Increase in thyroid follicular cell tumors in nelfinavir-treated rats observed in a 2-year carcinogenicity study is consistent with a rat-specific mechanism of thyroid neoplasia.

The carcinogenic potential of nelfinavir mesylate (nelfinavir) was evaluated in a 2-year oral (gavage) study on Sprague-Dawley rats at dose levels of 0 (control), 0 (vehicle control), 100, 300 and 1000 mg/kg per day. At the end of the treatment, increased incidences of thyroid follicular cell hyperplasia and neoplasms were observed at 300 (males) and 1000 mg/kg per day (both sexes). There were no other treatment-related effects and no tumors at other sites. Results from previous studies indicated a number of effects in the liver and thyroid, as well as metabolic profiles that suggested nelfinavir might cause thyroid hyperplasia/neoplasia secondary to hormone imbalance by altering thyroid hormone disposition. To investigate this hypothesis, the effects of nelfinavir on gene expression in rat hepatocytes and liver slices (in vitro), thyroxine plasma clearance, and thyroid gland function were evaluated. Compared to controls, gene expression analyses demonstrated an increased expression of glucuronyltransferase (UDPGT) and CYP450 3A1 in nelfinavir-treated rat hepatocytes and liver slices. In rats treated with nelfinavir (1000 mg/kg per day) for 4 weeks, liver weights and centrilobular hepatocellular hypertrophy were increased and minimal to mild diffuse thyroid follicular cell hypertrophy and follicular cell hyperplasia were evident in the thyroid gland. Thyroid-stimulating hormone (TSH) levels were significantly increased (three-fold), while tri-iodothyronine (T3)/tetra-iodothyronine (T4) and reverse T3(rT3) levels were unchanged, indicating that a compensated state to maintain homeostasis of T3/T4 had been achieved. Plasma 125I-thyroxine clearance was increased and the plasma thyroxine AUC0-48 was decreased (24%) compared to control. In conclusion, these data indicate that thyroid neoplasms observed in the nelfinavir-treated rats were secondary to thyroid hormone imbalance. Increased thyroxine clearance contributes to the effects of nelfinavir on thyroid gland function and is probably a result of UDPGT induction that leads to elevated TSH levels in the rat and eventual thyroid neoplasia. These results are consistent with a well-recognized rat-specific mechanism for thyroid neoplasms.

NMR of biofluids and pattern recognition: assessing the impact of NMR parameters on the principal component analysis of urine from rat and mouse.

The ability to interpret metabolic responses to toxic insult as expressed in altered urine composition and measured by NMR spectroscopy is dependent upon a database of proton NMR spectra of urine collected from both control and treated animals. Pattern recognition techniques, such as principal component analysis (PCA), can be used to establish whether the spectral data cluster according to a dose response. However, PCA will be sensitive to other variables that might exist in the data, such as those arising from the NMR instrument itself. Thus, studies were conducted to determine the impact that NMR-related variables might impart on the data, with a view towards understanding and minimizing variables that could interfere with the interpretation of a biological effect. This study has focused on solvent suppression methods, as well as instrument-to-instrument variability, including field strength. The magnitude of the NMR-induced variability was assessed in the presence of an established response to the nephrotoxin bromoethanamine. Changes caused by the model toxin were larger and easily distinguished from those caused by using different solvent suppression methods and field strengths.

In vitro photogenotoxic activity of clinafloxacin: a paradigm predicting photocarcinogenicity.

Fluoroquinolone antiinfective drugs exhibit phototoxic, photogenotoxic, and photocarcinogenic activities in experimental systems which may be interrelated. Clinafloxacin (CLX), a new fluoroquinolone, is a potent antiinfective agent being developed for use in life-threatening infections. While this drug has previously been demonstrated to be phototoxic, this report evaluated the photogenotoxic and photocarcinogenic potential of CLX. When Skh-1 mice were administered CLX in the presence of ultraviolet light (UVA) at the maximum tolerated dose expected for a photocarcinogenicity bioassay, induction of DNA strand breakage was noted in keratinocytes isolated from these animals. When compared with other well-studied fluoroquinolones in vitro, CLX and Lomefloxacin (LMX) were equally effective in producing chromosome damage and DNA strand breakage in Chinese hamster ovary (CHO) cells exposed to UVA. Treatment of CHO cells with CLX in the presence of UVA also resulted in hydroxyl radical formation. However, coincubation of CHO cells with CLX and various antioxidants markedly reduced hydroxyl radical formation, but inhibited photogenotoxicity only to a limited extent. Thus, while reactive oxygen species contribute to the photogenotoxic activity of CLX, other factors may be involved. Since CLX exhibits both phototoxic and photogenotoxic activity, we predict that CLX would be photocarcinogenic in vivo. The present study suggests that under conditions of human exposure, the potential risk for CLX-induced photocarcinogenicity is small.

