Chronic Toxicology Studies Research Articles

Analysis of rodent growth data in toxicology studies.

To evaluate compound-related effects on the growth of rodents, body weight and food consumption data are commonly collected either weekly or biweekly in toxicology studies. Body weight gain, food consumption relative to body weight, and efficiency of food utilization can be derived from body weight and food consumption for each animal in an attempt to better understand the compound-related effects. These five parameters are commonly analyzed in toxicology studies for each sex using a one-factor analysis of variance (ANOVA) at each collection point. The objective of this manuscript is to present an alternative approach to the evaluation of compound-related effects on body weight and food consumption data from both subchronic and chronic rodent toxicology studies. This approach is to perform a repeated-measures ANOVA on a selected set of parameters and analysis intervals. Compared with a standard one-factor ANOVA, this approach uses a statistical analysis method that has greater power and reduces the number of false-positive claims, and consequently provides a succinct yet comprehensive summary of the compound-related effects. Data from a mouse carcinogenicity study are included to illustrate this repeated-measures ANOVA approach to analyzing growth data in contrast with the one-factor ANOVA approach.

Safety and Advantages of Bacillus thuringiensis-Protected Plants to Control Insect Pests

Plants modified to express insecticidal proteins from Bacillus thuringiensis (referred to as Bt-protected plants) provide a safe and highly effective method of insect control. Bt-protected corn, cotton, and potato were introduced into the United States in 1995/1996 and grown on a total of approximately 10 million acres in 1997, 20 million acres in 1998, and 29 million acres globally in 1999. The extremely rapid adoption of these Bt-protected crops demonstrates the outstanding grower satisfaction of the performance and value of these products. These crops provide highly effective control of major insect pests such as the European corn borer, southwestern corn borer, tobacco budworm, cotton bollworm, pink bollworm, and Colorado potato beetle and reduce reliance on conventional chemical pesticides. They have provided notably higher yields in cotton and corn. The estimated total net savings to the grower using Bt-protected cotton in the United States was approximately $92 million in 1998. Other benefits of these crops include reduced levels of the fungal toxin fumonisin in corn and the opportunity for supplemental pest control by beneficial insects due to the reduced use of broad-spectrum insecticides. Insect resistance management plans are being implemented to ensure the prolonged effectiveness of these products. Extensive testing of Bt-protected crops has been conducted which establishes the safety of these products to humans, animals, and the environment. Acute, subchronic, and chronic toxicology studies conducted over the past 40 years establish the safety of the microbial Bt products, including their expressed insecticidal (Cry) proteins, which are fully approved for marketing. Mammalian toxicology and digestive fate studies, which have been conducted with the proteins produced in the currently approved Bt-protected plant products, have confirmed that these Cry proteins are nontoxic to humans and pose no significant concern for allergenicity. Food and feed derived from Bt-protected crops which have been fully approved by regulatory agencies have been shown to be substantially equivalent to the food and feed derived from conventional crops. Nontarget organisms exposed to high levels of Cry protein are virtually unaffected, except for certain insects that are closely related to the target pests. Because the Cry protein is contained within the plant (in microgram quantities), the potential for exposure to farm workers and nontarget organisms is extremely low. The Cry proteins produced in Bt-protected crops have been shown to rapidly degrade when crop residue is incorporated into the soil. Thus the environmental impact of these crops is negligible. The human and environmental safety of Bt-protected crops is further supported by the long history of safe use for Bt microbial pesticides around the world.

Response of the pituitary and thyroid to tropic hormones in Sprague-Dawley versus Fischer 344 male rats.

