[in Japanese]

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A 90-day toxicity inhalation study was conducted in Wister WU rats with bitumen fumes. The composition of the fume was aimed to be similar to the exposure atmosphere released during road paving. The objective of this study was to determine the concentration levels and maximally tolerated dose for a future carcinogenicity study. Male and female rats, approx. 10 weeks old, were exposed to clean air, or to target concentrations of 4, 20, and 100mg/m³ THC (total hydrocarbon), 6 hrs/day, 5 days/week for 14 weeks. In the male hight dose group a decrease of body weight up to 10% versus controls was observed. Milder effects on body weight (approx. 5%) were noted in all female bitumen groups. A statistically significant lower food consumption was noted in the male high dose group.Statistically significant test substance-related changes were only observed in histopathology, i.e. the nasal and paranasal cavities from rats of the bitumen high dose group. Eosinophilic cytoplasmic inclusions (hyalinosis) were found exclusively in nasal epithelial cells of the high dose group. In addition, degenerative hyalinosis associated with focal/multifocal basal cell hyperplasia was observed in the olfactory/respiratory transition areas of high-dosed males. Other significant treatment-related changes were multifocal mucous (goblet) cell hyperplasia and multifocal mucosal inflammatory cell infiltration.Under the conditions of this subchronic study an NOAEL of 20mg/m³ THC was derived for bitumen fumes. Thus, target concentrations of 4, 20, and 100mg/m³ THC were defined for a chronic study focussing on examination of potential carcinogenicity; further endpoints are BAL and cell proliferation measurements of nose and lungs. In addition, as mechanistic endpoints the analysis of DNA adduct formation and gene expression profiling have been included.Results of the chronic study, together with epidemiological data, may allow the definition of a health-based threshold value for bitumen road paving workplaces. These studies are sponsored by ARBIT, Hamburg, Germany.

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[in Japanese]

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Toxicology has advanced a long way since Paracelsus determined that experimentation is essential in the examination of responses to chemicals. Driven by advances in technology and the pursuit of the 3R's, specialised investigative techniques are no longer the domain of the scientist in a fundamental research laboratory. They now form part of many Regulatory Toxicology studies.During the 1980's Regulatory Toxicology studies were very standard regardless of the compound being tested. The investigative nature of the studies extended only to effects on body weight, food consumption, clinical observations and histopathology. Twenty years later a 'standard' study has seen at least the addition of ECG and Blood Pressure recordings, extensive plasma profiling of the drugs absorption and excretion, determination of P450 enzyme activities and advanced histopathological examination.In addition, an increasing number of studies are seeing the introduction of specialised and highly targeted dosing techniques, non-invasive but highly informative techniques such as Echocardiography and the more in-depth analysis of blood or urine for the determination of cell subsets or biomarkers.This poster will examine some of these new techniques and the contribution they are making to advancing the knowledge of Toxicologists and determining the safety of chemicals.

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Deoxynivalenol (DON), a trichothecene mycotoxin produced from Fusarium spp, can cause toxic effects in both animals and humans. Wheat is one among a variety of cereal grains that Thai people consume daily. Therefore, randomized samplings of commercially available wheat grains (n= 100) from Bangkok markets were conducted, in order to investigate contaminated levels of DON. Collected samples were analyzed for DON concentration using enzyme-linked immunosorbent assay (ELISA). The results showed low levels of DON in the range of 0 - 0.88 ppm (average = 0.165 ppm).