Principles and practices of integrating genotoxicity evaluation into routine toxicology studies: a pharmaceutical industry perspective.

In this article, an integrated in vivo genotoxicity testing philosophy and a practical approach, as applied to pharmaceuticals, are described. Recently, there has been an effort to integrate the rodent (primarily rat) micronucleus assay with routine 2-4-week toxicokinetic studies. This approach has several advantages: 1) it utilizes the general principles of toxicology that govern the overall toxicity profile of a test substance; 2) factors such as the dose and/or route of drug administration, drug metabolism, principles of toxicokinetics, and saturation of defense mechanisms are considered in evaluating genotoxicity; 3) it uses the concept of administering multiple tolerable doses aiding in achieving steady state plasma drug levels, which is more relevant for risk assessment compared to high acute doses; and 4) it helps minimize the amount of drug, number of animals used, and other resources. This integration approach can be extended to other toxicology studies and other relevant genotoxicity endpoints may be assessed. Based on the experience in our laboratory, integrating micronucleus assessment in routine toxicology testing is promising and should be utilized when practical.

Use of acridine orange in: flow cytometric evaluation of erythropoietic cytotoxicity.

Cytotoxic insult to bone marrow frequently impairs the proliferating and maturational abilities of erythroid cells. Typically, a ratio of enucleated, immature polychromatic erythrocytes (PCE) to mature normochromatic erythrocytes (NCE) is used to assess cytotoxicity in the micronucleus (MN) assay. The effects of cyclophosphamide (CP) on PCE/NCE ratio in rat bone marrow and spleen were assessed by a newly developed flow cytometric procedure using glutaraldehyde-fixed, acridine orange (AO)-stained cells, and compared to manual scoring of PCE/NCE in Wright stained slides. Comparison of methods showed that manual and flow cytometric determination of PCE were not statistically different. Several other parameters of cytotoxicity could be simultaneously assessed because the method allowed use of unfractionated whole bone marrow/spleen cell samples. Absolute numbers of total nucleated cells (TNC), a ratio of TNC to total erythrocytes (TE), and determination of RNA content within the PCE population demonstrated dose- and time-dependent effects with CP treatment. Shifts in RNA content were particularly sensitive, correctly identifying all CP-treated from control specimens, even in those samples where PCE/NCE ratio was similar. The AO methodology provided a more rapid, statistically-superior, and thorough approach in the assessment of bone marrow and spleen cytotoxicity than the conventional manual method of scoring PCE/NCE ratio alone.

Use of acridine orange in: flow cytometric assessment of micronuclei induction.

The micronucleus assay is a widely accepted method for evaluation of clastogens and aneugens. In the current study, acridine orange (AO) supravital staining was adapted for flow cytometric usage to assess micronucleated cells in rat bone marrow and spleen. Cyclophosphamide was used as a positive control test compound and results were compared to manual scoring in Wright-stained slides. In bone marrow, both manual and flow cytometric methods demonstrated positive dose response-trends for micronucleated polychromatic erythrocytes (MNPCE). Significant elevations in MNPCE were observed at all doses of cyclophosphamide, and comparisons between methods in bone marrow were not statistically different. The flow cytometric method was more sensitive in spleen samples, showing dose- and time-related increases in micronuclei compared with manual scoring. AO proved to be a sensitive discriminator of RNA and DNA, allowing distinct separation of polychromatic erythrocytes (PCE), normochromic erythrocytes (NCE), total nucleated cells (TNC), and micronucleated populations within both PCE and NCE regions. These results support the use of AO-based flow cytometry to provide a rapid and sensitive indicator of micronuclei inducers.

