Report of a panel discussion on how to run an effective Animal Care and Use Committee.

There was a consensus between the Panel and the participants in the audience as follows: All animal use should be reviewed by an Animal Care Committee. The policies and procedures of that Committee must, in the letter and in the spirit of federal guidelines, be developed by each institution, or group of institutions, mindful of its own operational practices and should apply to all research using animals, irrespective of the source of funds. The ACC will be accountable to the public at large for the conduct of animal research. To obtain the support of the community, the ACC must have the active support of the institution, both administratively and financially, and its own academic community who must be involved in ACC activities and, where possible, in the development of its policies and procedures. Education is essential. The Committee must understand its obligations and duties especially as they relate to confidentiality and internal freedom of discussion (this applies particularly to lay members). The faculty, staff, and students must understand the protective role of the Committee for both the community and animal subjects. The institutional community must be prepared to accept the responsibilities of animal use and so accept the need for education and updating in appropriate techniques and experimental design. Individual institutions and organizations, such as the Scientists Center for Animal Welfare, can and will provide information and guidance to those seeking assistance.

The role of the LD50 determination in drug safety evaluation.

Acute toxicology studies in animals are essential to drug development. Often such experiments seek to establish precisely the median lethal dose (LD50) in rodents. As scientific needs rarely require an exact value, practices and regulations should be changed to provide the option of obtaining adequate information on the acute toxicity of a drug with fewer animals than the precise LD50 test demands.

[Animal toxicity studies of probucol (author's transl)]. / Toxicologie animale du probucol.

The oral LD50 of probucol in rats and mice was found to be higher than 5 000 mg/kg. Subacute toxicity studies failed to show any toxic effect in rats and monkeys which received 3 000 and 200 mg/kg/24 h respectively for 90 days. Chronic toxicity studies were carried out in rats during 2 years and in monkeys during 8 years. Daily oral doses of up to 800 mg/kg in rats and 500 mg/kg in monkeys had no demonstrable toxic effects; in particular, there were no differences on electron microscopy between liver sections of monkeys treated for 8 years and those of untreated monkeys. In dogs, probucol proved non toxic when given for 14 days. However, during more prolonged administration (90 days) sudden death occurred in some animals, which was unrelated to dosage or to duration of treatment. Additional studies showed that probucol sensitizes dog myocardium to epinephrine, thereby inducing ventricular fibrillation. This effect was not observed in other animal species and is considered as specific to dogs. A special study was performed in monkeys, which received high doses of probucol (equivalent to 4-15 times the human dose) associated with an atherogenic diet containing 100 times more cholesterol than their normal diet and large quantities of fats. Some monkeys fainted and died; ECG tracings revealed no other abnormality than an increase in the length of QTc. Cardiac toxicity cannot be extrapolated from animal to man, since heart monitoring failed to show any abnormality in patients treated with probucol. No mutagenic or carcinogenic effects were observed in rats, and teratological and reproduction studies carried out in rats and rabbits gave negative results.

[Comparative study of the effects of probucol, fenofibrate and clofibrate on liver ultrastructure in rats (author's transl)]. / Etude comparative de l'action du probucol, du fénofibrate et du clofibrate sur l'ultrastructure hépatique chez le rat.

The effects on rat liver of probucol (250 or 500 mg/kg/24 h), clofibrate (250 mg/kg/24 h) and fenofibrate (100 mg/kg/24 h) were compared after 28 days' treatment. Th bodyweight of animals treated with probucol 250 mg/kg/24 h has increased, as compared with a control group of untreated animals. The increase in liver weight and live weight/bodyweight ratio was greater in rats treated with clofibrate or fenofibrate than in rats treated with probucol. Cholesterol and triglyceride plasma levels were lower in clofibrate-treated animals. There were no changes in SGOT and SGPT in any of the three groups. Alkaline phosphatases were increased only in rats treated with clofibrate (these enzymes were not assayed in rats which received fenofibrate). Electron microscope study of liver sections showed the following changes in clofibrate--and fenofibrate-treated rats as compared with the control group: increase in smooth-surfaced endoplasmic reticulum, ribosome detachment, slight glycogen depletion and increase in the number of peroxysomes. In the probucol-treated group, apart from an occasional increase in smooth-surfaced endoplasmic reticulum, liver cells were identical with those of the control group; in particular, there was no increase in the number of peroxysomes.

Eight-year toxicity study in monkeys and reproduction studies in rats and rabbits treated with probucol.