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This poster presents the development of Cryo Tissue Micro Arrays (cTMA) specifically for the assessment of tissue cross reactivity (TCR) in the screening of potential therapeutic biopharmaceuticals.Due to the differing nature of biopharmaceuticals the Preclinical testing program needs to be individually designed, as it is generally not appropriate to follow the pharmaceutical safety testing program traditionally used for small molecules. One aspect of this program is the assessment of TCR on cryo preserved tissues.Classical Histological techniques for the preparation of cryo preserved tissues generally relies on a single tissue sample per slide and although numerous methods have been used to increase the number of tissue samples presented on a slide, the number of tissue samples presented has been limited.In 1998, Kononen et al. first described tissue micro array technology and although subsequent emphasis has been with tissue micro arrays prepared from fixed paraffin processed tissues using manual tissue arrayers (Beecher Instruments Inc), there has been little interest with the preparation of cTMA. Problems encountered in the preparation of cTMA's include the maintenance of tissues to remain frozen and morphology associated with the preparation of cryo-sections from cryo preserved tissues.With this in mind, a technique has been developed where up to 200 tissue cores of 1.5mm in diameter can be presented on a single slide.This has allowed for the high throughput screening of Biopharmaceuticals to assess potential TCR as well as the efficient analysis of biomarkers as potential indicators of toxicology.

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All components in the microarray workflow are important for performance of a microarray system. An optimized microarray processing protocol including hybridization and wash steps can significantly improve the reproducibility, accuracy, system noise, and sensitivity of the platform. This study compared a new microarray processing protocol with Agilent standard protocol as well as several other hybridization and wash conditions on Agilent 60-mer oligonucleotide microarrays. Different hybridization and wash protocols tested in this study differ in buffers and temperatures and were evaluated for system performance. Among them, the new protocol recommended by Agilent provided lower system noise, more accurate log ratio, and greater reproducibility.

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The Immunotoxicology Technical Committee (ITC) was created in 1990 and is the oldest component of the scientific program of the Health and Environmental Sciences Institute (HESI), the global branch of the International Life Sciences Institute (ILSI). The mission of the ITC is to identify and address scientific issues related to the development and application of immunotoxicology to public health and human health risk assessment; to promote the understanding and appropriate use of immunotoxicologic data to protect human health; and to contribute substantively to the scientific decision-making processes relative to the development of guidelines and regulations for immunotoxicology testing at the local, national and international levels. Over the last 15+ years, the mission of the ITC was addressed with the support of global member companies reflecting the agrichemical, chemical, consumer products, petrochemical and pharmaceutical industries, and significant input from academic and government colleagues. Through this effort, a firm foundation is fostered to decide how and where to best allocate scarce research resources in immunotoxicology. The focus of this presentation will be to highlight some of the recent activities of the ITC including the following: a better understanding of how to increase research opportunities to address immune-mediated drug hypersensitivity reactions (IDHR); a proposed testing framework for developmental immunotoxicology; an identification of the priority research needs in respiratory allergy reflecting protein-specific, chemical-specific, and drug-specific issues; an assessment of preclinical data needs for human immunogenicity of large molecules; an appreciation of clinical immunotoxicology-liabilities and ‘bridges’ between nonclinical and clinical approaches; and an update on the progress of creating a nonhuman primate immunotoxicology database. The success of the HESI ITC is based on an approach where international experts from academia, industry and government come together in a neutral forum and a cooperative atmosphere to engage in an open and transparent exchange of scientific ideas.

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Zearalenone (ZEA) and aflatoxins (AF) are secondary metabolites produced from Fusarium graminearum and Aspergillus flavus, respectively. In this investigation, 100 maize samples were collected from animal farms in the middle part of Thailand. Then, the ZEA and AF levels were mainly detected using enzyme-linked immunosorbent assay (ELISA). The results showed that ZEA levels varied from 0 - > 1000 ppb ( < 10 ppb= 13%, 11-40 ppb= 38%, 41-100 ppb= 25%, 101-300 ppb= 12%, 301 – 1000 ppb= 10% and > 1000 ppb= 2%) and AF levels varied from 0 - > 40 ppb ( < 2 ppb= 34%, 2.1-5 ppb= 33%, 5.1-10 ppb= 17%, 10.1-20 ppb= 8%, 20.1-40 ppb= 5% and >40 ppb= 3%). The ZEA and AF levels that were higher than the maximal limit of detection by ELISA, it would be confirmed using High Pressure Liquid Chromatography (HPLC) with fluorescence detector. In addition, possible relationship between the levels of ZEA and AF was also determined.

