bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Simple Method for Cytotoxicity Studies of Non-hydrosoluble Substances. Possible Application as an Alternative to the Draize Test for Cosmetics and Toiletries
Authors: Boue-Grabot M. , Halaviat B. , Pinon J. François 
Published: August 2020

The use of a microporous membrane as an interface between a cell culture and a substance dissolved into a non-aqueous, non-cytotoxic medium made it possible to study the cytotoxicity of non-hydrosoluble substances, without addition of any adjuvant for dispersion or solubilisation. The LC50s of raw materials widely used in cosmetic and pharmaceutical products were established on skin human fibroblasts. This method was used for the study of cosmetics and toiletries which could be tested directly in their actual galenic forms. A relationship between exposure time and cytotoxicity was established for 50 products and a cytotoxicity index was calculated. The results were compared with those of the Draize irritation test on the rabbit eye. This comparison showed a very good correlation between the calculated cytotoxicity index and the ocular irritation index (p < 0.001). From the results of this preliminary study, it appears that the method could provide an alternative to animal studies on the ocular tolerance of cosmetics and toiletries.
Inhibition of Protein Synthesis after Exposure of Caco2 Cells to Heavy Metals
Authors: Scarino Maria Laura , Poverini Roberta , Di Lullo Giuseppe , Bises Giovanna 
Published: August 2020

The toxicity of two essential trace elements, the heavy metals zinc and copper, has been studied in Caco2 cells, a human colon carcinoma cell line able to differentiate in culture into mature enterocytes. The ID50 for zinc and copper in Caco2 cells was determined from dose-effect curves. The ID50 is the same (500μM) for zinc and copper in undifferentiated cells, but in differentiated cells the ID50 for zinc (1mM) is higher than the ID50 for copper (600μM). Total protein synthesis is more severely affected by both metals in undifferentiated Caco2 cells than in differentiated cells. This difference could be explained by the different intracellular accumulation of the two metals at the two stages of differentiation. Copper inhibits total protein synthesis more than zinc, at both stages of differentiation. A protective role of zinc-induced metallothionein is discussed in relation to these results.
ECITTS: An Integrated Approach to the Application of In Vitro Test Systems to the Hazard Assessment of Chemicals,
Authors: Walum Erik , Balls Michael , Bianchi Vera , Blaauboer Bas , Bolcsfoldi George , Guillouzo Andre , Moore Gregory A. , Odland Lena , Reinhardt Christoph , Spielmann Horst 
Published: August 2020

As a result of a workshop held at Täljöviken, Åkersberga, Sweden, on 27–29 May 1991, a multicentre collaborative research project was established, with the purpose of developing the concept of integrated in vitro toxicity testing. The first priority was the selection of tests within eight appropriate areas: basal cytotoxicity, irritancy, developmental toxicity, hepatotoxicity, nephrotoxicity, immunotoxicity, neurotoxicity and biokinetics. An ideal battery of tests for each area was identified. Since it was realised that it would not be feasible to include the full ideal list of tests in the project, a minimum test list was also agreed. For each area, ten calibration chemicals were selected. From these 80 compounds, 30 were selected for inclusion, together with 20 of the OECD test programme chemicals, in a first test set of chemicals. The toxicity of these 50 test set chemicals will be investigated in the minimum integrated test scheme. The aim of the project is to employ non-animal methods to assess the toxicological properties of chemicals, and to improve this assessment through the use of knowledge about mechanisms of toxic action. The information produced will contribute to the establishment of a more-scientific and more-efficient toxicological procedure for hazard assessment. Questions concerning which parameters need to be investigated and combined to make hazard assessments, and which parameters relevant to in vivo toxicity can be determined in non-whole animal test systems, will also be addressed.
The Eye (Rabbit/Human): Parameters to be Measured in the Field of Ocular Irritation
Authors: Hubert François 
Published: August 2020