Functional and subcellular organelle changes in isolated rat and human hepatocytes induced by tetrahydroaminoacridine.

Tacrine (tetrahydroaminoacridine) is a reversible cholinesterase inhibitor used for the treatment of Alzheimer's disease. This drug causes an elevation of serum aminotransferases in a limited population of patients. Several in vivo studies failed to elucidate the mechanism for the enzyme elevation but previous in vitro studies have indicated defects in mitochondrial function. In this study, electron microscopic, histochemical, and confocal microscopy techniques were used with primary hepatocyte cultures from humans and rats to examine the sequence of early cellular changes after tacrine exposure. Changes included ribosome alterations as early as 1-2 h following tacrine exposure at concentrations ranging between 0.1 and 1.0 mM. Mitochondrial membrane potential was also altered as indicated by decreased rhodamine 123 uptake with time. Cellular lysosome content increased as indicated by increased staining of fluorescein isothiocyanate (FITC)-conjugated dextran. The results of acid phosphatase histochemistry correlated with the FITC-dextran findings. Additionally, tacrine-related degranulation and vesiculation of the endoplasmic reticulum paralleled the ribosomal and mitochondrial changes. These subcellular changes were reproducible in rat and human hepatocytes, showing for the first time that human hepatocytes can be altered by tacrine. The molecular mechanism of the organelle changes is unknown at this time. Also, the relationship between these subcellular changes in isolated hepatocytes and the transaminase elevation noted in human populations treated with tacrine needs to be clarified.

Preclinical toxicology studies with the angiotensin-converting enzyme inhibitor quinapril hydrochloride (Accupril).

Acute, subacute, and chronic toxicity studies, carcinogenicity bioassays, and reproductive and genetic toxicology studies were performed with quinapril, an ACE inhibitor used in the treatment of hypertension. Acute toxicity is minimal in rodents, and repeated dosing elicits gastric irritation, juxtaglomerular apparatus (JGA) hypertrophy and hyperplasia and tubular degenerative changes in the kidney, and reduced red cell parameters and heart weights in rodents and/or dogs. Other manifestations of toxicity, including hepatic lesions in dogs, reduced offspring weights in rats, marked sensitivity of the rabbit, and clastogenic effects at cytotoxic doses in the in vitro V79 chromosome aberration assay, have been reported with other drugs of this class.

In vitro induction of micronuclei and chromosome aberrations by quinolones: possible mechanisms.

The bacterial gyrase inhibitors, ciprofloxacin and PD 124816, were tested for clastogenic and aneugenic activity in V79 Chinese hamster lung cells in vitro. Cells were exposed for 3 h, washed free of drug, and subcultured for assessment of various endpoints. For structural chromosomal aberration (SCA) analysis, cells were incubated for 18 h, and treated with Colcemid for 2 h before harvest. For micronucleus (MN) analysis, treated cells were incubated with cytochalasin B (CYB) for 16 h. Aneugenicity was assessed by utilizing antikinetochore antibody to detect kinetochore-containing (K +) MN. Both quinolones induced significant increases in SCAs and MN, indicating clastogenic activity. With both compounds, however, the MN response was apparent at lower doses, and remained much higher throughout the dose range than the SCA response. The induced MN were predominantly K --, indicating that aneugenicity was not playing a major role in their induction. A possible explanation for the chromosome effects is that cross-reactivity of the gyrase inhibitors with mammalian topoisomerase II interferes with the separation of chromatids at anaphase leading to chromosome breaks and MN. Quinolones are known to inhibit resolution of the normally transient topoisomerase II-DNA cleavable complex, which may result in chromosome stickness. Thus, SCAs detected in metaphase cells may be attributed to quinolone-induced inhibition of topoisomerase II prior to mitosis while MN arise in binucleated cells as a result of this effect which interferes with chromatid separation during anaphase.

Comparison of tacrine-induced cytotoxicity in primary cultures of rat, mouse, monkey, dog, rabbit, and human hepatocytes.