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During regulatory toxicity testing studies using rodents, test article is commonly administered admixed with powdered diet in order to ensure a stable plasma level. Circadian variation in feeding behaviour may results in alteration of exposure to test article.This poster reports characterisation of the circadian variation of food intake in rats maintained under typical toxicity laboratory husbandry conditions. The results are of particular use in interpreting data from investigations such as measurements of systemic exposure to test article, and helping determine the timing of such investigations during the working day.

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This purpose of this study was to validate the procedures used in the radio-telemetry monitoring of blood pressure, ECG, heart rate, body temperature and motor activity in rats with indwelling femoral vein catheters. 12 rats were simultaneously, surgically implanted with indwelling femoral vein catheters and radio-telemetry TL11M2-C50-PXT implants. For blood pressure measurements 6 of the animals were catheterised via the aorta and the other 6 animals via the femoral artery.Clonidine hydrochloride at dose levels of 0.1, 0.15 and 0.2 mg/kg/day given as a 72hour continuous intravenous infusion caused dose dependent reductions in blood pressure, heart rate and body temperature. No effects on any of the parameters were noted following the continuous intravenous infusion of physiological saline at dose volumes of 2, 4, 6 and 8 mL/kg/h. Nifedipine given as a 10mg/kg oral dose produced a dose dependent reduction in blood pressure, an increase in heart rate and associated ECG changes. D-Amphetamine sulphate given as a 3mg/kg subcutaneous injection resulted in an increase in blood pressure, heart rate, body temperature and motor activity. Rats implanted with both indwelling intravenous catheters and radio-telemetry implants serve as a useful tool for assessing the cardiovascular profile of intravenously administered test items

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This validation study was undertaken to demonstrate the ability of the DSI Telemetry system to detect physiological changes induced by test items of known pharmacological activity in the Beagle dog. For this purpose 4 adult Beagle dogs were surgically implanted with DSI TL11 M2-D70-PCT radio-telemetry devices. Following at least 3 weeks recovery from surgery the animals were assigned to the validation which comprised of 3 phases. Phase 1- Sotalol given orally at doses of 0, 4, 8 and 16 mg/kg. Phase 2 - Nifedipine given orally at doses of 0, 1, 2.5 and 5 mg/kg. Phase 3 – Cisapride given orally at doses of 0, 2, 4 and 8 mg/kg. All doses were administered at 1 to 2 day intervals. Systolic, diastolic and mean arterial blood pressure(MAP) , heart rate , Lead II ECG variables (PR, QT, QTcV and RR interval and QRS duration) and core body temperature were measured continuously for 1h prior to dosing and for up to 6h post dose. The animals were monitored for the recording period by digital CCTV. Data acquisition and analysis were performed using the Notochord HEM 3.5/DSI OpenART 2.3 system. Sotalol and Cisapride both produced dose dependent increases in QTcV. Nifedipine produced a dose dependent increase in heart rate and reduction in MAP. The Inveresk dog telemetry set up provides a scientifically robust system for conducting GLP studies.

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[in Japanese]

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This abstract describes the use of surgically prepared tethered rats to evaluate the anti-tumour efficacy effects of a protein kinase inhibitor. Female nude rats were subcutaneously inoculated with HCT 116 human colon colorectal carcinoma cells, surgically implanted with indwelling femoral vein catheters and monitored for tumour growth. Following a suitable tumour growth period thirty two animals were allocated to 3 treatment groups (0.5, 1 and 2mg/kg/h) and a control group. The treatment regimen comprised of 2 cycles of 72 hours of continuous intravenous infusion at a rate of 4ml/kg/h followed by a 4 day post dose observation period. For comparative purposes, an additional 4 animals were allocated to a fifth group and dosed by daily intraperitoneal injection (50mg/kg/day) over 2 cycles. The reduction in tumour growth noted for animals dosed at 2mg/kg/h by continuous intravenous infusion was comparable to that of the animals dosed by intraperitoneal injection (50mg/kg). Reductions in neutrophils and reticulocytes were evident for animals in the treated groups 48 hours after dosing of the first cycle and evidence of recovery was noted prior to commencement of the second cycle. Tumour mass weights measured at necropsy showed a dose related reduction for animals dosed by continuous intravenous infusion.

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