T-2 Toxin (12-13 epoxytrichothecene mycotoxin) induces apoptosis in basal keratinocytes when topically applied to the dorsal skin of Wistar-derived hypotrichotic WBN/ILA-Ht rats. In the present study, the effects of T-2 toxin on rat keratinocyte primary cultures were examined. Keratinocytes which were obtained from newborn Wistar rats and cultured by the method of Reynwald and Green (14) were used after the third passage. Keratinocyte medium containing 0.25μg/ml of T-2 toxin dissolved in DMSO or solvent alone was added to four-days-cultures and incubated at 37°C. At 0.5, 1, 3, 5, 7, and 9 hours after treatment (hr), feeder layer was separated from T-2 toxin-treated and control flasks, and cells were trypsinized. Cell viability was estimated by trypan blue exclusion method. In addition, RNA was obtained and RT-PCR was perfomed. Samples obtained from slide cultures at 3, 6, 9 and 12hr were fixed in 4% paraformaldehide or 2.5% glutaraldehide for morphological examination. After T-2 toxin application, cell viability decreased to 40% at 12hr. Pyknotic or karyorrhectic nuclei with cell body shrinkage were found in small sized keratinocytes at 6 hr, and their number increased until 12hr. These small sized keratinocytes showed ultraestructural changes characteristic for apoptosis; condensation of nuclear chromatine and phagocytosis of apoptotic bodies by neibour keratinocytes. At the same points, large squamous keratinocytes showed degeneration characterized by intracytoplasmic edema. The expression of apoptosis-related genes (c-fos and c-jun) and cytokines mRNA, TNF-α and IL-1β markedly increased prior to the development of apoptosis. These findings suggest that c-fos and c-jun oncogenes and TNF-α and IL-1β may play an important role in the development of T-2 toxin-induced apoptosis in basal keratinocytes.

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Over recent years there as been an increase in concern over the potential effects of chemicals on the human infant and adolescent. For pharmaceuticals agents, this increase in concern has led to the need to evaluate in young animals the effects of therapeutic agents intended for the treatment of the human young or agents which may be administered to this age group. For environmental chemicals the wider concern over the potential for life stage sensitivity has been directly translated into regulatory guidelines that address aspects of this potential issue, for example the need to assess the developmental neurotoxicity of pesticides.In order to scientifically and properly assess the effects of chemicals on the neonatal and young animal, it is essential to understand a number of key areas that influence study design. The normal development of the test animal species (rodent or non rodent) should be understood and how this may differ from the post natal development of the human. Any differences in kinetics and exposure between the young and the adult animal should be known and used in dose level selection. Having used this understanding to correctly design the study, of most importance is experience in successfully dosing young animals. At CTL we have extensive expertise in understanding comparative developmental biology, understanding comparative exposure and using our experience in dosing neonatal rodents and non rodents to conduct scientifically and technically robust assessments of juvenile toxicity.

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This poster is the 3rd part of our positive- and vehicle-control validation studies in murine local lymph node assay (LLNA). Based on our vehicle-control test results, ethanol-water systems were found to be suitable for LLNA tests. One of the most useful vehicles is the ethanol water (70/30, v/v)(EtOH 70%) solution.
In order to provide sufficient scientific rationale, a further investigation and comparison of the allergenic potency and local clinical signs caused by the positive control substance alpha-hexylcinnamaldehyde (HCA) have been made. In 6 parallel LLNA validation studies wiht 96 CBA/CaOlaHsd mice performed at RCC, 6 different solvent systems, 5 of them recommended by OECD Guideline 429, have been assayed.
In order to rationalize and quantitatively analyze the test results, some relevant physiochemical properties of the 6 organic solvents have been compared, and some statistic analyses have also been performed with the data obtained from 6 different vehicle systems.

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The common marmoset has been used extensively in biomedical research for more than 40 years and more recently, studies in this species have been used as part of the safety assessment to support clinical trials and registration of new pharmaceuticals. The common marmoset is an easily-bred primate that typically weighs 300-500g, approximating to the size of an adult rat. Consequently, it is a popular model for research and many technologies and methodologies used in the rat can also be performed in the marmoset. The dog is the non-rodent species of choice for pre-clinical toxicology studies but there may be reasons why, for certain products, it is unsuitable. For protein-based biopharmaceuticals the most appropriate non-rodent species is often the primate. Certain features of the common marmoset give it some advantages over the other commonly used primates (Cynomolgus and Rhesus monkey). For example, it becomes sexually mature at approximately 14-18 months (compared to approximately 4-years in the cynomolgus and rhesus monkey), therefore all studies can be conducted in sexually mature animals. Its small size also permits performance of some techniques, such as whole-body autoradiography, that are not practical in larger primates. The increasing use of the common marmoset has resulted in the availability of extensive background data and a summary of data for the most commonly measured parameters (haematology, plasma biochemistry, urinalysis, electrocardiography and organ weights) is presented. The extensive information available for the common marmoset and its many similarities to man make it an ideal primate model for pre-clinical studies in drug development.