Modulation of endocrine function is frequently a confounding factor in the interpretation of chronic rodent toxicology studies. Of particular interest are agents that cause deviation of thyroid hormone homeostasis and result in thyroid cancer for rodents. An endocrine challenge test (ECT), commonly used to study endocrine organ health in human and veterinary medicine, quantifies the response of the thyroid to tropic hormones. This study compared the response of Fischer (F344) and Sprague-Dawley (SD) rats to a thyrotropin-releasing hormone (TRH) ECT and a thyroid-stimulating hormone (TSH) ECT and characterized the dose-response curve. TSH, thyroxine (T4), triiodothyronine (T3), and prolactin responses were characterized for several doses of TRH over a 4-h time period. Animals were equipped with intra-atrial cannulae and were free moving at all times during blood sampling. Both strains of rats responded to intravenous TRH by releasing TSH into their blood in a dose-responsive fashion. At doses of > or = 100 ng, TSH concentrations were increased by more than 2-fold at 2 min. Concentrations reached a maximum at 15 min for doses of 100 ng/100 g body weight (bw) to 5000 ng/100g bw. The effective dose 50 (ED50) of TRH (that dose causing release of half maximal TSH concentrations) was 61 ng in F344 rats and 78 ng in SD rats. The ED75 was 173 ng and 217 ng/100 g bw, respectively. The response of T4 and T3 after TRH ECT and TSH ECT was highly variable. F344 rats responded with an increase in levels of both hormones, starting at 60 min and continuing through 240 min. In SD rats, the presence of a thyroid hormone response (T4) was present, although that of T3 was not clear. These data provide essential information for design of toxicology studies focused on the effects of toxicants and drugs on the pituitary-thyroid axis.

Toxicology in the Drug Discovery and Development Process

AbstractThe primary objective of toxicology studies in the drug development process is to evaluate the safety of potential drug candidates using relevant animal models and validated procedures. The ultimate goal is to interpret the animal data to assess risk for human subjects/patients. Therefore, the toxicologist must be aware of the international guidelines for safety evaluation in conjunction with the use of traditional and nontraditional models such as transgenic animals. This overview discusses the components of a typical toxicology profile including safety pharmacology, genetic toxicology, acute and subchronic toxicology, chronic toxicology, absorption, distribution, metabolism and excretion (ADME) studies, reproductive and developmental toxicology, and an evaluation of carcinogenic potential. (Due to the wide variety of therapeutic targets, toxicology profiles may differ from drug to drug.) Also, regulatory guidelines for toxicology testing are explained in relation to the drug discovery and development process.

PIRLINDOLE: A SELECTIVE REVERSIBLE INHIBITOR OF MONOAMINE OXIDASE A. A REVIEW OF ITS PRECLINICAL PROPERTIES

Pirlindole is a tetracyclic compound that has been characterized as a potential antidepressant drug. It has pharmacological characteristics in common with both tricyclic antidepressants and classical irreversible monoamine oxidase inhibitors. Its main mechanism of action consists of a selective and reversible inhibition of monoamine oxidase A. Secondarily, it exerts an inhibitory effect on noradrenaline and 5-hydroxytryptamine reuptakes. It has no effect on the dopaminergic and cholinergic systems. It has only a low potential for amplifying tyramine and noradrenaline pressor effect, which makes one expect that it will not be at the basis of a ‘cheese effect’. Pirlindole has an absolute bioavailability of between 20 and 30% due to an extensive first-pass effect. Orally, theTmaxvaries between 2.5 and 6 h in the rat and 0.8 and 2 h in the dog. Two phases of elimination (7.5 and 34–70 h) are measured in the rat and three phases in the dog (1.3, 10.8 and 185 h); it is extensively metabolized. The rat eliminates mainly unconjugated products while the dog eliminates mostly conjugated products. Acute and chronic toxicological studies have not revealed potentially dangerous effects of the drug at the usual doses. It does not present measurable mutagenic, clastogenic or carcinogenic properties. Thus, pirlindole shows pharmacological, pharmacokinetic and toxicological properties which make it suitable for the management of depression.