The so-called Draize eye test, the offical method used worldwide for the evaluation of ocular irritancy, has been heavily criticised in recent years concerning its scientific credibility and its extensive use of animals. Numerous methods have been, or are being, developed with the aim of complementing and/or replacing the Draize test. Regardless of their nature, these new techniques will have to be validated to guarantee their scientific credibility. Used singly or in batteries, the alternative tests will have to evaluate all the components of ocular toxicity, and in particular the reversible or non-reversible nature of the observed effects.
In Vitro Systems for the Study of Cereal Proteins Toxic in Coeliac Disease
Authors: De Vincenzi Massimo , de Ritis Giorgio , Dessi Maria R. 
Published: August 2020

For a number of years, we have been carrying out investigations intended to clarify the nature of the toxic gliadin peptides, and whether different species and/or cultivars of wheat are tolerated to varying extents by people suffering from coeliac disease. Our investigations have been carried out using three main biological systems: 1) cultures of biopsy specimens of human intestinal mucosa; 2) cultures of rat fetal intestine; and 3) cultures of erythroid K562(S) cells. Using these systems, we have tested several peptide mixtures prepared from gliadin and non-gliadin fractions extracted from wheats having various degrees of ploidy and from other cereals. Fraction preparation has been carried out by means of sequential digestion with pepsin and trypsin under experimental conditions simulating protein digestion in man. The results obtained indicate that the pathological symptoms associated with intolerance to gluten are largely due to the cytotoxic activity of wheat gliadin peptides toward immature enterocytes.
Cytotoxicity Studies of Anchorage-independent LS-L-929 Mouse Fibroblasts Using Membrane Integrity, ATP Content and ATP/ADP Ratio as Determinants: Including the Xenobiotic Effects of Components in an Experimental Shampoo Formulation
Authors: Kemp Richard B. , Anton Andreas , Carlsson Marie , Hirt Doris H. 
Published: August 2020

Cell death (CD50), as measured by loss of membrane integrity using fluorescein diacetate and ethidium bromide, and reduction in ATP content (ATP50), were used as endpoints in a comparative study of the cytotoxicity of 40 chemicals (supplied by FRAME) to anchorage-independent LS-L-929 mouse fibroblasts. The rank order correlation of the two tests was strong (r = 0.98), but the ATP50 values only showed a weak correlation with rat acute lethality (LD50) data (r = 0.43). More-mechanistic endpoints were sought in order to characterise cellular energy status, and the effects of acetaminophen, acetylsalicylic acid, diazepam, digoxin and ethanol on ATP/ADP ratio, phosphorylation potential and adenylate energy charge were examined in detail. The first two ratios were very sensitive to toxic insult to the cells and gave results which more closely mimicked differences in human acute oral lethality than did the ATP50 and CD50. In addition, there was evidence that digoxin and diazepam affected oxidative phosphorylation, which suggested an alteration to mitochondrial function. Energy charge appeared less sensitive to the xenobiotics, probably owing to the dampening effect of the denominator in the ratio. The effects of individual components of an experimental detergent-based formulation on cell death were examined. It was found that one detergent gave different results, depending on the presence or absence of a second detergent. A commercial cream containing varying amounts of glyceryl monostearate contaminated with varying levels of sodium dodecyl sulphate gave results which reinforced the point that cytotoxic assays are valuable in quality assurance.
Testing Acute Toxicity in the Embryo of Zebrafish, Brachydanio rerio, as an Alternative to the Acute Fish Test: Preliminary Results
Authors: Schulte Christoph , Nagel Roland 
Published: August 2020