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In this study, 6 main study groups of 10 F344 rats/sex were given 0,300,600,900,1200,1500mg/kg Myristica Fragrans Houttuyn for 90 days. Special endpoints in this study were included sperm morphology and vaginal cytology. All animals survived to the scheduled termination. In clinical signs, there were treatment-related salivation at dosage of 600, 900, 1200 and1500mg/kg after injection. Irregular estrous cycle and long anestrous period were observed in 900, 1200, 1500mg/kg treatment group. Absolute organ weights of liver were increased in male 1200, 1500 and female 900, 1200, 1500mg/kg treatment group. Necropsy findings showed uterus atropy in female 1500mg/kg treatment group. Histopathological findigs showed basophilic tubular change of kidney in all male treatment group compared than control group. In liver, mild cellular infiltration, microgranuloma, necrosis, hyperplasia, sinusoidal dilatation were showed dose-dependently in male and female treatment group. Atropy of uterus and ovaries were showed dose-dependently in female treatment groups. Based on these results, NOEL is considered to be 300mg/kg in male and female.

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In previous 2-year studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) conducted by the NTP using female Harlan Sprague-Dawley rats, acinar-cell vacuolation, atrophy, inflammation, and arteritis developed at high incidence, and a rare occurrence of pancreatic acinar-cell adenomas and carcinomas was noted. In this investigation, we sought to identify the mechanism involved in the early formative stages of acinar-cell lesions. Pancreas from animals treated for 14 and 31 weeks with 100 ng TCDD/kg body weight or corn oil vehicle was examined immunohistochemically and/or morphometrically. Acinar-cell kinetics was analyzed using staining with hematoxylin and eosin and proliferating cell nuclear antigen. Expressions of cytochrome P450 (CYP) 1A1 and aryl hydrocarbon receptor (AhR) were evaluated to assess direct effects of TCDD exposure. CCK-A receptor (CCKAR) and duodenal cholecystokinin 8 (CCK) revealed the associations of dioxin treatment with hormonal changes. Amylase localization showed acinar structural changes that could affect enzymatic production. Increased apoptotic activity in acinar cells occurred in 14- and 31-week-treated animals, with an increase in proliferative activity in the latter. Also in the latter, in the vacuolated acinar cells, CYP1A1 was overexpressed, while statistically significant decreases in expressions of AhR, CCKAR, and amylase occurred. The intensity of CCKAR expression increased in nonvacuolated acinar cells; a decrease in the size of CCK-positive epithelial cells occurred in duodenum. Our findings indicate that dioxin-induced acinar-cell lesions might be related to a direct effect of TCDD on the pancreas. Increase in CYP1A1 and decrease in CCKAR expressions in vacuolated acinar cells may be involved in the pathogenesis of pancreatic lesions.

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The telemetric measurement of cardiovascular and respiratory parameters in conscious, freely moving animals is an essential component of safety pharmacology studies. It is well known that heart rates as well as blood pressure values are different when measured in freely moving animals compared to restrained and/or in anaesthetized animals. For these reasons, data obtained by telemetry are considered to reflect the physiological condition. It was the aim of this study to evaluate an ITS system for the measurement of aortic blood pressure, ECG waveforms, temperature and intra-thoracic-pressure (ITP). Overall, six mature male cynomolgus monkeys (> 6.0 kg) were implanted with telemetry devices. Two male animals were dosed on two successive days with sotalol, ketamine, theophylline and chlorpromazine, respectively with saline as the corresponding vehicle. Biopotential signals were measured two hours before dosing and for a 6 hour period thereafter. Ketamine at 20 mg/kg caused a marked decrease in body temperature and in maximum ITP. QT-interval was markedly prolonged, heart rates and mean-arterial-blood-pressure were decreased at 10 mg/kg sotalol. Respiratory rate was decreased whereas the minimum ITP was slightly elevated following 25 mg/kg chlorpromazine administration. Theophylline at 20 mg/kg elicited no effect. On repeated occasions, pair- and single-housing of the two animals were compared: Heart rates as well as respiratory rates were reproducibly lower during pair-housing compared to single housing, whereas blood pressure remained unaltered. In conclusion, the ITS system is a valid tool for the selective detection of drug induced changes for cardiovascular/ respiratory parameters in conscious, freely moving monkeys. The ITS approach appears also feasible for animals under group housing conditions.