Improvement of bioavailability of the HIV protease inhibitor SC-52151 in the beagle dog by coadministration of the CYP3A4 inhibitor, ketoconazole

1. SC-52151. an HIV protease inhibitor, is mainly metabolized by CYP3A4 and is poorly bioavailable after oral administration. After i.v. administration of SC-52151 to the female beagle dog (2 5 mg kg), SC-52151 was rapidly eliminated in plasma with an elimination half-life of about 1 h, a plasma clearance of 44 ml min kg and an apparent steady-state volume distribution of 2 2 litre kg. The high value of plasma clearance of SC-52151 suggests an extensive hepatic first-pass metabolism since SC-52151 is highly protein bound and does not partition itself into red blood cells. 2. The extensive hepatic first-pass metabolism was reduced by coadministration of a CYP3A4 inhibitor, ketoconazole. 3. Dogs were dosed daily with ketoconazole at dose of 100 mg ketoconazole per dog (10 mg kg) for 5days prior to theinitiationof coadministration of SC-52151 for 15 days. The doses used for SC-52151 was 0, 60 and 120 mg SC-52151 kg day (divided t.i.d., 8-h dosing interval). Coadministration of ketoconazole improved the bioavailability of SC52151 from 4 1 to 9 6% and also improved the C of SC-52151 from 0 41 to 0 83 mu g ml. max 4. Although the absolute bioavailability of SC-52151 was still low (10%), the C max and AUC achieved in this study were satisfactory for conducting chronic toxicology studies. No toxicity associated with the coadministration of ketoconazole was evident. Results from this study suggest that coadministration of ketoconazole might be a practical approach to increase the exposure of SC-52151 in both preclinical and clinical studies.

Research and development of cancer chemopreventive agents in China

Since the late 1970s, a comprehensive search for cancer chemopreventive agents has been established in our Institute. A series of new retinoids have been synthesized and screened on the basis of established methodologies of experimental chemoprevention in vitro as well as in vivo. Pharmacological studies demonstrated that N-4-(carboxyphenyl)retinamide (RII) induces cell differentiation of HL-60 cells and inhibits dimethylnitrosamine-induced carcinogenesis of the forestomach in mice, 7,12-dimethylbenz[a]anthracene (DMBA)-induced papilloma in mouse skin, and DMBA-induced carcinogenesis of the buccal pouch in Syrian golden hamsters. It significantly promoted lymphoblastic transformation and activated macrophages. In further studies, RII significantly inhibited ornithine decarboxylase activity. After 6 months of chronic toxicological studies in rats and dogs, RII was recommended for clinical trial. Phase II studies found that RII is effective in treating oral and vulvar leukoplakia. It is also effective in treating myelodysplastic syndrome and dysplasia of uterine cervix. The chalcone retinoidal compounds were discovered when the search for new retinoids with less toxicity and higher potency led to third-generation retinoids, which were synthesized and screened. Structure-activity relationship studies found that 3,5-di-tert-butyl-4-methoxy-4-carboxyl chalcone (R9158) is the most active inhibitor of a variety of cancer cells. It has no effect on the Colony Forming Unit-Granulocyte/Macrophage (CFU-GM) of bone marrow in mice. In in vivo studies, R9158 showed a remarkable inhibition of chondrosarcoma in rats. It had no cross-resistance to vincristine, but was cross-resistant to all-trans retinoic acid. Red ginseng, a processed Panax ginseng, is considered a typical tonic in traditional Chinese medicine. Our studies demonstrated that red ginseng extract inhibited DMBA-induced skin papilloma significantly. Experiments showed that glycyrrhetinic acid inhibited croton oil-induced ear edema in mice. It also inhibited epidermal ornithine decarboxylase as well as the rapid DNA damage induced by the carcinogen benzo[a]pyrene (B[a]P). Our pharmacological studies demonstrated that Chinese gallotannin inhibited the malignant transformation of B[a]P-induced V79 cells in vitro and B[a]P-induced pulmonary adenoma in A/J mice in vivo significantly.

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.

Spontaneous hemangioendothelial cell hyperplasia of the heart in B6C3F1 female mice.

Spontaneous hemangioendothelial cell hyperplasia of the heart was found in 12 of 3,910 female B6C3F1(C57BL/6CrSlc x C3H/HeSlc) mice that were used in chronic toxicology and carcinogenicity studies at the An-Pyo Center (Shizuoka, Japan). Histologically, the lesions appeared diffusely in the heart as widened vascular spaces lined by a single layer of endothelial-like cells with enlarged nuclei between the muscle fibers. These proliferating cells were identified as hemangioendothelial cells by immunohistochemical techniques, lectin-binding patterns, and electron microscopic examination. Morphological differences were found between this spontaneous hemangioendothelial hyperplasia and that which has been reported as a preneoplastic lesions of hemangiosarcoma. Spontaneous hemangioendothelial cell hyperplasia of the heart occurs rarely; however, it was interesting that spontaneous hemangioendothelial cell hyperplasia was only seen in females with adenocarcinomas of the mammary gland.

