Histologic and histochemical changes in articular cartilages of immature beagle dogs dosed with difloxacin, a fluoroquinolone.

The histologic and histochemical features of quinolone-induced arthropathy were studied using 14 skeletally immature Beagle dogs (3 to 4 months old) dosed orally with difloxacin at 300 mg/kg body weight once daily for 1, 2, 5, or 7 days. A placebo was given to eight other age-matched Beagle dogs that served as controls. A scoring technique that included lesion size and histologic features was used to determine the progression of lesions. Articular-epiphyseal cartilage complexes on the femoral and humeral heads and tibial tarsal bone were identified as predilection sites. Within predilection sites on femoral and humeral heads, lesions developed in specific areas. Lesions appeared within 2 days of the onset of treatment, and lesion scores increased with time. Grossly, the lesions were raised, fluid-filled vesicles on the articular surface. Histologic changes included vesicle formation with loss of proteoglycan, clumping of unmasked collagen, and degeneration and necrosis of chondrocytes. In lesions with higher scores, chondrocytes were often in clusters or they were undergoing metaplasia toward spindle-shaped cells. Although dissolution of matrix and necrosis of chondrocytes were typical of all lesions, smaller lesions had histologically normal chondrocytes adjacent to small vesicles. In sections stained with toluidine blue, proteoglycan was aggregated with collagen fibrils or was absent from the matrix adjacent to vesicles. Unique features, such as biomechanical forces, may predispose specific areas of articular cartilage to develop lesions.

One-month inhalation toxicity study of tulobuterol hydrochloride in rats and dogs.

Tulobuterol hydrochloride (HCl) has beta 2-adrenergic agonist activity and is under development for use in the treatment of chronic obstructive lung disease. The purpose of this study was to determine the toxicity of inhaled tulobuterol HCl in rats and dogs. Rats were whole-body exposed to aerosol gravimetric concentrations of 0, 0.03, 0.22, or 1.1 mg/liter of tulobuterol HCl, 60 min/day for 28 days. Dogs were exposed (via insufflation) to estimated daily doses of 0, 0.2, 1.0, or 6.0 mg/kg for an equal period. Plasma levels of tulobuterol were determined following exposure on Days 1, 8, and 28 using a high-pressure liquid chromatographic method developed for this study. Results indicated that plasma tulobuterol levels were highly correlated with tulobuterol doses (p less than 0.001 for rats and dogs). No dose-related changes in body weight food consumption, hematological, or serum chemistry parameters were observed in either species. Anterior nasal cavity lesions were observed by light microscopy in rats exposed to 0.22 and 1.1 mg/liter tulobuterol HCl at an incidence of 14 and 93%, respectively. These lesions involved the nasal septum, turbinates, and/or the dorsolateral wall of the nasal cavity and consisted of suppurative rhinitis and necrosis. The corresponding mean plasma tulobuterol levels on Day 28 in mid- and high-dose rats were approximately 1000 and 15,000 ng/ml. Nasal lesions were not observed in rats allowed to recover for 2 weeks. No gross or microscopic lesions were detected in lungs or other tissues of either species.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative vestibular toxicity study with 3-O-demethylfortimicin A disulfate and gentamicin sulfate in cats.

3-O-demethylfortimicin A disulfate (ODMF), a novel aminocyclitol antibiotic, was administered subcutaneously for three months to groups of male and female cats at 15, 30 or 60 mg base/kg/day. Gentamicin sulfate (GS) at doses of 6 and 13 mg base/kg/day served as a reference compound. Signs of vestibular toxicity were considered to include persistent unsteady gait and stance, impaired righting reflex and abnormally diminished postrotatory vestibular nystagmus. Renal toxicities produced by ODMF and GS were also determined and compared. ODMF at 15 and 30 mg base/kg/day produced no signs of vestibular toxicity, while a dosage of 60 mg base/kg/day of ODMF produced vestibular toxicity in 7/10 cats. Three affected male cats died or were killed in moribund condition between study days 49 and 64. Vestibular toxicity was observed in 10/10 cats treated with GS at 13 mg base/kg/day and in 3/10 cats at 6 mg base/kg/day. All ten cats treated with GS at 13 mg base/kg/day died or were killed in moribund condition between study days 30 and 81. The deaths and moribundity in cats treated with ODMF or GS were attributed to renal toxicity. The vestibular toxicity and nephrotoxicity produced by ODMF and GS were more severe in male cats than in females. In conclusion, ODMF given at doses up to 60 mg base/kg/day for three months induced comparatively less vestibular toxicity and renal pathology than did GS at a dose of 13 mg base/kg/day.

Toxicological aspects of cyclamate and cyclohexylamine.