Tacrine is the first drug approved for the treatment of Alzheimer's disease. Approximately 50% of patients treated with tacrine develop elevated serum aminotransferase levels, an indication of potential hepatotoxicity. The mechanism of human hepatoxicity has been difficult to study, because of the absence of an animal model. Therefore, this study compared the cytotoxicity induced by tacrine in primary rat, mouse, monkey, dog, rabbit and human hepatocytes to determine differences in response to tacrine between species in vitro. Cytotoxicity was assessed by determination of extra- and intracellular lactate dehydrogenase. The ratio of intracellular enzyme to total enzyme (i.e. intracellular and extracellular) was used to represent the viabilities of the cultures. Concentration-dependent cytotoxicity occurred after four and 24-hour exposure over a tacrine concentration range of 0 to 380 micrograms/ml. Cytotoxic potency of tacrine in hepatocytes from human, dog, mouse and rat was not significantly different; monkey hepatocytes appeared slightly more sensitive, while rabbit hepatocytes appeared slightly less sensitive than human hepatocytes. Increased time of exposure to tacrine decreased the concentration necessary to induce a cytotoxic response. This in vitro model suggests only minimal differences in sensitivity to tacrine-induce cytotoxicity; therefore, cytotoxicity in primary cultures of hepatocytes from various species would appear to be related to common metabolite(s) and/or mechanism of cellular injury.

Cytotoxicity study of tacrine, structurally and pharmacologically related compounds using rat hepatocytes.

Tacrine is the first drug approved for the treatment of Alzheimer's disease. Approximately 50% of patients treated with tacrine develop elevated serum aminotransferase levels, as an indication of potential hepatotoxicity. However, acute and chronic studies with a limited number of animal models have not demonstrated hepatotoxicity. The present study compared the cytotoxicity in hepatocyte cultures of tacrine with structurally (proflavine and 9-aminoacridine) or pharmacologically similar compounds (physostigmine), as well as structurally modified tacrine to determine if there was a structure activity relationship with regards to toxicity. Cytotoxicity was assessed by determination of extra- and intracellular amounts of lactate dehydrogenase. Cytotoxicity was assessed after a four-hour exposure over a test compound concentration range of 0 to 3 mM. Concentration-dependent cytotoxicity occurred with tacrine and all structurally related compounds. Physostigmine which is pharmacologically similar, but structurally different, did not induce cytotoxicity. Cytotoxic potency did not appear to be related to acetylcholinesterase inhibitory activity, while compounds with acridine structures induced cytotoxicity. Thus, in this in vitro model, cytotoxicity appears to be related to structure and not pharmacological action. Results of this study indicate that compounds structurally related to tacrine are cytotoxic because of the heterocyclic ring structure. Neither unsaturation of an aromatic ring of the heterocyclic compound, amino substitution of the heterocyclic rings, N-hydroxylation of the amino group, nor ring hydroxylation dramatically alter cytotoxicity.

Genotoxicity evaluation of selenium sulfide in in vivo and in vivo/in vitro micronucleus and chromosome aberration assays.

Selenium monosulfide (SeS) was reported to be carcinogenic to livers of male and female rats and livers and lungs of female mice. However, its genotoxicity profile in short-term assays is somewhat equivocal. A multiple endpoint/multiple tissue approach to short-term genetic toxicity testing has been developed in our laboratory. In the present paper, the effect of SeS in in vivo and in vivo/in vitro micronucleus and chromosome aberration assays in rat bone marrow and spleen are reported. In the in vivo assay, small but statistically significant increases in bone marrow micronucleated polychromatic erythrocytes (MNPCEs) were observed 24 h after treatment of rats with 50 mg/kg SeS and 48 h after treatment with 12.5 mg/kg. A significant decrease in the PCE/total erythrocyte (TE) ratio, indicative of cytotoxicity, was observed at the 50 mg/kg dose at the 24-h timepoint. In spleen, no increases in MNPCEs or decreases in the PCE/TE ratios were observed. No evidence of a significant increase in aberrations was observed in bone marrow or spleen. In the in vivo/in vitro assay, no increase in micronucleated binucleated cells or cells with aberrations was observed in SeS-treated rats. The small but statistically significant increases in MN observed in the in vivo study are considered likely not to be biologically significant since no dose-response was observed and all the values obtained were within historical control range in our laboratory. Given the overall genetic toxicity profile of SeS, it appears that SeS may be a weak mutagen and that differences between testing protocols may be very important in determining whether or not it is found to be negative or positive. Histological evidence was obtained in this study that suggests that the liver is the acute target organ of SeS in rats. Given the fact that SeS is selectively hepatocarcinogenic, we are currently testing the hypothesis that the genotoxicity of SeS in rats may be more readily detectable in liver than in bone marrow or spleen.