Five cases of spontaneous hemangioendothelial cell tumor of the heart in B6C3F1 mice.

Spontaneous hemangioendothelial cell tumor of the heart was found in five of 4, 160 male and 3, 910 female B6C3F1 (C57BL/6CrSIc×C3H/HeSlc) mice which were used in chronic toxicology and carcinogenicity studies at the An-Pyo Center. Two cases were diagnosed histologically as benign hemangioma and the other three cases were diagnosed as hemangiosarcoma. Hemangioendothelioma of the bone marrow, liver, and spleen were also observed in two out of three cases with hemangiosarcoma of the heart, and these cases were recognized as systemic, multiple vescular cell tumor of B6C3F1 mice. The tumor cells were identified as hemangioendothelial cells by immunohistochemical techniques and lectin binding patterns. The finding of five cases of spontaneous hemangioendothelial cell tumor of the heart in this report is considered to be extremely rare.

Inhaled Nitric Oxide in Adult Respiratory Distress Syndrome and Other Lung Diseases

In 1987, nitric oxide was reported to be an endothelium-dependent relaxing factor. When inhaled as a gas at low levels, nitric oxide selectively dilates the pulmonary circulation. Significant systemic vasodilation does not occur because nitric oxide is inactivated by rapidly binding to hemoglobin. In an injured lung with pulmonary hypertension, inhaled nitric oxide produces local vasodilation of well-ventilated lung units and may "steal" blood flow away from unventilated regions. This reduces intrapulmonary shunting and may improve systemic arterial oxygenation. In patients with adult respiratory distress syndrome, inhaled nitric oxide reduces pulmonary hypertension and improves arterial oxygenation without reducing systemic arterial pressure. Tachyphylaxis to nitric oxide inhalation has not been observed. While additional chronic toxicology studies need to be performed, significant pulmonary toxicity has not been observed at low inhaled concentrations (< 80 parts per million by volume). Potentially, inhaled nitric oxide may be a valuable therapy in patients with adult respiratory distress syndrome.

Comparison of the effects of propylthiouracil, amiodarone, diphenylhydantoin, phenobarbital, and 3-methylcholanthrene on hepatic and renal T4 metabolism and thyroid gland function in rats

We studied the effects of propylthiouracil (PTU), amiodarone (AMIO), diphenylhydantoin (DPH), phenobarbital (PB), and 3-methylcholanthrene (MC) on thyroid histomorphology, on the hepatic and renal enzymes involved in endogenous and exogenous metabolism, and on the plasma levels and pharmacokinetics of thyroid hormones after 7 and 14 days of treatment. PTU and PB, by decreasing both serum tetraiodothyronine (T4) and triiodothyronine (T3), induced a massive increase in serum thyrotropin (TSH) and thus induced thyroid hypertrophy. AMIO and MC, by decreasing respectively serum T3 and T4, also induced an increase of TSH, but to a lesser extent, not sufficient to induce thyroid hypertrophy. Hepatic 5′-deiodinase activity was decreased in all treated rats. Inhibition of this enzyme by PTU was demonstrated in vitro: AMIO also decreased the enzyme activity by a still unelucidated mechanism, which probably requires intact cell plasma membranes, whereas in PB- and MC-treated rats the decrease in enzyme activity certainly resulted from decreased serum concentrations of T4. In PTU-treated rats, and probably in MC-treated rats, decreases in circulating thyroid hormones were primarily due to impairment of synthesis and/or of secretion by the thyroid. In contrast, in PB-treated rats, the decrease in serum thyroid hormone levels seems to be due to increased excretion of these hormones, as T4 serum clearance was significantly increased. PB, a microsomal enzyme inducer, increased the cytochrome b 5 and P450 content as well as the cytochrome P450-dependent O-depentylation of pentoxyresorufin. The other type of enzyme inducer, MC, did not affect cytochrome b 5 and P450 levels, but did increase the cytochrome P450 dependent O-deethylation of ethoxyresorufin. PB increased the glucuronidation of morphine, whereas MC increased the glucuronidation of 1-naphthol. However, serum T4 clearance, mainly determined by its hepatic conjugation rate, was increased only in PB-treated rats. It appears from this study that the close metabolic relationship between the liver/kidney and the thyroid should be taken into consideration when the findings of chronic toxicology and carcinogenicity studies are interpreted.

