in vitro alternatives

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Applications of the Neutral Red Cytotoxicity Assay to In Vitro Toxicology

Harvey Babich and Ellen Borenfreund

A concerted effort is currently in progress to develop alternatives to the use of live animals for the acute toxicity testing of xenobiotics. To this end, the neutral red in vitro cytotoxicity assay was developed which, although initially based on the use of mammalian cells in culture, has also been adapted for ecotoxicity studies using fish cells in culture. The neutral red assay is based on the binding of neutral red, a weakly cationic supra vital dye, to the lysosomal matrix of viable cells after their incubation with toxic agents. Spectrophotometric quantitation of the extracted dye with a scanning microtitre well reader at 540nm was found to be linear with the number of surviving, viable cells. The assay has been used to determine the relative acute cytotoxicities of a broad spectrum of chemical test agents, to establish structure-toxicity relationships for series of related chemicals, to study metabolism-mediated cytotoxicity, to evaluate interactions between combinations oftest agents, to evaluate differential and selective toxicities of cancer chemotherapeutics and other pharmaceuticals, and to study temperature-toxicity interactions.
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Harvey Babich & Ellen Borenfreund

A concerted effort is currently in progress to develop alternatives to the use of live animals for the acute toxicity testing of xenobiotics. To this end, the neutral red in vitro cytotoxicity assay was developed which, although initially based on the use of mammalian cells in culture, has also been adapted for ecotoxicity studies using fish cells in culture. The neutral red assay is based on the binding of neutral red, a weakly cationic supra vital dye, to the lysosomal matrix of viable cells after their incubation with toxic agents. Spectrophotometric quantitation of the extracted dye with a scanning microtitre well reader at 540nm was found to be linear with the number of surviving, viable cells. The assay has been used to determine the relative acute cytotoxicities of a broad spectrum of chemical test agents, to establish structure-toxicity relationships for series of related chemicals, to study metabolism-mediated cytotoxicity, to evaluate interactions between combinations oftest agents, to evaluate differential and selective toxicities of cancer chemotherapeutics and other pharmaceuticals, and to study temperaturetoxicity
interactions.
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The Mouse Bioassay for Diarrhetic Shellfish Poisoning: A Gross Misuse of Laboratory Animals and of Scientific Methodology

Robert D. Combes

The UK shellfish industry has recently been affected by the statutory closure of several cockle beds, following the detection of samples causing rapid and severe reactions in the regulatory approved test for diarrhetic shellfish poisoning (DSP) toxins, the mouse bioassay (MBA). It is contended that these socalled atypical results are due to procedural artefacts of the MBA; so far, several studies have failed to identify their cause. This paper critically assesses the development, regulatory use and methodological deficiencies of the MBA. It also discusses how testing for DSP toxins could and should have been improved and made more humane by applying the Three Rs concept of Reduction, Refinement and Replacement, and by the proper validation of the test method used. It is concluded that the MBA should not have been developed for the routine screening of shellfish samples, as it has a substantially severe endpoint and is not used as part of a tiered-testing strategy with non-animal methods. Moreover, during the UK monitoring programme for DSP toxins, the assay has been used without an optimised and universal protocol, and apparently without due regard to the principles of basic scientific methodology. In view of this, the atypical results obtained for cockle samples cannot be relied on as evidence of a human health hazard. It is recommended that the use of the MBA should be discontinued as soon as possible, in favour of other methods, especially those involving non-animal techniques. In the short-term, these methods should be based on analytical chemical detection systems and the essential availability of the relevant pure toxin standards. The lack of any known toxins in samples should be taken as evidence of lack of contamination. The suitability of the existing non-animal methods needs to be assessed as a matter of urgency. It is crucial that all new methods should be properly validated, and that their acceptability for their stated purposes should be endorsed by recognised criteria and validation centres, before being recommended to, or required by, regulatory agencies. In this way, the possibility that scientifically unsuitable methods will once again be used for monitoring for the contamination of shellfish with toxins can be avoided. This gross misuse of laboratory animals and ill-judged application of science should never be allowed to occur again.
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Ammonia-containing Industrial Effluents, Lethal to Rainbow Trout, Induce Vacuolisation and Neutral Red Uptake in the Rainbow Trout Gill Cell Line, RTgill-W1

Vivian R. Dayeh, Kristin Schirmer, and Niels C. Bols

Nine samples of whole effluent from the operation of an industrial plant over the course of one year, were tested on rainbow trout for lethality and on the rainbow trout gill cell line, RTgill-W1, for metabolic activity, plasma membrane integrity, and lysosomal activity, as measured by using the alamar Blue (AB), 5-carboxyfluorescein diacetate acetoxymethyl (CFDA-AM), and neutral red (NR) assays, respectively. None of the nine samples caused a loss of plasma membrane integrity, and only two caused a transitory decline in metabolism. Three samples caused massive vacuolisation in RTgill-W1 cells, which was accompanied by increased uptake of NR, and only these three samples were lethal to the rainbow trout. The addition of ammonia to RTgill-W1 cultures also induced vacuolisation and NR uptake, with little change in plasma membrane integrity or metabolism. Subsequently, the effluent source was identified as a nitrogen product producer, and variable levels of ammonia were found in the nine samples. Three of the four samples with the highest non-ionised ammonia levels were those which were toxic to rainbow trout and which caused vacuoles in RTgill-W1 cells. The close correlation between rainbow trout-killing and RTgill-W1 vacuolisation by the effluents, suggests that vacuolisation of RTgill-W1 cells could be used to indicate effluents which would be toxic to rainbow trout as a result of their ammonia content.
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Animal Use in the Chemical and Product Manufacturing Sectors — Can the Downtrend Continue?

Rodger Curren

During the 1990s and early 2000s, a number of manufacturing companies in the cosmetic, personal care and household product industries were able to substantially reduce their use of animals for testing (or to not use animals in the first place). These reductions were almost always the result of significant financial contributions to either direct, in-house alternatives research, or to support personnel whose duties were to understand and apply the current state-of-the-art for in vitro testing. They occurred almost exclusively in non-regulatory areas, and primarily involved acute topical toxicities. Over the last few years, the reduction in animal use has been much less dramatic, because some companies are still reluctant to change from the traditional animal studies, because systemic, repeat-dose toxicity is more difficult to model in vitro, and because many products still require animal testing for regulatory approval. Encouragingly, we are now observing an increased acceptance of non-animal methods by regulatory agencies. This is due to mounting scientific evidence from larger databases, agreement by companies to share data and testing strategies with regulatory agencies, and a focus on smaller domains of applicability. These changes, along with new emphasis and financial support for addressing systemic toxicities, promise to provide additional possibilities for industry to replace animals with in vitro methods, alone or in combination with in silico methods. However, the largest advance will not occur until more companies commit to using the non-animal test strategies that are currently available.
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