In the late 1960s the artificial sweetener cyclamate was implicated as a bladder carcinogen in rats. This finding and other concerns about its safety ultimately led to a ban on cyclamate in the U.S. and restrictions on its use in many other countries. Since that time, the carcinogenic potential of cyclamate and cyclohexylamine, its principal metabolite, has been reevaluated in a group of well-controlled, well-designed bioassays that have failed to substantiate the earlier findings. This review of the published and unpublished literature on cyclamate attempts to evaluate the carcinogenicity question and other important aspects of the toxicity of cyclamate and cyclohexylamine, including their effects on various organ systems, their genotoxic potential, and their effects on reproduction. In addition, the physiological disposition of cyclamate is reviewed, with particular attention directed toward the site and extent of its conversion to cyclohexylamine.

Acute, subchronic, and chronic toxicity studies with 3-O-demethylfortimicin A disulfate, a new aminocyclitol antibiotic.

The acute LD50 for 3-O-demethylfortimicin A disulfate (ODMF) in mice and rats were 419 and 778 mg activity/kg (dosages are expressed in terms of antibiotic activity (potency), rather than on a weight basis) for single-dose im administration and, 90 and 96 mg activity/kg for single-dose iv administration, respectively. No drug-related gross or microscopic lesions were found in rabbits given single iv infusions of ODMF at dosages of 10 to 400 mg activity/kg. Minimal to mild muscle irritation was seen in rabbits given im concentrations of 3.8 or 7.5% ODMF at dosages of 48 or 93 mg ODMF activity/kg. In 1-month iv studies in dogs treated with ODMF at dosages of 0.4, 1, 4, or 8 mg activity/kg/day, and in concurrent studies in rats treated with ODMF dosages of 1, 3, 6, or 12 mg activity/kg/day, treated animals remained essentially free of adverse effects. In 1-month im studies in dogs treated with ODMF at dosages of 1, 4, 8, or 16 mg activity/kg/day, no renal lesions occurred after an ODMF dosage of 1 mg activity/kg/day. Concurrent im studies in rats treated with ODMF at dosages of 6, 12, 24, or 48 mg activity/kg/day showed that ODMF dosages of 6 and 12 mg activity/kg/day did not produce renal lesions. In 6-month chronic im studies in dogs with ODMF dosages of 0.5, 1, or 4 mg activity/kg/day or gentamicin sulfate (GS) dosages of 2 mg activity/kg/day, and in concurrent studies in rats treated with ODMF dosages of 0.5, 2, or 6 mg activity/kg/day or GS dosages of 3 mg activity/kg/day, less severe local irritation and nephrotoxicity occurred after treatments with ODMF than with GS. In both rats and dogs treated by either the iv or the im route of administration, higher concentrations of ODMF and GS were found in the kidneys than in the sera. Mean serum and tissue concentrations of GS were higher than those of ODMF. Local tissue irritation and nephrotoxicity were lower with ODMF than with GS on a milligram activity per kilogram basis.

The hepatotoxicity of valproic acid and its metabolites in rats. I. Toxicologic, biochemical and histopathologic studies.

Valproic acid (VPA), its unsaturated metabolites and pent-4-enoate (4-PA) were studied for potential hepatotoxicity in rats. 4-PA, 4-en-VPA and 2,4-dien-VPA were potent inducers of microvesicular steatosis in young rats. Microvesicular steatosis induced by the 4-en-VPA was accompanied by ultrastructural changes characterized by myeloid bodies, lipid vacuoles and mitochondrial abnormalities. Myeloid bodies and lipid vacuoles were seen to a lesser extent in 2,4-dien-VPA and 4-PA-treated rats. VPA failed to induce discernible liver lesions in young rats even at near lethal doses of 700 mg per kg per day. The drug did, however, induce hepatic lipid accumulation in mature rats and in young rats dosed concomitantly with phenobarbital. beta-oxidation inhibition and several other biochemical alterations were observed in rats dosed with VPA, its unsaturated metabolites and 4-PA. It was suggested that beta-oxidation inhibition observed in both VPA and en-metabolite-treated rats occurred by different mechanisms. VPA inhibits by a transient sequestering of CoA while the CoA esters of some en-VPA-metabolites, particularly 4-en-VPA, inhibit specific enzyme(s) in the beta-oxidation sequences.

The hepatotoxicity of valproic acid and its metabolites in rats. II. Intermediary and valproic acid metabolism.