Chronic toxicology and carcinogenesis studies of Telone II by gavage in Fischer-344 rats and B6C3F1 mice.

Telone II (technical grade, 1,3-dichloropropene), a soil fumigant, was evaluated in chronic toxicology/carcinogenicity studies using Fischer-344 (F344) rats and B6C3F1 mice of both sexes. Doses administered were 0, 25, or 50 mg/kg to rats and 0, 50, or 100 mg/kg to mice. Telone II was given in corn oil by gavage 3 times per week for 104 wk. Ancillary studies were conducted to determine time-related effects, in which dose groups containing 5 male and 5 females rats were killed after receiving Telone II for 9, 16, 21, 24, or 27 mo. The primary organs affected were the forestomach (rats and mice), urinary bladder (mice), lung (mice), and liver (rats). Compound-related non-neoplastic lesions included basal-cell or epithelial hyperplasia of the forestomach (rats and mice), epithelial hyperplasia of the urinary bladder (mice), and hydronephrosis (mice). Neoplastic lesions associated with administration of Telone II included squamous-cell papillomas of the forestomach (male and female rats, female mice), squamous-cell carcinomas of the forestomach (Male rats, female mice), transitional-cell carcinomas of the urinary bladder (female mice), alveolar/bronchiolar adenomas (female mice), and neoplastic nodules of the liver (male rats). Although cis- and trans-1,3-dichloropropene are the principal components of Telone II, the presence of 1% epichlorohydrin, a direct-acting mutagen and carcinogen added as stabilizer, may have influenced the development of forestomach lesions. The results of the ancillary studies supported the findings of the carcinogenesis studies and demonstrated the time-dependent development of lesions in the forestomach (basal-cell hyperplasia and squamous-cell papilloma). Under the conditions of these gavage studies, Telone II was shown to be carcinogenic in male and female F344 rats and female B6C3F1 mice. Although the study in male B6C3F1 mice was considered inadequate because of the low survival resulting from suppurative inflammation of the heart (myocarditis) in the control group, there was some indication of Telone II-related increases of transitional-cell carcinomas of the urinary bladder, squamous-cell papillomas of the forestomach, and alveolar/bronchiolar adenomas and carcinomas of the lung.

The value of chronic animal toxicology studies of pharmaceutical compounds: A retrospective analysis

Extensive animal studies are carried out during the development of new medicines to assess toxicity and predict their safety for use in man. There are, however, differences of opinion concerning the nature of safety evaluation studies required prior to marketing. Any prospect of rationalizing the number and design of animal studies must stem from a reappraisal of conventional animal testing procedures and better use of available data on the toxicity of compounds previously investigated. The Centre for Medicines Research has therefore established a toxicology databank containing comprehensive data from repeated-dose animal safety evaluation studies provided by pharmaceutical companies. Thirteen companies within the United Kingdom have provided toxicological data for 32 pharmaceutical compounds studied in the rat, dog, or primate, resulting in 45 case studies for which both short-term (≤6 months) and long-term (>6 months) tests had been completed. A comprehensive analysis of these studies has been carried out to determine what new findings, if any, become apparent in studies after 6 months. The results do not support the need for animal toxicity studies of longer than 6 months duration, apart from those designed to investigate carcinogenicity.

In vitro mutagenicity testing of a potent, new, benzoyl urea insect growth regulator

Bacterial mutagenicity assay to detect potential chronic toxicity of a potent, new, benzoyl urea insect growth regulator (CGA-112913 or IKI-7899, formerly UC-62644) was conducted using 5 histidine auxotrophs ofSalmonella typhimurium. Tests within the concentration range of 0.9–500 μg (saturating)/plate of the compound with and without the 5–9 mammalian metabolic activation system showed no mutagenic effects clearing the way for long-term chronic toxicology studies.