Use of cyclophosphamide as a positive control in dominant lethal and micronucleus assays.

Analyses of dominant lethal (DL) mutations and micronuclei (MN) are 2 important and widely used genotoxicity assays to measure drug-induced chromosome damage in germ cells and somatic cells, respectively. Cyclophosphamide (CP) has been widely used as a positive control in the single-dose mouse MN assay; however, its utility as a positive control for the DL assay has not been fully studied. In the present study, CP was tested in both assays under similar experimental conditions and MN seen in somatic tissue (bone marrow) were correlated with DL mutations seen in germinal tissue. In a dose-range finding study, groups of 5 male mice were dosed i.p. daily for 5 days at 0, 30 or 40 mg/kg CP and bone marrow was harvested 24 h later for MN assay. CP induced a dose-related increase (7- and 11-fold over control at 30 and 40 mg/kg) in micronucleated polychromatic erythrocytes (MNPCEs) and decreased %PCEs (to 60% and 54% of controls at 30 and 40 mg/kg, respectively). Based on this, a definitive DL and MN study was conducted using separate groups of 30 male mice at 0 and 40 mg/kg CP with a daily times 5 dosing regimen. For the MN assay, bone marrow was collected 24 h after the last dose from 5 animals and evaluated for MNPCEs and %PCEs. For the DL assay, each male was caged with 2 untreated females per week for 8 weeks to cover the postmeiotic germ cell stages. On day 17 after the initiation of breeding, the females were evaluated for the number of implantation sites and live, dead and resorbed implants. The results indicated that CP induced about a 17-fold increase in MNPCEs and a 46% decrease in PCEs in relation to controls. In the DL assay, CP produced a slight (13%) but statistically significant reduction in fertility index at week 7 of mating. Also, the total number of implants was significantly lower during weeks 1, 2, 3, 6 and 7 and the numbers of dead implants and postimplantation loss (PIL) were increased for weeks 1, 2 and 3 (55%, 71% and 34% PIL, respectively) over controls. These data clearly show that CP produced clastogenicity and some toxicity in both somatic tissue and germinal tissue. It was concluded that a dose of 40 mg/kg CP can be used as a positive control compound in the DL assay and in the multiple-dose marrow MN assay.

Simultaneous evaluation of dexamethasone-induced apoptosis and micronuclei in rat primary spleen cell cultures.

Apoptosis or programmed cell death is a biological event that is biochemically and morphologically distinct from cellular necrosis. Nonetheless, its relationship has not been studied in terms of a cytogenetic endpoint such as micronucleus formation. In the present study, based on cytological observations, the incidence of dexamethasone-induced apoptotic cells was related to the frequency of micronucleated cells in vitro. Rat primary spleen cells were grown in 6-well plates with RPMI 1640 media using concanavalin A and lipopolysaccharide as mitogens. At culture initiation, the test agent dexamethasone (10, 20 or 40 microM) and a cytokinesis inhibitor cytochalasin B (3 micrograms/ml) were added. Cultures were harvested 18 h and 40 h later. Slides were prepared and stained with Diff-Quik stain. Frequencies of apoptotic cells and micronucleated binucleate cells were enumerated cytologically based on 500 cells per treatment from the same slides. The results showed a dose-dependent increase in the number of apoptotic cells in rat spleen cultures treated with dexamethasone. At 18 h, the percentages of apoptotic cells were 0.8, 1.6, 3.4 and 4.4 with 0, 10, 20 and 40 microM dexamethasone, respectively. The corresponding percentages of apoptotic cells at 40 h were: 2.8, 2.6, 5.6 and 10.4. However, at the same concentrations of dexamethasone, the micronucleus frequency in binucleate cells remained relatively unchanged. The phenomenon of apoptosis induced by dexamethasone was confirmed biochemically based on a characteristic DNA 'ladder' pattern by gel electrophoresis. These data suggest that dexamethasone at the concentrations which induced apoptosis did not produce cytogenetic damage. Also, these findings indicate that micronucleus formation and nuclear changes leading to apoptosis are separate events and these endpoints may not be closely correlated for dexamethasone.

