation and up to 200 metaphases (100 from each of two duplicate cultures) were analysed for chromosome damage (400 cells analysed for the solvent control cultures). Aberrations were classified according to Savage (1976) into chromosome and chromatid type damage, with further subdivision into deletions and exchanges. Prior to chromosomal analysis the mitotic index (MI) for each culture was determined from a total of 1000 cells. The maximum test concentrations of lanthanum carbonate caused a 51% reduction in MI (at 550 µg/ml) in the absence of S9 mix and a 65% reduction in MI (at 5000 µg/ml) in the presence of S9 mix. Any increase in aberration frequency was compared with the negative controls using Fishers exact test (Armitage, 1971).
In vivo micronucleus assay in mouse bone marrow after oral administration of lanthanum carbonate
Lanthanum carbonate was administered to groups of five male and five female CD1 mice by oral gavage on a single occasion at doses of 800, 1250 and 2000 mg/kg. The highest dose is the maximum recommended in current guidelines for a non-toxic drug (Hayashi et al., 1994; OECD, 1997). Vehicle control groups received carboxymethylcellulose at an oral dose of 0.5% w/v and positive control groups received an i.p. dose of mitomycin C at 4 mg/kg. Groups of animals were killed 24, 48 and 72 h after treatment and bone marrow smears were prepared. The use of three sampling times is based on older recommendations (OECD, 1993). After staining, the incidence of micronuclei was scored in polychromatic erythrocytes (2000/mouse) and the ratio of polychromatic to normochromatic cells was also determined from a sample of 1000 total cells. Statistical analysis was performed using the statistical methods described in Statistical Evaluation of Mutagenicity Test Data (Kirkland, 1989).
In vivo micronucleus assay in rat bone marrow after i.v. administration of lanthanum chloride
Lanthanum chloride was administered to groups of six male Sprague–Dawley rats by a single i.v. administration at doses of 0.025, 0.05 or 0.1 mg/kg. The highest dose chosen for this study (0.1 mg/kg) was the maximum associated with linear pharmacokinetics in other experiments. Higher doses result in supraproportional plasma exposures that are unrepresentative of the exposure encountered in clinical use of orally administered lanthanum carbonate. Vehicle control groups received 0.9% w/v sodium chloride (physiological saline) i.v. at a dose volume of 5 ml/kg and positive control groups received an oral dose of cyclophosphamide at 20 mg/kg. Groups of animals were killed 24 and 48 h after treatment and bone marrow smears were prepared. The incidence of micronuclei was scored in polychromatic erythrocytes (2000/rat) and the ratio of polychromatic to normochromatic cells was also determined from a sample of 1000 total cells. Statistical analysis was performed using the statistical methods described in Statistical Evaluation of Mutagenicity Test Data (Kirkland, 1989).
In addition, satellite groups of five animals were given a single i.v. dose of each concentration of lanthanum chloride (and vehicle control), blood was taken 2, 15 and 60 min after administration and the plasma lanthanum concentration analysed using inductively coupled plasma mass spectrometry (ICP-MS).
In vivo assay for unscheduled DNA synthesis in rat liver after i.v. administration of lanthanum chloride
Examination of hepatocytes for DNA repair can provide a means of studying carcinogen–DNA adducts indirectly and thus provides a useful in vivo test. This is the principle behind the in vivo/in vitro rat liver assay for UDS (Kennelly et al., 1993), which was performed according to OECD Guideline 486 (OECD, 1997).
In this study lanthanum chloride was administered to groups of three male Han Wistar rats as 28 daily i.v. doses of 0.025, 0.05 or 0.1 mg/kg. This regimen ensured exaggerated exposure of the liver to lanthanum and hence stringent evaluation of its potential to induce genetic damage. Vehicle control groups received 0.9% w/v sodium chloride (physiological saline) i.v. at a dose volume of 5 ml/kg and positive control groups received an oral dose of 2-acetylaminofluorene at 75 mg/kg. On day 28 livers were perfused with collagenase 12–14 h after the last dose, hepatocytes were isolated and, after exposure to [3H]thymidine, the measurement of UDS was carried out by autoradiographic analysis. Three slides were prepared from each animal and, where possible, 100 morphologically normal cells were analysed from at least two slides using an image analysis system. For each slide the number of cells analysed was recorded and for each cell the nuclear area, nuclear grain count and cytoplasmic grain counts were measured. Using these data, the net grain (nuclear grain count – cytoplasmic grain count) and percentage of cells in repair (net grain 
5) were calculated. The mean and standard deviation of the nuclear area, nuclear and cytoplasmic grain counts and net grain were then calculated for each animal and the mean and standard error calculated for each group. The criteria for a positive response followed the recommendations of the UKEMS guidelines (Kennelly et al., 1993). Briefly, an animal or group is considered positive (in repair) when the mean net grain is 0 and the percentage of cells in repair is 20%.