Generating, Interpreting, and Reporting Information in Toxicity Studies

The toxicological evaluation of a compound involves a variety of acute, subchronic, and chronic toxicity studies which over the past decade have greatly increased in size and complexity. The combination of voluminous amounts of data collected in chronic toxicology studies and regulatory requirements for extensive documentation of the toxicology testing process presents an enormous burden when manual processes are used for data collection, storage, and analysis. Toxicology studies using relatively standardized experimental protocols present an ideal opportunity for nearly complete automation and computerization of the data acquisition, storage, analysis, and reporting processes. The complexity of toxicology data collection and the requirement for flexibility to accommodate special needs for individual experiments necessitate, however, the use of sophisticated computer software and hardware on a scale that is often underestimated at the outset of a toxicology computer project by both toxicologists and compu...

Urinary enzyme measurements as sensitive indicators of nephrotoxicity in chronic toxicological studies

Urinary enzyme measurements as sensitive indicators of nephrotoxicity in chronic toxicological studies

Absorption and Bioavailability of Captopril in Mice and Rats After Administration by Gavage and in the Diet

The absorption of captopril (I), a new antihypertensive agent, was studied in mice and rats at doses (50 and 1350 mg/kg) administered in the diet in chronic toxicological studies. 3H- or 35S-Labeled I was administered by gavage and in the diet to male and female animals in a two-way crossover study. Animals received daily doses of nonradiolabeled I in the diet for 25 days, except on Days 15 and 22 when radiolabeled I was administered either by gavage or in the diet. Absorption of the total radioactivity in 2-month-old mice averaged 49 and 48%, respectively, of the 50- and 1350-mg/kg doses given in the diet and 57 and 65%, respectively, of the doses given by gavage. The bioavailability of I in 2-month-old mice averaged 48 and 39% (diet) and 44 and 59% (gavage) of the 50- and 1350-mg/kg doses, respectively. In 2-month-old rats, absorption of the total radioactivity averaged 41% of the 50-mg/kg dose given in the diet. In 2- and 15-month-old rats, minimum absorption of the 1350-mg/kg dose averaged 36 and 45% (diet) and 51 and 71% (gavage), respectively; the minimum bioavailability averaged 29 and 29% (diet) and 39 and 44% (gavage), respectively. These studies demonstrate adequate absorption and bioavailability of I over a wide range of doses from the drug-diet mixtures and by young and old animals and also illustrate a useful experimental design for the estimation of relative oral absorption of a drug administered continuously in the diet over several days.

Contraceptive steroid toxicology in the Beagle dog and its relevance to human carcinogenicity

Problems associated with the use of the Beagle dog in chronic toxicological studies of contraceptive steroids are described. A short review is presented on the occurrence of spontaneous tumours in dogs and in bitches of various breeds. The current status of knowledge of canine reproductive hormones and endocrinology is outlined, together with effects of contraceptive steroids. The pathology and histological classification of spontaneous and induced mammary neoplasia in the dog is discussed and compared with breast cancer in women. A series of recommendations are included for future research in this field which it is hoped may resolve some of the outstanding issues and lead to a more suitable toxicological model for contraceptive steroids.

The Activities of 1-Hydrazinophthalazine (Ba-5968), a Hypotensive Agent

The pharmacological effects of 1-hydrazinophthalatine (Ba-5968) have been reported. Its two principal actions were hypotension and partial adrenolysis. Doses of from 0.25 to 1.0 mg./Kg. intravenously usually sufficed in the dog and cat to produce the maximal hypotensive response that the drug could effect. Further marked increases in the dose exerted no obvious effect. The hypotensive action of the drug was characterized by a slow development of the maximal effect after a significant latent period. The action is probably central since the drug little influenced the blood pressure of “spinal” cats and dogs and did not affect the flow in the perfused vascular bed of the rabbit's hind limb. Both acute and chronic toxicological studies have evidenced the safety and high therapeutic index of the compound.