An in vivo/in vitro method for assessing micronucleus and chromosome aberration induction in rat bone marrow and spleen. 1. Studies with cyclophosphamide.

The mouse micronucleus assay has long been used as an indicator of in vivo genotoxicity. Recently, it was shown that no single protocol is adequate to detect all clastogens. As a first step in developing a potentially more sensitive assay, micronucleus induction by cyclophosphamide (CP) was assessed in an in vivo/in vitro system using rat bone marrow and spleen cells. In each of two independent experiments, two rats/dose were treated i.p. with 0, 20, or 40 mg CP/kg and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested and slides prepared 24 h after initiation, while spleen cells were harvested at 48 h. One thousand cells/tissue/group were scored for cell cycle kinetics and 1000 binucleate (BN) cells were scored for micronuclei. In addition, spleen cells were concurrently assayed for chromosome aberrations. A dose-related cell cycle delay was observed in both tissues in both experiments. Bone marrow showed a 6% average background frequency of micronucleated BN cells, while the low dose induced an average of 20%, and the high dose 31%. For spleen, the average control frequency of micronucleated BN cells was 3%, the low dose induced a 40% average frequency, and the high dose 65%. Also in splenocytes, a dose-dependent increase in chromosome aberrations was observed, with an almost 40-fold increase observed over the control value at the high dose. Thus, the in vivo/in vitro approach described here shows great potential in detecting drug induced genotoxicity. Also, spleen appears more sensitive than bone marrow to CP.

An in vivo/in vitro method for assessing micronucleus and chromosome aberration induction in rat bone marrow and spleen. 2. Studies with chlorambucil and mitomycin C.

An in vivo/in vitro system using rat bone marrow cells and spleen cells to assess micronucleus (MN) and structural chromosome aberrations (SCA) simultaneously (Moore et al., 1995) was further developed. In two separate experiments, two rats/dose/experiment were treated i.p. with 0, 5, 10 and 15 mg chlorambucil (CA)/kg or with mitomycin C (MMC) at 0, 1, 2, 4 mg/kg (experiment 1) or 0, 4, 6, and 8 mg/kg (experiment 2) and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested 24 h after establishment of cultures, while spleen cells were harvested at 48 h. In addition, spleen cells were concurrently assayed for chromosome aberrations. With the MN endpoint, spleen cells appeared more sensitive than bone marrow cells to the effects of CA due both to a lower background and an increased response. For MMC, bone marrow cells exhibited both a higher background of MN and a greater numerical response than did spleen cells. However, on the basis of a fold-increase over control values, spleen cells were more sensitive than bone marrow cells. In general, the MN endpoint appeared more sensitive than the SCA in spleen cells after treatment with CA or MMC. Thus, the approach described here shows greater potential in detecting genotoxicity.

The genotoxicity profile of atorvastatin, a new drug in the treatment of hypercholesterolemia.