The role of metabolites in valproic acid (VPA)-associated hepatotoxicity was studied in rats. The most steatogenic mono-unsaturated metabolite, 4-en-VPA, caused the greatest changes in indicators of beta-oxidation inhibition (dicarboxylic aciduria, beta-hydroxybutyrate reduction); however, the biochemical effects were much less pronounced than those reported for hypoglycin. Steatosis in VPA-treated rats occurred only at nearly lethal doses. Phenobarbital induction was confirmed as a predisposing factor; however, it appeared not to greatly enhance production of 4-en-VPA or its recognized metabolites, which collectively comprised only 0.5% of the dose. Elevated oxo-VPA metabolites in serum and 2-propylglutarate in liver were associated with toxicity. Among the newly discovered minor metabolites with possible biologic effects were diols (suggesting epoxide precursors) and a series of dienes and trienes. The rarity of severe human hepatotoxicity indicates that, normally, beta-oxidation inhibition is compensated, and cellular defense mechanisms prevail over reactive metabolites. This requires adequate nutrition; on the other hand, severe glycogen depletion may promote toxicity by compromising glucuronidation, the major clearance route. Other literature comments are also supported: (i) caution is indicated for patients with various unusual congenital disorders (e.g., organic acidurias or other mitochondrial defects), and (ii) monotherapy obviates both the predisposition to toxicity and the requirement of large doses to produce therapeutic levels.

Carcinogenic evaluation of estazolam via diet in CD strain Sprague-Dawley rats and B6C3F1 mice for 2 years.

A comparative toxicity and carcinogenicity study was carried out for 2 years with estazolam, a benzodiazepine, via diet in Sprague-Dawley rats (0.5, 2, and 10 mg/kg/day) and in B6C3F1 mice (0.8, 3, and 10 mg/kg/day). In rats, no biologically significant changes were seen with respect to mortality, clinical signs, food consumption, or occurrence of palpable masses. Body weight gain in females (10 mg/kg/day) was depressed 12.6% and reflected a maximum-tolerated dosage (females). Spontaneous and incidental nonneoplastic lesions were consistent with aging in this species and unrelated to drug treatment. No biologically significant differences in tumor incidences occurred. Mice were more responsive to estazolam as suggested by (1) increased mortality (males) at 10 mg/kg/day, (2) increased food consumption and body weight gains (females), (3) withdrawal signs characterized by hyperactivity/aggressiveness and convulsions, and (4) appearance of dose-related nodular hyperplasia of the liver due to the relatively high dosages used coupled with the propensity of benzodiazepines to enhance liver enzyme induction. Several spontaneous benign and malignant tumors observed in all groups were not considered to be drug related. Based on the findings in these studies, estazolam was not considered to be carcinogenic when administered via diet to either rats at 0.5, 2, and 10 mg/kg/day or to mice at 0.8, 3, and 10 mg/kg/day for 2 consecutive years.

Aspects of the metabolism of valproic acid.

The metabolic routes of valproic acid (VPA) were studied by i.p. administration of the mono-unsaturated and hydroxylated metabolites to rats. Conjugation with glucuronic acid was a major metabolic route for VPA and its metabolites. Conjugation with glycine was a minor route for VPA, but was of more importance with the unsaturated metabolites. The hydroxylated metabolites, which were further oxidized to oxo-derivatives and subsequently to the dicarboxylic acids, were not metabolically dehydrated to form unsaturated metabolites. Multiple metabolic pathways, including dehydrogenation, isomerization, hydration, hydroxylation, reduction and epoxidation were inferred from the metabolites obtained after dosage of the unsaturated metabolites. Six dien-VPA metabolites were detected in VPA-treated rats, four of which are present in patients. It was concluded that 3-en-VPA and 4-en-VPA pathways, originating through dehydrogenation, are distinct from the omega- and omega-1-hydroxylation pathways. Enzyme induction from co-administration of phenobarbital caused enhancement of the minor omega-1-oxidation pathway, yet the largest effect on clearance came from increases in glucuronidation. Mitochondrial processes were unaffected, resulting in decreased contribution to the total clearance.

Heinz bodies, methemoglobinemia, and hemolytic anemia induced in rats by 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline.

Sprague-Dawley CD strain rats were given 18, 35, 70, or 140 mg/kg/day of 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline by gavage for 2 weeks. Heinz bodies were seen in the erythrocytes of rats given 140 mg/kg/day. Dose-related increases in methemoglobin were found at 35 mg/kg/day or more. Hemolytic anemia was characterized by dose-related decreases in hematocrit, hemoglobin, and total erythrocyte count. Reticulocytosis, decreased myeloid:erythroid ratio, splenomegaly, extramedullary hematopoiesis, increased serum total bilirubin, and icterus were also observed. This compound was found to oxidize oxyhemoglobin to methemoglobin in vitro, suggesting that the parent compound is capable of causing the hematological changes observed in vivo without conversion to active metabolites.

Terazosin: intravenous safety evaluation in rats.