In addition, satellite groups of five animals were dosed for 28 days with each concentration of lanthanum chloride in order to provide plasma and liver samples for lanthanum analysis using ICP-MS.
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Results |
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In vitro genotoxicity assays
In the reverse mutation assay (Ames test) using bacterial cells there were no increases in revertant numbers as a result of lanthanum carbonate treatment for any of the S.typhimurium strains or for E.coli WP2 uvrA in either the absence or presence of S9 mix (Tables I and II). In the case of E.coli WP2 uvrA (pkm101), small but statistically significant increases in revertant numbers were observed in the first experiment after treatment with drug at 500 and 2500 µg/plate in the absence and presence of S9 mix. The increases were <2-fold, did not appear to be dose-related and all revertant numbers were within the historical control ranges for this bacterial strain within this laboratory. In addition, the increases were not reproducible in a second experiment in the absence and presence of S9 mix. It is also noteworthy that uniformly negative results were obtained with S.typhimurium TA102. This strain is known to have a very similar sensitivity and reversion profile to E.coli WP2 uvrA (pkm101). For these reasons it was concluded that lanthanum carbonate was non-mutagenic under the conditions of this test.
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In the in vitro cytogenetics assay using CHO cells there were chemically induced, statistically significant increases in the number of aberrant cells at several concentrations in the absence and presence of S9 mix (Tables III and IV). These effects, although small and not dose-related, were shown to be reproducible in several experiments. In the first experiment treatment with lanthanum was associated with statistically significant increases in the number of aberrant cells at several concentrations in the absence of S9 mix and at the highest test concentration in the presence of S9 mix. Increases were obtained whether gaps were included or not in the calculation of total aberration frequencies. In the second experiment statistically significant increases were again observed, but only at the highest concentration (500 µg/ml) in the absence of S9 mix and at 2500 and 5000 µg/ml in the presence of S9 mix. In this experiment a second, later harvest time was included and a statistically significant increase in aberrant cell frequency was observed at 5000 µg/ml in the presence of S9 mix. Lastly, in the third experiment statistically significant increases in the frequency of aberrant cells (including and excluding gaps) were obtained at test concentrations of 400, 450 and 550 µg/ml in the absence of S9 mix. However, the majority of these increases fell within the historical control range for this cell line (0–6%) and were only apparent at test concentrations where toxicity was on the borderline of the acceptable range for this experimental design (i.e.
50% reduction in MI relative to concurrent vehicle controls) (Figure 1). There was also considerable precipitation of the test material at these concentrations. Consequently, the results from this assay were judged to be equivocal and probably the result of cell toxicity or the confounding effects of precipitation.
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In the hgprt gene mutation assay using CHO cells adequate toxicity (i.e. between 80 and 90% reduction in survival in terms of colony-forming ability) was achieved in the first experiment performed in the absence of S9 mix. Although the same dose range was used in a second experiment in the absence of S9 mix, the drug failed to induce the same degree of toxicity on this occasion (i.e. only 38% reduction in survival was achieved). In the presence of S9 mix there was little or no toxicity at the highest dose of 5000 µg/ml for either experiment. However, in this case 5000 µg/ml is the maximum dose recommended for non-toxic compounds in this assay (OECD, 1997
). There were no significant effects on mutation frequency in the presence of S9 mix. In the absence of S9 mix increases in mutation frequency were obtained at isolated concentrations in the first experiment (Table V). However, there was no obvious dose–response relationship and the effects were not reproducible in a second experiment, indicating that they had arisen by chance and do not indicate a mutagenic effect due to lanthanum carbonate. It is known that this assay can be subject to statistical insensitivity and inherent variability when insufficient cells are carried through the expression period or plated for mutation selection.
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In vivo genotoxicity assays
In the mouse bone marrow micronucleus assay after oral administration of lanthanum carbonate all treatment and vehicle control groups exhibited normal frequencies of micronucleated polychromatic erythrocytes (MNPCE), with the exception of the vehicle control group (male mice) sampled at 48 h (Table VI). In this group there was a complete absence of micronuclei for all of the five animals analysed. This resulted in statistically significant differences being recorded for the mean MNPCE in the male groups sampled at 48 h after doses of 1250 and 2000 mg/kg. For these two groups the MNPCE frequencies fell well within the historical control range for this strain of mouse. Consequently, this result was not considered to be biologically relevant and was the result of the unusually low micronucleus frequency recorded for the control animals at this sample time.
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It can be concluded that lanthanum carbonate does not induce micronuclei in mouse bone marrow after administration of doses up to the maximum practicable oral dose of 2000 mg/kg.
