It seems desirable to conduct as many function tests as possible in repeated dose toxicity studies, but is it practicable? The current status of conducting function tests in repeated dose toxicity studies in Japan was investigated by a literature survey of more than one thousand papers published in seven Japanese toxicology journals during the past 10 years and by a questionnaire survey directed to toxicologists among the Japan Pharmaceutical Manufacturers Association (JPMA) member companies. The function tests often carried out in repeated dose toxicity studies were, for example: 1) electro-retinography (ERG) and visually evoked potential (VEP) for visual test and tonometer for intraocular pressure; 2) auricular reflex, evoked response audiometry (ERA) and auditory bfainstem response (ABR) to sound stimuli; 3) respiration and heart rate, electrocardiogram (ECG) and blood pressure by noninvasive cuff methods or using electronic devices such as telemetry; 4) body temperature, spontaneous motility and some adaptation tests (using rotarod and sloped plate in rats); 5) indocyanin (ICG) or bromosul-fophtalein (BSP) for hepatic test; 6) phenolsulfonphtalein (PSP) and creatinine clearance for renal test; and 7) immunoglobulin, leukocyte phagocytosis, lymphocyte blastgenesis and natural killer cell (NK) for immuno-reaction test. Limitations to conducting function tests in repeated dose toxicity studies and issues to be resolved were discussed, based on questions and suggestions given by the respondents to the questionnaire. Although it certainly seemed desirable to conduct as many function tests as possible in repeated dose toxicity studies, most of the function tests so far introduced into toxicity studies were not satisfactory, because those tests could not be carried out under the restricted conditions of repeated dose toxicity studies, and not much reliable data from function tests were obtainable. A variety of function tests should firstly be incorporated into single dose toxicity studies together with development of a new concept for methodology in safety pharmacology.

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Many cell functions and aspects of cell configuration are linked with changes in cytoskeletal proteins, by intracellular signal transduction. Histochemical and pathophysiological changes caused by toxins or drugs affect on dynamic aspects of cytoskeletal proteins. Here we review that biochemical and molecular biological properties of the microtubules and microfilaments and the relationships between these cytoskeletal proteins and the toxic effects of drugs and toxins. The intracellular signaling pathway makes use of cytoskeletal proteins.

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Cytotoxic effects of nitrobenzene (NB) on spermatogenesis of mature Sprague-Dawley (Crj:CD) rats were analyzed by measuring the DNA content distribution and testicular weight at 1, 2, and 3 weeks of daily oral dose of NB (60 mg/kg/day). Rats at the age of 9 weeks were used because the ratios of 1C, 2C, S, 4C to total testicular cells were stabilized after the age of 52 days. Within a week of administration, a large number of 1C cells were lost but 2C cells proliferated, resulting in little change of testicular weight. In another week that followed, the number of 1C cells and testicular weight were decreased, but the ratio of S-4C cells was increased, indicating that an appreciable number of 2C cells could progress to the 4C compartment. The data indicated that (1) 1C cells were destroyed, and (2) meiosis of secondary spermatocytes was suppressed, but (3) NB had little effect on the spermatocytes prior to the early pachytene stage. This interpretation was reinforced by the observation that (4) the ratio of 1C cells returned to a nearly normal level during a recovery period of 2 weeks. In conclusion, flow cytometry could offer an efficient method for the quantitative analysis of male reproductive toxicity.

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The dog has been used as an experimental animal in emesis research. In this study, we analyzed the emetic effects of ipecac syrup using a smaller animal, the ferret, and compared its response to that of the dog. Dogs and ferrets were divided into 4 groups (n=4, each). Each group was given either 0.1, 0.25, 0.5, or 1.0 ml/kg of ipecac syrup, and the latency and numbers of retching and vomiting were recorded. Animals given an equal volume of saline served as controls. The numbers of vomiting and retching increased dose-dependently in both dogs and ferrets, and there was no difference in latency and numbers of vomiting between them. The numbers of retching were greater in ferrets than in dogs at ≥0.25 ml/kg. Taking these results into consideration, the ferret seems to be as useful as the dog in studies on emetic effects of ipecac syrup.

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The protective effect of 1, 3-dithia-2-thioxo-cyclopent-4-ene (DT827A) and its two derivatives of 4-phenyl-1, 3-dithia-2-thioxo-cyclopent-4-ene (DT827B) and 4-(4-fluorophenyl)-1, 3-dithia-2-thioxo-cyclopent-4-ene (DT827C) on liver injury induced by carbon tetrachloride (CCl₄) and orotic acid was studied using male rats. The approximate lethal doses were about 100mg/kg for DT827A-treated animals and more than 800mg/kg for the other two compounds-treated groups. Single oral administration of the three test compounds at the dose levels of 2 and 10 mg/kg 1 hour before CCl₄ exposure revealed a protective effect on the findings of centrolobular necrosis, balloon cells and macrophage infiltration in histopathological findings in livers in the order of DT827B-treated rats > DT827A-treated rats > DT827C-treated rats. Repeated oral administration of the compounds at the dose levels of 2 and 10 mg/kg/day for 10 consecutive days revealed a protective effect against liver injury on the findings of centrolobular necrosis, balloon cells and macrophage infiltration in the order of DT827B-treated rats > DT827A-treated rats ≒ DT827C-treated rats. Simultaneous administration of the compounds at the dose level of 10 mg/kg/day together with a high sucrose diet containing orotic acid for 12 days revealed an inhibitory effect on fatty liver formation in the order of DT827B-treated rats > DT827C-treated rats > DT827A-treated rats. A hepatoprotective potential of the DT827 series compounds was suggested under the conditions of these studies, and DT827B was considered to be the most effective.