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Cardiac thrombosis, especially atrial thrombosis, accounts for more deaths in the world than any other disease, and major conditions in which thrombosis plays a principal role include several cardiovascular diseases, such as atrial fibrillation, myocardial infarction and stroke. Results from the induction of chemical thrombosis in rodents are useful to identify substances in our environment that may contribute to cardiac thrombosis. We evaluated atrial thrombosis induced by chemical exposures in rodents, compared it to similarly induced lesions reported in the literature, from the results of more than 500 chemicals in National Toxicology Program (NTP) database, and summarized a putative pathogenesis. The incidences of atrial thrombosis were increased in high-dosed groups in the NTP rodent studies of 13 compounds: 2-butoxyethanol, C.I. Direct Blue 15, bis(2-chloroethoxy)methane, diazoaminobenzene, diethanolamine, 3,3'-dimethyl-benzidine dihydrochloride, hexachloroethane, isobutene, methyleugenol, oxazepam, C.I. Pigment Red 23, C.I. Acid Red 114, and 4,4'-thiobis(6-t-butyl-m-cresol). The left atrium was the main localization in spontaneous and chemically induced thrombosis. The literature survey suggested that chemical-induced atrial thrombosis might be closely related to myocardial injury, endothelial injury, circulatory stasis and/or hypercoagulability. Impaired atrial mechanical activity, such as atrial fibrillation, could cause stasis of blood within the left atrial appendage, contributing to left atrial thrombosis.

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Alzheimer's disease describes a human degenerative brain disorder with an age-related increasing incidence. The present work was undertaken to evaluate whether aged cynomolgus monkeys (Macaca fascicularis) exhibit alterations indicative of Alzheimer's disease. Twenty cynomolgus monkeys (Macaca fascicularis) aged 2 - >20 years were examined for the presence of β-amyloid peptides 1-40 (Aβ40) and 1-42 (Aβ42), and the presence of phosphorylated and hyperphosphorylated tau protein species. Analyses were conducted in both cerebrospinal fluid (CSF) and brain tissue. Evaluations comprised similarities of parameters/alterations between the cynomolgus monkey and human and the relationship between age and amyloid/tau expression pattern. With advancing age, increasing Aβ42 content and altered Aβ42/Aβ40 ratio for CSF were observed in animals older than 15 years. These peptides were largely comparable to those found in aged human beings. In addition, tau protein was present at a low constant level. Development of plaque burden (including vascular amyloidosis) and tau pathology were detected by immunocytochemistry, silver stainng and standard staining protocols. Hyperphosphorylated tau protein and tau protein aggregates were present in nerve cells and in oligodendrocytes. Western blot evaluation revealed differences in tau phosphorylation in distinct brain areas. Insoluble tau protein migrated as bands at relative molecular masses of 64, 69 and 74 kD. This correlates well with the protein pattern found in some human tauopathies. In conclusion, the expression and distribution pattern of β-amyloid and tau parameters was similar for cynomolgus monkey and human. Our findings suggest that the (aging) cynomolgus monkey provides a potentially relevant model for the development of therapeutic drugs against human amyloidopathies and tauopathies.

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We examined the structure-phototoxicity relationship for fluoroquinolone antimicrobial agents (quinolones) using female albino Balb/c mice. First of all, to obtain an optimum dosage level for induction of phototoxicity, sparfloxacin was intravenously administered once at 10, 30 or 100 mg/kg to female mice, followed immediately by ultraviolet-A irradiation for 4 h (21.6 J/cm²). The auricular thickness was measured 4, 24, 48, 72 and 96 h later, and then the histopathological examination of the auricle was performed. As results, the auricular thickness increased from 30 mg/kg, and edema, cellular infiltration, epidermal necrosis and focal loss of the auricle were also morphologically observed. On the basis of these information, ciprofloxacin, enoxacin, fleroxacin, gatifloxacin, lomefloxacin, norfloxacin and ofloxacin were given intravenously to mice at a fixed dose of 100 mg/kg to compare their potential phototoxicities. Certain quinolones caused the auricular lesions in the following rank order (no to severe change): vehicle control (no-phototoxicity) = gatifloxacin = ofloxacin < ciprofloxacin = norfloxacin < enoxacin = fleroxacin < lomefloxacin = sparfloxacin. From the view point of the structure-phototoxicity relationship, quinolones possessing a fluorine, nitrogen or hydrogen at the 8 position of the quinolone ring evoked phototoxicity in the mouse auricle. These results demonstrate that phototoxicity induced by quinolones would be related to the property of a substituent at the 8 position of the quinolone ring.

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