It is a requirement that parenteral medicines be tested for pyrogens (fever causing agents) using one of two animal-based tests: the rabbit pyrogen test and the bacterial endotoxin test. ...Understanding the human fever reaction has led to novel non-animal alternative tests based on in vitro activation of human monocytoid cells in response to pyrogens. Using 13 prototypic drugs, clean or contaminated with pyrogens, we have validated blindly six novel pyrogen tests in ten laboratories. Compared with the rabbit test, the new tests have a lower limit of detection and are more accurate as well as cost and time efficient. In contrast to the bacterial endotoxin test, all tests are able to detect Gram-positive pyrogens. The validation process showed that at least four of the tests meet quality criteria for pyrogen detection. These validated in vitro pyrogen tests overcome several shortcomings of animal-based pyrogen tests. Our data suggest that animal testing could be completely replaced by these evidence-based pyrogen tests and highlight their potential to further improve drug safety.
Good Cell and Tissue Culture Practice (GCCP) 2.0 is an updated guidance document from GCCP 1.0 (published by ECVAM in 2005), which was developed for practical use in the laboratory to assure the ...reproducibility of in vitro (cell-based) work. The update in the guidance was essential as cell models have advanced dramatically to more complex culture systems and need more comprehensive quality management to ensure reproducibility and high-quality scientific data. This document describes six main principles to consider when performing cell culture including characterization and maintenance of essential characteristics, quality management, documentation and reporting, safety, education and training, and ethics. The document does not intend to impose detailed procedures but to describe potential quality issues. It is foreseen that the document will require further updates as the science and technologies evolve over time.
Abstract Inorganic arsenic (iAs) is a human carcinogen, well known as a clastogenic compound. To evaluate the molecular mechanism of arsenite (iAsIII ) toxicity, we investigated the effects on cell ...growth and apoptosis, telomere length, telomerase expression, as well as the formation of reactive oxygen species (ROS) in male and female human cord blood cells in vitro . Incubation with iAsIII at the concentration of 0.0001 μM increased telomerase mRNA and protein expression maintained both telomere length and cellular growth, and induced mRNA over-expression of the two oncogenes ras and myc. Our results suggest that female cord blood cells are more sensitive than male ones to iAsIII induced telomerase stimulation at low concentrations, possibly related to the increased expression of ras and myc oncogenes. On the contrary, at the concentration of 1 μM, iAsIII decreased telomerase expression and telomere length, and induced apoptosis, necrosis and production of reactive oxygen species. Buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, markedly increased the percentage of apoptotic cells, suggesting that GSH is fundamental for detoxification of iAsIII in cord blood cells. The reactive oxygen species (ROS) scavenger, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), protected cord blood cells from iAsIII toxicity, and prevented telomere shortening and telomerase down-modulation. It can be concluded that telomerase expression and telomere length are associated with iAsIII induced cell death, via production of reactive oxygen species, as well as with iAsIII induced effects on cell differentiation processes and rate of cell growth.
Abstract Inorganic arsenic (iAs) and its metabolites are transferred to the foetus through the placental barrier and this exposure can compromise the normal development of the unborn. For this ...reason, we assessed the toxicity of sodium arsenite (iAsIII ) and its metabolites dimethylarsinic acid (DMAV ), monomethylarsonic acid (MMAV ) and monomethylarsonous acid (MMAIII ) on human haematopoietic cord blood cells and murine bone marrow progenitors in vitro , looking at the effects induced at different concentrations in the two genders. The expression of two enzymes responsible for arsenic biotransformation arsenic methyltranferase (AS3MT) and glutathione S -transferase omega 1 (GSTO1) was evaluated in human cord blood cells. Cord blood and bone marrow cells were exposed in vitro to iAsIII at a wide range of concentrations: from 0.0001 μM to 10 μM. The methylated arsenic metabolites were tested only on human cord blood cells at concentrations ranging from 0.00064 μM to 50 μM. The results showed that iAsIII was toxic on male and female colony forming units to about the same extent both in human and in mouse. Surprisingly, very low concentrations of iAsIII increased the proliferation rate of both human and murine female cells, while male cells showed no significant modulation. MMAV and DMAV did not exert detectable toxicity on the cord blood cells, while MMAIII had a marked toxic effect both in male and female human progenitors. AS3MT mRNA expression was not induced in human cord blood cells after iAsIII exposure. GSTO1 expression decreased after MMAIII treatment. This study provides evidence that exposure to iAsIII and MMAIII at μM concentrations is associated with immunosuppression in vitro.
This chapter focuses on practical aspects of conducting prospective in vitro validation studies, and in particular, by laboratories that are members of the European Union Network of Laboratories for ...the Validation of Alternative Methods (EU-NETVAL) that is coordinated by the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). Prospective validation studies involving EU-NETVAL, comprising a multi-study trial involving several laboratories or "test facilities", typically consist of two main steps: (1) the design of the validation study by EURL ECVAM and (2) the execution of the multi-study trial by a number of qualified laboratories within EU-NETVAL, coordinated and supported by EURL ECVAM. The approach adopted in the conduct of these validation studies adheres to the principles described in the OECD Guidance Document on the Validation and International Acceptance of new or updated test methods for Hazard Assessment No. 34 (OECD 2005). The context and scope of conducting prospective in vitro validation studies is dealt with in Chap. 4 . Here we focus mainly on the processes followed to carry out a prospective validation of in vitro methods involving different laboratories with the ultimate aim of generating a dataset that can support a decision in relation to the possible development of an international test guideline (e.g. by the OECD) or the establishment of performance standards.
PDF