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Eighty Sprague-Dawley rats, consisting of 40 animals of each sex, were examined for age-related lesions after a 13-week food restriction. From 6 weeks of age, four groups of 10 male and 10 female rats each were fed normal pelleted chow, ad libitum for the control group and approximately 85%, 70% and 55% of feed consumption in the control group, for 13 weeks. Estrous cycles in females were examined during the 3rd through 12th week of the study, and hematological, blood biochemical and pathological examinations of all animals were carried out after the 13-week feeding. Estrous cycles were prolonged or stopped in most females of the 55% feeding group from the 7th week of restricted feeding and atrophy of the theca granulosa cells with decreased cytoplasmic lipid droplets was found in the ovaries at necropsy. In the males of the 55% feeding group, Leydig cell atrophy was found in the testes. The groups at 70% and 85% feeding showed no abnormality in hematological, blood biochemical and pathological observations. On the other hand, decreases in plasma lipid peroxide levels were noted in the 70 and 85% feeding groups. These results suggest that 70% and 85% feedings seem suitable for minimizing development of age-related changes without nutritional disorders.

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The present experiments were undertaken to clarify the differences in humoral immune response to two β-lactam antibiotics, benzylpenicillin potassium (PcG) and cephalothin sodium (CET), in guinea-pig strains. Guinea pigs of three different strains, Hartley, Strain 2 and Strain 13, were immunized subcutaneously with PcG (10mg/body) 1 or 3 times a week, 10 times in total, or with CET (20 mg/body) 3 times a week, 12 times in total. Humoral immune responses to the two antibiotics were assessed by passive cutaneous anaphylaxis (PCA) and active systemic anaphylaxis (ASA) reactions. The relative intensities of the responses to PcG and CET detected by PCA reaction were Hartley > Strain 2 > Strain 13. On the other hand, Hartley and Strain 13 guinea pigs displayed high responses to the two antibiotics by ASA reaction, and Strain 2 exhibited a relatively low response. Based on these results, it was clarified that the Hartley strain, which is the most common strain used in ordinary allergenicity tests, showed the highest response to the two antibiotics tested in PCA and ASA reactions.

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The purpose of this study was to morphologically assess a possible mechanism for caffeine-induced ocular hypertension. Taking into consideration the relationship between the secretion of aqueous humor and the ultrastructure of the ciliary body, the time course of the morphological features in the ciliary epithelium when caffeine was administered intravenously to male Wistar rats was investigated by electron-microscopy. These morphological findings were also compared with the changes in the intraocular pressure (IOP). A significant increase in IOP was noted 15 min and 1 hr after a single dosing of caffeine alone. This change disappeared in all animals within 2 hr after dosing. The IOP in the animals receiving caffeine and the β-blocker befunolol, which lowers the IOP by inhibiting aqueous humor secretion, decreased significantly from 15 min after dosing, and this change persisted 2 hr after dosing. In electron-microscopy 15 min and/or 1 hr after dosing with caffeine, a slight dilatation in the lateral intercellular spaces near the basement membrane of the non-pigmented ciliary epithelium was observed and the interdigitations between the non-pigmented epithelial cells were intact. Reversal of these changes was observed 2 hr after dosing. On the other hand, the lateral intercellular spaces between the non-pigmented epithelial cells were markedly dilated and the interdigitations were disorganized following dosing with caffeine alone and in combination with befunolol. These results described here indicate that the intravenous administration of caffeine causes ocular hypertension and also changes in the non-pigmented ciliary epithelium, suggesting an enhancement of aqueous humor transportation. This paradigm in the rat is considered to be useful to further assess caffeine-induced ocular hypertension and for use as an animal model in glaucoma research associated with an aqueous humor secretion.

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Repeated blood collection from pregnant rats was examined for effects on fetuses. Blood was collected four times from the same pregnant rat (0.5 ml each time and 2 ml in total). In Experiment 1, blood was first collected on day 9 of gestation and then repeatedly at 1, 4 and 18 hr intervals. In Experiment 2, blood was collected once on day 6 of gestation and then repeatedly on days 9, 12 and 15. Decreases in body weight and food consumption of the pregnant animals were noted in both experiments and considered due to restraint and needle puncture. In neither experiment could any effects be found on day 20 of gestation with regard to viability, fetal weight, placental weight or gross malformation. The results indicated that this regimen of blood collection could be used in developmental toxicity assessment studies.

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