Probucol [4,4'-(isopropylidenedithio)bis(2,6-di-tert-butylphenol)], a hypocholesterolemic agent, was given orally to male and female rhesus monkeys at 0, 60, 125, 250, and 500 mg/kg . d for more than 8 yr without adverse effect. Of 40 monkeys on test, 14 were killed for interim studies (wk 81 and 102), 21 were maintained for more than 8 yr, and 5 were submitted for necropsy for conditions unrelated to treatment. Monitored parameters included growth rate, demeanor, hematology, clinical chemistries, urinalyses, ophthalmoscopy, organ weights, and gross, histopathologic, and electron microscopic evaluations. Bone marrow smears at the conclusion of the test revealed no differences between control and treated animals. Electron microscopy of liver specimens from monkeys treated for 8 yr revealed comparable hepatocellular ultrastructure in control and treated monkeys. Probucol was given orally at 0, 100, 500, and 1000 mg/kg to Sprague-Dawley rats during appropriate time intervals to evaluate effects on fertility and postnatal development. The same dose levels were given during organogenesis, or in some cases prior to breeding and throughout organogenesis, to Sprague-Dawley rats and New Zealand white rabbits. No adverse effects on fertility or postnatal development were observed in rats, and no evidence of teratogenicity was observed in either species. The results indicate that probucol does not affect reproduction of rats, lacks teratogenic potential in the species studied, and is nontoxic to subhuman primates treated for more than 8 yr.

Comparative ultrastructural study of rat hepatocytes after treatment with the hypolipidemic agents probucol, clofibrate, and fenofibrate.

Experimental findings are reported for two similarly conducted studies. One was designed to compared rat liver cell ultrastructure during and after 91 d of dosing with probucol, a hypocholesterolemic agent, and clofibrate, a hypolipidemic drug known to elicit marked alteration of rat hepatocellular morphology. The second was designed to similarly assess male rats after 28 d of treatment with the hypolipidemic agent fenofibrate. Diet mixes for these studies were prepared to attain dosage levels of approximately 500 mg/kg . d for probucol, 250 mg/kg . d for clofibrate, and 100 mg/kg . d for fenofibrate. Control rats were given untreated basal ration. Weekly adjustments in dietary concentrations were made in accordance with group mean food consumption and body weight changes. Probucol and clofibrate treatments produced statistically significant reductions in mean serum cholesterol levels of both sexes after 28 and 91 d of dosing. Only male rats were given fenofibrate, and they exhibited statistically significant cholesterol reductions after 28 d. Clofibrate and fenofibrate administration resulted in marked increases in liver weight/body weight ratios. Probucol had no statistically significant effects on liver weight/body weight ratios after 28 and 91 consecutive days of treatment. Light microscopy of liver sections stained with hematoxylin and eosin revealed an abnormal amount of cytoplasmic granularity within hepatocytes from rats given clofibrate and fenofibrate. The granules were identified by electron microscopy and cytochemistry as enlarged, proliferated peroxisomes--a known rat hepatocellular response to treatment with many hypolipidemic drugs. In addition, ultrastructural cytochemistry suggested reduced amounts of catalase in individual peroxisomes after clofibrate and fenofibrate dosing. Liver tissue from rats given probucol showed no abnormal cytoplasmic granularity and, ultrastructurally, no peroxisomal changes. Liver tissues from probucol-treated rats revealed features similar to those encountered in tissues from untreated control animals. It was concluded that the hypocholesterolemic response elicited by probucol treatment does not involve significant changes in rat liver cell morphology.

Dose-dependent pharmacokinetic profile of silvex following intravenous administration in rats.

The pharmacokinetic profile of silvex was defined in rats after single intravenous doses of 5 and 50 mg/kg 14C ring-labeles silvex. Clearance of silvex from plasma at the 5 mg/kg dose was linear with a half-life of 16.2 hr, while clearance at the 50 mg/kg dose was nonlinear. Activities of 14C recovered in excreta were 94.1 and 95.1% of the administered doses at 5 and 50 mg/kg, respectively. Excreta was collected for 192 hr at 5 mg/kg and 216 hr at 50 mg/kg. Urinary excretion of 14C activity accounted for 80.5 and 68.7% of the administered dose at 5 and 50 mg/kg, respectively; fecal excretion accounted for 13.7 and 26.4% of the administered dose at 5 and 50 mg/kg, respectively. Urinary excretion of silvex is saturated at the 50 mg/kg dose. Significant amounts of silvex are excreted in bile and undergo enterohepatic circulation. Concentrations of silvex in liver and kidney are higher than those in fat, brain, and muscle 8 and 216 hr after administration. In a companion oral study, silvex was extensively if not completely absorbed. The pharmacokinetic data presented indicate that statistical projection of experimental results with large doses of silvex to predict the hazard of exposure to small amounts is not justified because the capability to excrete silvex in urine has been saturated.