While HMG-CoA reductase inhibitors such as fluvastatin, lovastatin, pravastatin and simvastatin demonstrate lack of in vitro and in vivo mutagenicity and clastogenicity in bacterial and mammalian cells, long term rodent carcinogenicity studies resulted in an increased incidence in neoplasms at high doses. These effects may be attributable to an exaggeration of the desired biochemical effect of the drug and/or a tumor promoting effect. The genotoxicity of atorvastatin, a newly developed HMG-CoA reductase inhibitor, was evaluated in a variety of test systems. In bacterial mutagenicity tests, the E. coli tester strain WP2(uvrA) and S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 were exposed to concentrations of atorvastatin as high as 5000 micrograms/plate both in the absence (S9-) and presence (S9+) of metabolic activation. Atorvastatin was not mutagenic in either E. coli or S. typhimurium. Chinese hamster lung V79 cell cultures were exposed to atorvastatin at concentrations of 50-300 micrograms/ml (S9-) and 100-300 micrograms/ml (S9+) and structural chromosome aberrations were assessed. Mutation at the hgprt locus was assessed at concentrations of 100-300 micrograms/ml (S9-) and 150-275 micrograms/ml (S9+). Atorvastatin was neither mutagenic nor clastogenic in the absence or presence of S9. The lack of in vitro genotoxicity was corroborated in vivo in a mouse micronucleus study in which single oral doses of atorvastatin were administered to male and female CD-1 mice at 1, 2500, or 5000 mg/kg. No biologically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48, or 72 h postdosing were observed. Thus, atorvastatin, as with the other tested HMG-CoA reductase inhibitors, is not genotoxic.

Concurrent analysis of cytogenetic damage in vivo: a multiple endpoint-multiple tissue approach.

Simultaneous assessment of in vivo micronucleus and chromosome aberration endpoints has been described in the rat and the mouse. Bone marrow and spleen cells were utilized for genotoxicity assessment. A cellulose column methodology was used in the micronucleus assay (where applicable) to eliminate nucleated cells and facilitate cytogenetic scoring. The test agents--cyclophosphamide, chlorambucil, and acrylamide--produced qualitatively comparable results between micronucleus and chromosome aberration endpoints and varied slightly on a quantitative basis depending on the type of test agent and tissue studied. The results from test agents such as cyclophosphamide, chlorambucil, acrylamide, dimethylnitrosamine, vincristine, and x-rays indicated that bone marrow cytogenetic results are similar to spleen and that the spleen tissue is at least as sensitive as the bone marrow. The concurrent analysis of cytogenetic damage in vivo using a multiple endpoint-multiple tissue approach described here has the following advantages: a) reducing the overall animal usage, b) clarifying marginal micronucleus and/or chromosome aberration data, c) correlating cytogenetic results from multiple endpoints and multiple tissues, and d) helping the investigation of the mechanism of action of test agents and their potential target organs. Also, the multiple endpoint-multiple tissue approach can be extended to other endpoints, tissues, and species (where appropriate and practical) to obtain detailed genotoxicity information.

Mammalian cell gene mutation assays working group report.

As part of the International Workshop on Standardization of Genotoxicity Test Procedures, in Melbourne, 27-28 February 1993, various international guidelines were examined with respect to protocol issues in the area of mammalian cell gene mutation assays. The working group on mammalian cell gene mutation assays discussed a wide range of protocol issues related to study design; in most cases the recommendations are reasonably consistent with existing guidelines. Agreement was reached on several issues as follows. The upper limit of concentration for testing non-toxic substances should be 10 mM or 5 mg/ml, whichever is lower. For testing toxic substances the criteria of an acceptable upper limit of concentration should yield 10-20% survival. Any of several established mammalian cell mutation assays (L5178Y TK+/-, CHO/HPRT, AS52/XPRT, V79/HPRT) can be used to evaluate mutagenesis in mammalian cells; the ouabain (Na/K-ATPase) system is not an acceptable mutation assay for routine evaluation of mutagenesis in mammalian cells. Ability to recover small colonies must be convincingly demonstrated when using the L5178Y TK+/- mouse lymphoma assay. In the mouse lymphoma assay (L5178Y TK+/-), colonies in positive controls and at least two (if available) representative positive doses of the test compound should be sized if a positive response is seen; in the event of a negative response due to the test compound, colony sizing of the positive control is necessary to validate the conduct of the assay. Testing both in the presence and absence of S9 metabolic activation is necessary. It was not possible to come to a firm conclusion about the length of treatment. There was a general agreement that extended treatment times (> 2 cell cycles) often bear more disadvantages than advantages and should only be used with adequate justification. It is not necessary to repeat clear positive or clear negative tests when the assay has been adequately performed; this recommendation differs significantly from the UK guidelines. If treatment groups are not replicated, the numbers of doses tested should be increased; this recommendation differs significantly from the UK guidelines. Each laboratory should establish a historical database for the performance of a given assay in that laboratory.