International regulatory uses of acute systemic toxicity data and integration of new approach methodologies.

Chemical regulatory authorities around the world require systemic toxicity data from acute exposures via the oral, dermal, and inhalation routes for human health risk assessment. To identify opportunities for regulatory uses of non-animal replacements for these tests, we reviewed acute systemic toxicity testing requirements for jurisdictions that participate in the International Cooperation on Alternative Test Methods (ICATM): Brazil, Canada, China, the European Union, Japan, South Korea, Taiwan, and the USA. The chemical sectors included in our review of each jurisdiction were cosmetics, consumer products, industrial chemicals, pharmaceuticals, medical devices, and pesticides. We found acute systemic toxicity data were most often required for hazard assessment, classification, and labeling, and to a lesser extent quantitative risk assessment. Where animal methods were required, animal reduction methods were typically recommended. For many jurisdictions and chemical sectors, non-animal alternatives are not accepted, but several jurisdictions provide guidance to support the use of test waivers to reduce animal use for specific applications. An understanding of international regulatory requirements for acute systemic toxicity testing will inform ICATM's strategy for the development, acceptance, and implementation of non-animal alternatives to assess the health hazards and risks associated with acute toxicity.

The importance of metrological tools to implementation of alternative method OECD TG 428.

Usually, if percutaneous absorption tests are conducted in accordance with OECD Guideline 428, in vitro determination is accepted by mainly regulatory agencies. In this paper, we focus on the lack of comparability of the results regarding the permeation parameter/flow rate, although it is widely discussed in the literature. This work sought to evaluate the absorption of caffeine using Franz-type diffusion cell with porcine ear skin samples, varying the storage duration and the way to handle them. Metrological tools were used for caffeine quantification such as certified reference material candidate, calibrated instruments, and validated methodology. Our results corroborate with the recommendation that membranes should be freshly prepared or frozen for short periods. Samples frozen for approximately one year should not be used because they present high cutaneous absorption. The results obtained for the absorption rate (J) are comparable to the results obtained by previous studies using similar experimental conditions. The evidence of the barrier characteristic promoted by the stratum corneum and the effect promoted by the storage time is shown through J = 6.25 ± 0.48 µg/cm2/h. We demonstrated the importance of metrological tools to guarantee reproducibility and comparability of the results between different laboratories.

Carotenoids from UV-resistant Antarctic Microbacterium sp. LEMMJ01.

The Microbacterium sp. LEMMJ01 isolated from Antarctic soil does not belong to any of the nearest species identified in the RDP database. Under UV radiation (A, B and C wavebands) the survival fractions of Microbacterium sp. cells were much higher compared with wild-type E. coli K12A15. Especially remarkable for an Antarctic bacterium, an expressive resistance against high UV-B doses was observed. The increased survival of DNA repair-proficient E. coli grown overnight added of 0.1 mg/ml or 1 mg/ml of the whole pigment extract produced by Microbacterium sp. revealed that part of the resistance of Microbacterium sp. against UV-B radiation seems to be connected with photoprotection by its pigments. Scanning electron microscopy revealed that UV-A and UV-B ensued membrane alterations only in E. coli. The APCI-MS fingerprints revealed the diagnostic ions for neurosporene (m/z 580, 566, 522, 538, and 524) synergism for the first time in this bacterium by HPLC-MS/MS analysis. Carotenoids also were devoid of phototoxicity and cytotoxicity effects in mouse cells and in human keratinocytes and fibroblasts.

FluxCTTX: A LIMS-based tool for management and analysis of cytotoxicity assays data.

BACKGROUND: Cytotoxicity assays have been used by researchers to screen for cytotoxicity in compound libraries. Researchers can either look for cytotoxic compounds or screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical. These assays may be used as an alternative to animal experimentation and are becoming increasingly important in modern laboratories. However, the execution of these assays in large scale and different laboratories requires, among other things, the management of protocols, reagents, cell lines used as well as the data produced, which can be a challenge. The management of all this information is greatly improved by the utilization of computational tools to save time and guarantee quality. However, a tool that performs this task designed specifically for cytotoxicity assays is not yet available. RESULTS: In this work, we have used a workflow based LIMS -- the Flux system -- and the Together Workflow Editor as a framework to develop FluxCTTX, a tool for management of data from cytotoxicity assays performed at different laboratories. The main work is the development of a workflow, which represents all stages of the assay and has been developed and uploaded in Flux. This workflow models the activities of cytotoxicity assays performed as described in the OECD 129 Guidance Document. CONCLUSIONS: FluxCTTX presents a solution for the management of the data produced by cytotoxicity assays performed at Interlaboratory comparisons. Its adoption will contribute to guarantee the quality of activities in the process of cytotoxicity tests and enforce the use of Good Laboratory Practices (GLP). Furthermore, the workflow developed is complete and can be adapted to other contexts and different tests for management of other types of data.

Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells.

In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.