alternative tests

/Tag:alternative tests

The Bioavailability of Substances Administered to Chick Embryos: The Maximum Effective Route of Administration

Drahomír Veselý, Doubravka Veselá and Richard Jelínek

Toxicokinetic studies are of key importance in both the design and the interpretation of developmental toxicity studies. The aim of this study was to determine concentrations of test substances within the chick embryo following the administration schedule recommended in the chick embryotoxicity screening test (CHEST). The concentration–time relationships were investigated by using four labelled substances with various physicochemical and embryotoxic properties ([14C] sodium acetate, [14C] palmitic acid, [3H] cortisol and [3H] cytosine arabinoside). These labelled chemicals were mixed with cold substances and singly administered at two dose levels to chick embryos on days 2, 3 and 4 of incubation. Extrachorial and subgerminal routes were used on day 2, and extrachorial and intra-amniotic applications were chosen on days 3 and 4. The concentration of labelled chemical present within the embryo was assessed at predetermined intervals by scintillation fluorimetry (from 6 minutes to 96 hours after administration), and used for estimating the concentration curves. Regardless of the substance, dose and application route, the concentration curves exhibited a characteristic pattern, reaching their peaks within the first 6 hours, and dropping down to near zero 48–96 hours after administration. The decrease followed the first order law, demonstrating that, within the CHEST system, the avian embryo does not act as a closed system. With regard to the total amount of substance entering the embryo, extrachorial administration appeared to be superior to subgerminal administration on day 2. Intra-amniotic administration was superior to extrachorial administration on days 3 and 4. These differences were most pronounced after administration of lipid-soluble palmitic acid. The concentrations within embryonic tissues were directly dosedependent. After consideration of all these findings, we concluded that the CHEST system probably has closer similarity to the toxicokinetics of exposure of mammalian embryos (i.e. reaching a peak and then a gradual decline over time) than any other in vitro test of developmental toxicity, where the chemical is simply added to culture media. Several practical recommendations for improving the CHEST system were derived.
You need to register (for free) to download this article. Please log in/register here.

The Detection of Differentiation-inducing Chemicals by using Green Fluorescent Protein Expression in Genetically Engineered Teratocarcinoma Cells

Sarah Adler, Martin Paparella, Cristian Pellizzer, Thomas Hartung and Susanne Bremer

The murine embryonal teratocarcinoma cell line, P19, was genetically manipulated in order to provide preliminary information on compounds that induce differentiation. Without chemical induction, P19 cells remain in an undifferentiated state, but can be induced to differentiate into specific cell types. For example, dimethyl sulphoxide (DMSO) induces cardiac and skeletal muscle differentiation, whereas retinoic acid stimulates neuronal differentiation. P19 cells were transfected with a construct containing a segment of the murineTert (mTert) promoter sequence combined with the green fluorescent protein (GFP) gene, which acts as a reporter gene. mTert expression, the reverse transcriptase component of murine telomerase, is closely linked to telomerase activity and is down-regulated during differentiation. Three retinoids and DMSO induced the differentiation of P19 cells, which was determined by a reduction in mTert_GFP expression, detected by flow cytometry and confocal microscopy as independent methods of detection. A test substance, ethanol, and a control substance, saccharin, did not cause a decrease in mTert_GFP expression. In addition, it could be demonstrated that the mTert_GFP test detects developmentally relevant effects at non-cytotoxic concentrations. The ID50 values derived for the reduction of mTert_GFP expression were lower than the IC50 values detected with the MTT test, by a factor of 21.4 for all-trans retinoic acid, 12.7 for 9-cis retinoic acid, 29.6 for 13-cis retinoic acid, and 8.7 for DMSO. In comparison to the IC50 value for the P19 cell line, a similar IC50 value was obtained with 3T3 cells for ethanol, but there was a 2-fold
increase for DMSO. The retinoids were not cytotoxic to 3T3 cells at the concentrations tested. This newly developed test is capable of detecting differentiation-inducing compounds at non-cytotoxic concentrations within 4 days. It offers a method for detecting chemicals with specific toxicological mechanisms, such as the retinoids, which could provide additional information in embryotoxicity testing as different promoters could be employed. Here, we report the use of this novel test system for the successful analysis of DMSO and three retinoids with different in vivo teratogenic potentials.
You need to register (for free) to download this article. Please log in/register here.

Working With Other Partners — The Retail Sector

Petrina Fridd

Animal testing to demonstrate the safety of cosmetic and toiletries has been used for many years. However, such testing, now viewed by many consumers as an emotive issue, has been banned within the UK, and is severely restricted in the European Union. Consumer knowledge and understanding have been challenged by the use of focus group studies, which showed that consumers were often confused and potentially misled by claims on labels and in leaflets. Despite the fact that some pressure groups had taken positive action, even holding store demonstrations, in attempts to stop such testing or claims, there was still a legal requirement to demonstrate the safety of products, and this involved the use of animal testing. Nevertheless, there was great pressure to move away from animal testing, clarify marketing and pack claims, and provide general transparency to the consumer. Therefore, the decision was taken to actively seek out and work with organisations which were working toward the validation of alternative testing. A number of potential organisations were approached and, after careful consideration of their respective aims and aspirations, it was decided that, for the retailer in question, FRAME was the most appropriate organisation with which to work.
You need to register (for free) to download this article. Please log in/register here.

Integrated Testing Strategies for Toxicity Employing New and Existing Technologies

Robert D. Combes and Michael Balls

We have developed individual, integrated testing strategies (ITS) for predicting the toxicity of general chemicals, cosmetics, pharmaceuticals, inhaled chemicals, and nanoparticles. These ITS are based on published schemes developed previously for the risk assessment of chemicals to fulfil the requirements of REACH, which have been updated to take account of the latest developments in advanced in chemico modelling and in vitro technologies. In addition, we propose an ITS for neurotoxicity, based on the same principles, for incorporation in the other ITS. The technologies are deployed in a step-wise manner, as a basis for decision-tree approaches, incorporating weight-of-evidence stages. This means that testing can be stopped at the point where a risk assessment and/or classification can be performed, with labelling in accordance with the requirements of the regulatory authority concerned, rather than following a checklist approach to hazard identification. In addition, the strategies are intelligent, in that they are based on the fundamental premise that there is no hazard in the absence of exposure — which is why pharmacokinetic modelling plays a key role in each ITS. The new technologies include the use of complex, three-dimensional human cell tissue culture systems with in vivolike structural, physiological and biochemical features, as well as dosing conditions. In this way, problems of inter-species extrapolation and in vitro/in vivo extrapolation are minimised. This is reflected in the ITS placing more emphasis on the use of volunteers at the whole organism testing stage, rather than on existing animal testing, which is the current situation.
You need to register (for free) to download this article. Please log in/register here.