Acute toxicity testing in fish is a standard method used in estimating the influences of chemicals on aquatic vertebrates. The ecotoxicological data obtained from acute toxicity tests in fish are, however, not sufficiently reliable to justify the continued use of this test. Fertilised eggs of zebrafish ( Brachydanio rerio) were used to test the acute toxicity of chemicals. They were chosen because the development of B. rerio has been studied extensively and information already exists concerning the normal development of this species. The following parameters of the development of B. rerio were observed: coagulation of the egg, gastrulation, number of somites, movement, development of organs, pigmentation, heartbeat and circulation. Some of these toxicological endpoints indicate lethality. In addition, various interactions between the test chemical and the embryos can be measured by investigating these parameters. Six chemicals (2,4-dinitrophenol, 4,6-dinitro-o-cresol, malathion, carbaryl, phenol and 4-nitrophenol) were selected for testing in the embryos of zebrafish within the first 48 hours of their development. The toxicities of the test chemicals to zebrafish embryos were compared with their acute toxicities to adult fish. Further investigations with more chemicals are in progress. The effective concentrations (EC50) and the lowest effect concentrations (LOEC) investigated in this preliminary study were comparable to the LC50 values for adult fish. The use of this test system as an alternative method to testing toxicity in adult fish is discussed.
The Calorimetric-respirometric Ratio: Its Potential as a Cytotoxicity Test
Authors: Kemp Richard B. , Stephansen Catherine , Mohamed Sajid , Meredith R.W. John 
Published: August 2020

The ratio between heat flux and oxygen flux, the calorimetric ratio, is an enthalpy budget device used to identify anaerobic pathways in the presence of respiration. Ratios that are more exothermic (i.e. more negative) than the average for catabolic substrates (-450kJ mol O2 ± 5%; Thornton's rule), are usual for cells established in culture, including suspension-adapted LS-L929 mouse fibroblasts. A common reason for this is a high level of glycolysis, to produce lactate, simultaneously with aerobic pathways. To test the idea that the calorimetric-respirometric (CR) ratio is a revealing cytotoxic endpoint, LS cells grown in serum-rich medium were insulted with known metabolic poisons. Malonate, 2,4-dinitrophenol (2,4-DNP) and a mixture of antimycin A and rotenone increased the CR ratio to degrees largely explained by greater lactate flux, the CR700 values being 22μM malonate, 56μM 2,4-DNP and, for the mixture, 2μM antimycin A and 5μM rotenone. Higher concentrations of 2,4-DNP gave an “exothermic gap” for which there was no explained pathway. Iodoacetate decreased the CR ratio while inhibiting glycolysis, a result which can be explained by the hypothesis that substrates available in the serum were degraded by mitochondrial pathways and thereby substituted for the normal input from the Krebs cycle, which had been arrested by pyruvate starvation. In a balanced salt solution containing only 5.5mM glucose, the metabolic rate slowed and the CR ratio was more exothermic (CR700 = 6μM), giving a “gap” for which there was no explanation. Ten MEIC chemicals gave CR700 endpoints which closely corresponded to the order of toxicity for a battery of tests using animal cells. The CR method thus provides a good basis for investigating the mechanisms by which chemicals have toxic effects on cells.
Alternatives in Safety Testing: Progress or Uselessness?
Authors: Roberfroid Marcel 
Published: August 2020

Safety testing is a major responsibility of toxicologists. Toxicology is not only a science but also an art. The science of toxicology characterises the toxic potential of a given chemical entity, i.e. the intrinsic property which allows it to react with, and/or to be transformed by, a particular biological system. Based on such scientific data, the art of toxicology has to predict the risk, i.e. the probability that a particular adverse event will occur during a stated period of time or result from a particular challenge. Until now, the science of toxicology has relied almost exclusively on animal tests, the protocols of which are described in directives and regulations. As stated in an Editorial in ATLA (1) the question that toxicologists now have to tackle is, “can non-animal toxicity studies become genuine replacement alternatives …” for assessing risk adequately? Indeed, the science of toxicology has developed, and continues to develop, new approaches (alternatives) to characterise, in well-defined in vitro models (including, for the first time, human models), the toxic potential of chemicals, namely, cytotoxicity, organ-specific effects, modulation of metabolic functions, interference with cell-mediated processes, metabolic activation, etc. But the question remains, what about the art of toxicology? Is it realistic to predict that such new scientific data will, in time, be accepted by regulators for risk evaluation? If these data are to be accepted, we believe that, instead of the present trend towards a regulation-required “protocol toxicology”, toxicologists will have to impose a stepwise decision-tier approach based on the systematic and sequential progression of scientifically justified and rigorously performed investigations, the results of which will be thoroughly and realistically evaluated by experts. It has to be recognised that scientific knowledge has advanced far enough to permit a focus on mechanisms, so that alternatives are fully accepted, no longer as a supplement to a check-list approach, but as a full part of the scientific expertise.
Reduction in Animal Use 35 Years after Russell & Burch's Principles of Humane Experimental Technique
Authors: Festing Michael F.W. 
Published: August 2020