Terazosin, an alpha-adrenergic antagonist, was administered as a 15 mg/ml solution to rats intravenously at a rate of 2 ml/min. Under these conditions the LD50 was 277 mg/kg for males and 293 mg/kg for females. When administered daily for 1 month at dosages of 0, 10, 40 or 150 mg/kg/day, the no-toxic-effect dosage was 40 mg/kg/day. Evidence of toxicity at 150 mg/kg included hypothermia and deaths. Death resulted from acute, exaggerated pharmacologic effects leading to cardiorespiratory failure. Evidence of sympatholytic activity observed at lower dosages included hypoactivity, blepharoptosis, ptyalism and splenic congestion.

Tulobuterol: one-month intravenous safety studies in rats and dogs.

Tulobuterol was given intravenously to rats and dogs at dosages of 1, 5, or 25 mg/kg/day and 0.6, 2, or 6 mg/kg/day, respectively. The no-toxic-effect dosages were 5 mg/kg/day in rats and 6 mg/kg/day in dogs. Two rats died at 25 mg/kg/day. Convulsions, jerking movements, hyperactivity, tremors, hypoactivity and ptyalism were observed in rats given 25 mg/kg/day. Restlessness, ptyalism and hypoactivity were also observed in dogs at 2 and 6 mg/kg/day. Cutaneous and/or mucosal erythema were observed in rats and dogs at all dosages. Increased body weight gain occurred in drug-treated rats and in mid- and high-dose female dogs. Slight elevations in serum creatinine and BUN were seen in rats and dogs at the highest dosages. Heart weights were increased in rats at all dosages after 1 month of treatment and in rats given 25 mg/kg/day after 2 weeks of recovery. There were no treatment-related morphologic changes in either species.

Cetaben versus clofibrate: comparison of toxicity and peroxisome proliferation in rats.

Cetaben and clofibrate were each administered to groups of 6 rats (3 male and 3 female) by gavage at dosages of 50 mg/kg per day and 200 mg/kg per day for 2 weeks. Cetaben caused 1 death at 200 mg/kg per day and decreases in body weight gain and food consumption at 50 mg/kg per day or more. There were no effects on body weight or food consumption in the clofibrate-treated groups. At 200 mg/kg per day cetaben and clofibrate induced comparable increases in liver weight and in numbers of liver peroxisomes while cetaben caused increases in liver catalase activity as well.

Buflomedil: three month intravenous safety evaluation in rats.

Buflomedil was administered intravenously to rats at dosages of 1, 4, 12 or 30 mg/kg/day for up to three months. The no-adverse-effect dosage was considered to be 12 mg/kg/day. At 30 mg/kg/day several deaths and clinical signs, including ataxia, decreased activity, dyspnea and jerking movements after dosing, were observed. These were considered to result from the acute, exaggerated pharmacologic effects of buflomedil. Body weight gain and food consumption were decreased after six weeks in males at 30 mg/kg/day. Increases in the relative weights of the kidneys, brain and testes of males at 30 mg/kg/day were correlated with decreased body weight gain in this group. There were no effects on hematology or serum chemistry parameters, and no morphologic changes were found.

Comparative effects of repeated and prolonged inhalation exposure of beagle dogs and Cynomolgus monkeys to anaesthetic and subanaesthetic concentrations of enflurane and halothane.

Male and female Beagle dogs and Cynomolgus monkeys were exposed to anaesthetic (1.5 MAC) and subanaesthetic (1/100 MAC) levels of enflurane and halothane for 3 hours on alternate days for 4 weeks. One-half of the animals were killed following the last exposure and the remainder after 4 weeks of recovery. The animals' condition was assessed during anaesthetic periods by measuring respiration, ECG, blood pressure, temperature and EEG. Haematology, urinalysis and clinical chemistry parameters were evaluated. Gross and microscopic pathological examinations were conducted at the end of the exposure and recovery periods. Two female monkeys in the mid- and high-dose halothane groups died during the study. No deaths were observed in the enflurane group. No quantitative differences were observed in respiration rate, heart rate, blood pressure and EEG activity of animals anaesthetized with enflurane or halothane. Muscle twitches were observed in some mid- and high-dose dogs inhaling enflurane, but not in monkeys. A number of liver function tests became abnormal in mid- and high-dose halothane-treated dogs and high-dose halothane-treated monkeys. This was not observed with enflurane. Histopathologic alterations were confined to the liver of animals exposed to halothane. In dogs, the lesions were characterized by centrilobular hepatocyte degeneration and/or necrosis, fibroblastic proliferation, hepatocyte enlargement, fat deposition and glycogen depletion; and in mid- and high-dose monkeys by moderate to marked hepatocyte vacuolation and fat deposition. Except for one high-dose dog, these lesions were not seen in animals killed after 4 weeks of recovery. No histopathologic alterations were observed with enflurane.