In the rat bone marrow micronucleus assay after i.v. administration of lanthanum chloride all treatment and vehicle control groups exhibited normal frequencies of MNPCE. It can be concluded that lanthanum chloride does not induce micronuclei in rat bone marrow after i.v. administration of doses up to 0.1 mg/kg (Table VII). It is noteworthy that the mean peak plasma lanthanum concentration (Cmax) at this dose was 2093 ± 106 ng/ml (Figure 2), which is >2000-fold higher than the steady state human plasma Cmax in dialysis patients receiving an oral dose of 3 g elemental lanthanum (as lanthanum carbonate).
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1 ng/ml.In the rat liver UDS assay animals treated daily for 28 consecutive days with i.v. bolus injections of lanthanum chloride at doses up to 0.1 mg/kg did not show any indication of UDS in hepatocytes isolated 12–14 h after the last dose (Table VIII). The mean Cmax for lanthanum was 3539 ± 333 ng/ml (Figure 2) and mean liver concentration 34.0 ± 3.7 µg/g wet weight, indicating that substantial systemic and target organ exposure was achieved during the study.
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Discussion |
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Lanthanum carbonate did not induce gene mutation in bacteria or at the hgprt locus in mammalian (CHO) cells. However, small but statistically significant and reproducible increases in chromosome damage were seen in vitro in the presence and absence of S9 mix in mammalian (CHO) cells. The magnitude of the effect was most pronounced in the absence of S9 mix. However, the majority of the increases fell within the historical control range for this cell line (0–6%) and were only apparent at test concentrations where toxicity was on the borderline of the acceptable range for this experimental design (i.e.
50% reduction in MI relative to concurrent vehicle controls). It is known that a number of indirect mechanisms can result in genotoxic artefacts in vitro, due to the use of high (non-physiological) concentrations of the chemical (Scott et al., 1991; Kirkland and Müller, 2000; Müller and Kasper, 2000). Lanthanum carbonate has a very low aqueous solubility and, although precipitation was noted only at the highest doses of the drug, this was a potential confounding factor in the interpretation of all the in vitro studies. It is of interest also that small increases in chromosomal aberrations in an in vitro mammalian cytogenetics assay have been reported for another phosphate binder, sevelamer (Renagel®, US Data Sheet), which may suggest class-related artefacts linked to a phosphate-trapping action. In the present investigation, however, it was not possible to conclude that the observed effects were entirely the result of artefact or overt cell toxicity.
Therefore, a detailed investigation of the potential in vivo genotoxicity of lanthanum was carried out using both the carbonate and chloride salts. In the latter case, i.v. micronucleus and UDS assays were performed to generate high levels of exposure to lanthanum in tissues (bone marrow and liver, respectively) and thus provide a more robust evaluation of any potential effects. Furthermore, as small amounts of absorbed lanthanum can be retained in bone and liver for prolonged periods (Damment and Gill, 2003; D'Haese et al., 2003), the liver UDS assay was performed using a multiple dose regimen involving daily administration of lanthanum for 28 consecutive days. This maximized exposure of the liver and the sensitivity to detect any potential for tissue-deposited lanthanum to cause genetic damage.
Uniformly negative results were obtained with both salts in these assays (mouse and rat bone marrow micronucleus tests and rat liver UDS assay). In the case of the i.v. studies the negative results were obtained after exposure to plasma lanthanum concentrations representing a 3000-fold multiple of the steady state Cmax measured in renal dialysis patients receiving therapeutic doses of Fosrenol® (doses up to 3 g/day, Cmax 1.06 ± 1.04 ng/ml) (Damment and Gill, 2003).
Lanthanum carbonate has been evaluated for its carcinogenic potential in lifetime studies in mice and rats and found to be negative at doses up to 13 times the human dose of 3 g/day on a mg/kg dose comparison (Damment et al., 2003). The mean liver lanthanum concentration in the UDS assay of 34.0 ± 3.7 µg/g wet weight was substantially higher than reported in the lifetime rodent studies (Damment and Gill, 2003), due to the i.v. route of administration. The absence of a carcinogenic response in rodent studies and of genetic toxicity at this high liver concentration suggests that tissue-deposited lanthanum does not present a latent hazard for patients taking therapeutic doses of the drug.
It can be concluded that lanthanum is not an in vivo genotoxin and that equivocal effects obtained in an in vitro cytogenetics assay using CHO cells have no biological relevance to the intended therapeutic use of lanthanum carbonate. These results provide further non-clinical evidence that lanthanum carbonate is safe for its intended use and are consistent with the good safety profile emerging from long-term studies in dialysis patients (D'Haese et al., 2003; Behets et al., 2004).
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Notes |
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* To whom correspondence should be addressed. Tel: +44 1256 894194; Fax: +44 1256 894703; Email: sdamment{at}uk.shire.com
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Received on October 14, 2004; accepted on November 19, 2004.
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