“Reduction” in animal use can be achieved by better experimental strategy and improved experimental design. In 1959, one strategy for discovering new drugs involved the random testing of thousands of chemicals in animal models of disease. This strategy was deplored by Russell & Burch in The Principles of Humane Experimental Technique (1959) as being inefficient and inhumane, and has now been almost completely superseded, following fundamental research resulting in an improved understanding of the mechanisms of drug action. Random screening is still done on a large scale, but by using in vitro methods. Good experimental design involves the control of variation. In 1959, it was not clear whether isogenic (inbred or F1 hybrid) strains of mice and rats were more uniform than outbred stocks and whether they should therefore be used preferentially in bioassays. It is now generally accepted that F1 hybrids and inbred strains tend to be more uniform, but, as precision also depends on sensitivity, which is unpredictable, there is no general rule for choosing the best strain for a bioassay. Choice should be based on pilot studies. However, the use of more-uniform, specific pathogen-free animals, which were only just becoming available in 1959, has certainly reduced the number of animals which are used. Other aspects of experimental design, such as the need to avoid bias, have a wide range of applicability; the importance of simplicity and the ability to calculate uncertainty are, apparently, not always appreciated by research scientists. The concept of “reduction” has been useful in focusing attention on the need for improved experimental design. In the long-run, animal welfare legislation and improved training for research scientists should lead to improvements, not only in the way in which animals are kept, but also in the way in which experiments are planned and analysed.
Animal Models and Alternatives in the Quality Control of Vaccines: Are In Vitro Methods or In Vivo Methods the Scientific Equivalent of the Emperor's New Clothes?
Authors: Hendriksen Coenraad , van der Gun Johan 
Published: August 2020

In the quality control of vaccine batches, the potency testing of inactivated vaccines is one of the areas requiring very large numbers of animals, which usually suffer significant distress as a result of the experimental procedures employed. This article deals with the potency testing of diphtheria and tetanus toxoids, two vaccines which are used extensively throughout the world. The relevance of the potency test prescribed by the European Pharmacopoeia monographs is questioned. The validity of the potency test as a model for the human response, the ability of the test to be standardised, and the relevance of the test in relation to the quality of the product are discussed. It is concluded that the potency test has only limited predictive value for the antitoxin responses to be expected in recipients of these toxoids. An alternative approach for estimating the potency of toxoid batches is discussed, in which a distinction is made between estimation of the immunogenic potency of the first few batches obtained from a seed lot and monitoring the consistency of the quality of subsequent batches. The use of animals is limited to the first few batches. Monitoring the consistency of the quality of subsequent batches is based on in vitro test methods. Factors which hamper the introduction and acceptance of the alternative approach are considered. Finally, proposals are made for replacement, reduction and/or refinement (the Three Rs) in the use of animals in the routine potency testing of toxoids.
The Misuse of Chimpanzees in Biomedical Experiments
Authors: Johnson Kirstin 
Published: August 2020

