Annual Meeting of the Japanese Society of Toxicology

Requirements on bioequivalence and relevant impurities are in place for the evaluation and registration of new compounds with biocidal or pesticidal activities. However, the registration requirements for established compounds from new suppliers or for established compounds produced by a different manufacturing process have been less clear and ambiguity exists as to how ‘equivalence of health hazards’ can unequivocally be demonstrated analytically and by toxicological assays. The case presented in this analysis focuses on the chiral pyrethroid transfluthrin (TFL) and chemically reactive impurities in low concentrations (<0.1%) difficult to capture analytically. This study compares reference TFL with commercialized generic TFLs from two alternative manufacturing sources in India and China. Despite their apparent high racemic purity these TFLs were biologically less effective, genotoxic in the Ames’ assay, demonstrated sensory lung irritation and lung/skin sensitization in specialized bioassays. Additionally, these TFLs demonstrated a high batch-to-batch variability. In contrast, the off-patent reference TFL was unequivocally negative in all assays. Tier I analytical assays failed to detect the most relevant, critical impurity in the absence of impurity-specific optimized analytical procedures. These findings suggests that a well-balanced, combined approach of analytical and toxicological assays provides the best means to assure objectively that all impurities deemed to be critical are identified and accounted for. In summary, this study demonstrates that ‘structural alert’-based toxicity tests proved to be more predictive than any indiscriminant battery of standard bioassays commonly applied to demonstrate equivalence. This applies specifically to chemically reactive impurities that may undergo site reactions during their analytical work-up.

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Phototoxicity sometimes has a strong impact on drug development. However, there is no validated in vivo system likes as 3T3 NRU PT test in vitro. This study focused on the usefulness of an in vivo phototoxicity model using Long-Evans (LE) rats for quantitative human risk assessment. Three phototoxic drugs for human, nabumetone, chlorpromazine and promethazine were selected because there were no positive results of these compounds in vivo phototoxicity reports using animals. Nabumetone (2000 mg/kg), chlorpromazine (40 mg/kg) and promethazine (200 mg/kg) were administrated once orally to female LE rats. The skin and eyes of LE rats were exposed by UVA at 10 J/cm2 around the Tmax of each compound. Daily observation of skin and eyes, ophthalmological examination 4 days after dosing, and blood sampling for toxicokinetics (TK) was also performed. No treatment-related phototoxic change was observed in rats with nabumetone. However, skin reaction was noted in rats treated with chlorpromazine or promethazine from Day 3. These results suggest that in vivo phototoxicity model using Long-Evans (LE) rats have enough sensitivity for human risk assessment.

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The integral assessment of neurobehavioral, cardiovascular and respiratory (NCR) system in telemetered conscious animals is becoming more widely employed in safety pharmacology studies. The purpose of this study was to assess the feasibility of performing a NCR safety pharmacology study with amphetamine and acepromazine, compounds known to modulate neurobehavior, when administered orally to conscious beagle dogs and to validate procedures.

0.5% methylcellulose (5 mL/kg), 1 mg/kg amphetamine (5 mL/kg) or 2-3 mg/kg acepromazine (two 10 mg tablets per animal) were administered orally to 4 male beagle dogs. The cardiovascular parameters (DSI ART: blood pressure, heart rate) waveforms), respiratory parameters (EMKA IOX RIP: respiratory rate, tidal volume and minute volume) and neurobehavior (EMKA IOX video) were monitored for 2 hours prior to and 24 hours following the dose administration. In addition, animals were observed for potential neurobehavioral effects using a standard observation battery which allows the assessment for both peripheral and central nervous system in dogs. At 2 and 24 hours postdose after the completion of the cardiovascular and respiratory data collection, the neurobehavioral assessments were performed while animals were in and out of their cages. Following amphetamine or acepromazine administration, the expected changes in NCR systems were observed, thus demoinstrated the validity of the procedures used.

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Abstract: In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line ), but also invasion of B16-BL6 cell in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially purified by filtration and hydrophobic column chromatography. Methods: B16-BL6, JYG-B or KPL-1 cells (2.5×10⁵cells/mL) were cultivated in 100μL of D-MEM in wells of 96-well microculture plate, with 10μL of extracts of the alga. The cell growth was assayed at 1, 2 and 3 days after the cultivation using a cell counting kit according to the instructions. Seven hundred microliters of B16-BL6 cell suspension (2.5×10⁵ cells) with algal extract (15μL) in D-MEM medium was added into the chamber, and fibronectin (10μg/mL) in the medium was added in the corresponding wells of 24-well plates. They were incubated in the CO₂ incubator for 24 hr. The melanoma cells which invaded through the filter to the outside surface of the chamber filter were counted after fixing with 70% methanol and staining with hematoxylin and eosin. Into the rail vein of female C57BL/6J mice (5 weeks old), 50,000 B16-BL6 cells were inoculated. Metastases of lung were monitored by counting lung-surface colonies of the mice 3 weeks after the inoculation. Results: The protein-like substance possibly affects the cancer cell directly (or indirectly) and inhibits its growth and metastasis. Further purification of the substance and in vivo studies will be done in next experiment to clarify the mechanism of the antimetastatic effects of a marine alga.

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