Field studies of the chimpanzee have earned our closest evolutionary relative extensive admiration and respect, but the very similarities we share have also exposed it to cruel exploitation in medical research laboratories. The scientific reasoning behind such use of chimpanzees for research into human diseases such as AIDS and hepatitis is weak, and the use of human volunteers, although with its own ethical dilemmas, is the only scientifically valid alternative method currently available. Once the ethical issues are explored, there is no doubt that inflicting such abominable treatment on such intelligent, social animals is morally unacceptable. Until these experiments stop completely, those interested in primate welfare should campaign to improve conditions, so that those chimpanzees in existing laboratories at least experience some small improvement in their lives.
Cryopreservation of Rat and Monkey Liver Slices
Authors: de Kanter Ruben , Koster Henk J. 
Published: August 2020

A method for the long-term storage of liver slices by cryopreservation was developed. The viability of liver slices was determined by analysing the leakage of alanine aminotransferase, urea production, and the metabolism of testosterone. Rat liver slices were found to be optimally cryopreserved by exposure for 30 minutes to 12% dimethyl sulphoxide (v/v) at 2°C before freezing. Subsequent direct immersion in liquid nitrogen was more effective than a cooling rate of ±1°C/mmute, which reduced viability. Storage at a temperature of -80°C lowered viability compared to storage at -196°C. These conditions for optimal cryopreservation were used to cryopreserve rat, rhesus monkey and cynomolgus monkey liver slices. The viability of these liver slices was maintained at: 74%, 86% and 85%, respectively, when alanine aminotransferase content was measured; 80%, 109% and 82%, respectively, when urea production was measured; and 109%, 60% and 85%, respectively, when the metabolism of testosterone was measured. Viability was maintained for at least one month. The results show that, by using the method presented here, liver slices from these species can be stored while maintaining viabilities similar to initial values. This method will facilitate the optimal use of liver slices and reduce the number of experimental animals used.
Intestinal Motility: A Computer Simulation of the Effects of Drugs on Colonic Peristalsis, for Teaching Undergraduate Pharmacology Students
Authors: Dewhurst David , Leathard Helen , Ullyott Richard 
Published: August 2020

An interactive computer-assisted learning (CAD program, which simulates experiments performed on segments of rat colon (in vitro) to study the pharmacology of intestinal motility, is described. The program covers: a) the actions of drugs that affect cholinergic excitatory neurotransmission in the colon; b) the effects of sympathomimetic amines; and c) an investigation of the mechanism of action of the laxative, phenolphthalein. The program can be run on any IBM-compatible PC, and makes use of text and high-resolution graphics to provide a background to the experiments and to describe the methodology. The screen display emulates a chart recorder, and simulated results, derived from actual data, are presented, in response to student selection of pre-determined experimental protocols from a menu. The program is aimed at undergraduate students, and is intended to support or replace a conventional laboratory practical class, while achieving the majority of the same teaching and learning objectives.
In Vitro-based and In Vivo-hased Simulations of Benzene Uptake and Metabolism in Rats
Authors: de Jongh Joost , Blaauboer Bas J. 
Published: August 2020

The metabolism of benzene was modelled in the rat by application of a physiologically based pharmacokinetic (PBPK) model. The model parameters were set by using reference physiological parameter values and reported partition coefficients from in vitro studies. Three sets of Vmax and Km values for benzene, derived from published in vitro studies, were substituted into the model while keeping all other model parameters constant. These model simulations were compared with two sets of empirical data on the metabolism or uptake of benzene after inhalation exposure. It was observed that the biotransformation parameter sets derived in vitro predicted all empirical data within a factor of two. In addition, it was observed that simulations across the two sets of empirical data which used biotransformation parameters obtained by fitting to one set of data to simulate the other set, led to results comparable to those in the in vitro-based simulations. It is concluded that the results of in vitro studies can be directly applied in a PBPK model in order to estimate the in vivo uptake and metabolism of benzene on the basis of previously determined model parameter assumptions. These results support earlier studies on the application of in vitro techniques for deriving PBPK model parameters. On the basis of other studies on the simulation of benzene kinetics, it is also concluded that additional studies are required to extend the validity of this approach for other compounds.