bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxicity of Non-hydrosoluble Substances towards Human Skin Fibroblasts Cultured on Microporous Membrane: A Model for the Study of Ocular Irritancy Potential
Authors: Boue-Grabot Marc , Halaviat Brigitte , Pinon Jean-François
Published: August 2020

The use of a microporous membrane as an interface between the cell monolayer and a substance dissolved in a non-aqueous, non-cytotoxic medium has permitted the study of the cytotoxicity of non water-soluble substances without the addition of any adjuvant for dispersion or solubilisation. The LC50 values of raw materials widely used in cosmetic and pharmaceutical products were established on human skin fibroblasts. This method was also applied to the study of cosmetic products. A relationship between exposure time and cytotoxicity was established for 169 products and a cytotoxicity index (MCI) was calculated. When the results were compared to the Draize maximal ocular irritation index (MaOI), a very good correlation was shown between MCI and MaOI.
Ocular Irritation: Present Cell Culture Models and Perspectives
Authors: Guillot Raymonde
Published: August 2020

Various cell culture models have been proposed as partial or total replacements for in vitro ocular irritation tests. The cytotoxic potential of substances can be evaluated according to various parameters for measuring cellular viability and metabolic capacity. Cell cultures have their limitations, but they also have advantages which justify the inclusion of some such models in a battery of in vitro tests that could replace the Draize test.
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.
An Interactive Computer Simulation of Experiments to Teach the Principles of Nutrient Transport in the Small Intestine
Authors: Dewhurst David , Hardcastle Jacqueline , Hardcastle Peter , Williams Alan
Published: August 2020

An interactive computer-assisted learning program to teach, by investigation, the characteristics of transport of nutrients by the rat small intestine, is described.
The Use of the Epithelial Kidney OK Cell Line for Studying the Nephrotoxicity of Anticancer Platinum Coordination Complexes
Authors: Toutain Hervé , Courjault Françoise
Published: August 2020

Nephrotoxicity is one of the most important dose-limiting side-effects of cis-diaminedichloroplatinum (II) (cDDP) in humans. Quiescent OK cells grown in hormonally-defined, serum-free medium in the total absence of antibiotics were used to study the in vitro nephrotoxicity of three platinum complexes which produce different renal toxicity in vivo: cDDP, its stereoisomer trans-diaminedichloroplatinum (II) (tDDP), and cis-diamine-1,1-cyclobutane-dicarboxylateplatinUm (II) (CBDCA). The uptake and cytotoxicity of these compounds at concentrations of 3-l,600μM and their impact on DNA and protein synthesis, glucose uptake, Na+-K+-ATPase and succinate dehydrogenase activities, as well as intracellular total glutathione level, were measured. The results showed that the cytotoxicity ranking of these three compounds, assessed by the LDH release method, was not in agreement with their in vivo nephrotoxic potentials (tDDP > cDDP > CBDCA00 in OK cells versus cDDP > CBDCA > tDDP in vivo). cDDP and tDDP showed similar uptakes at all the concentrations studied, which demonstrated that their cytotoxic potential was not directly related to intracellular levels of platinum. At non-cytotoxic concentrations, both cDDP and CBDCA decreased DNA and protein synthesis and, to a lesser extent, Na+-K+-ATPase activity, whereas no effect on glucose uptake and succinate dehydrogenase activity (a mitochondrial marker enzyme) was observed. Nevertheless, 5–10 times greater concentrations of CBDCA were required to induce effects similar to those induced by cDDP. Our results did not show a rapid and early depletion of intracellular glutathione after exposure to cDDP or CBDCA. tDDP exhibited the characteristics of a non-specific cytotoxic chemical which was unable to markedly inhibit the biochemical and functional parameters studied at non-cytotoxic concentrations. These results underline the key role of the inhibition of synthetic activities in the pathogenesis of cDDP-induced and CBDCA-induced nephrotoxicity in OK cells.
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.
Reductive Activation of Carbon Tetrachloride by Human Haemoglobin
Authors: Ferrara Roberta , Rezzadore Michela , Cazzaro Stefano , Tolando Roberto , Manno Maurizio
Published: August 2020

The reductive metabolism of carbon tetrachloride (CC14) by human haemoglobin (Hb) was observed in vitro by absolute absorption spectra recorded under anaerobic conditions. The following results were obtained: 1) a decrease of the 430nm peak typical of free reduced Hb (Hb2+); 2) the formation of a shoulder of absorbance, attributable to the production of a complex between Hb2+ and a metabolite of CC14 carbon monoxide (Hb-CO); and 3) the oxidation of some Hb2+ to methaemoglobin (Hb3+). The concentration of these three forms — Hb2+, Hb-CO and Hb3+ — during anaerobic incubation of Hb with CC14 was calculated algebraically from the absolute spectra. CO production was then calculated from the concentration of Hb-CO, using a suitable calibration curve. Interestingly, under identical experimental conditions, a substrate-dependent loss of Hb-derived haem, but not of Hb itself nor of haem-derived porphyrin fluorescence, was measured. Preliminary HPLC studies to clarify the discrepancy and, in particular, the role and fate of the haem group, showed two substrate-dependent modified haem products. The results indicate that human Hb is able to catalyse the reductive activation of CCl4, and suggest that, during the process, its prosthetic group haem may be modified by CC14 metabolites to products which maintain a tetrapyrrolic structure but are unable to react with pyridine.
The Evaluation of a Multi-endpoint Cytotoxicity Assay System
Authors: Renzi Daniela , Valtolina Marinella , Forster Roy
Published: August 2020

The aim of this study was to evaluate a multi-endpoint cytotoxicity screening method using V79 cells based on four different endpoints of cytotoxicity: trypan blue exclusion, reduction of XTT, neutral red uptake and total protein content. In addition, cell morphology was routinely observed after each treatment. Seven compounds were studied, which can be divided into five classes: protein synthesis inhibitors (cycloheximide, actinomycin D); inhibitors of cell division (bleomycin, vincristine); membrane-active compounds (Triton X-100); lysosomotropic agents (ammonium chloride); and general toxicants (sodium chloride). We obtained a variety of different toxicity profiles, which may be useful in defining the mechanisms of toxic action of these compounds. The multi-endpoint screening system proved to be readily applicable, robust and rapid, and gave reliable toxicity results over a wide range of chemical concentrations.
In Vitro Validation of Poly(Ester-Ether-Ester) Block Copolymers as Biomaterials
Authors: Guerra Rosella Sbarbati-Del , Cascone M. Grazia , Tricoli Mario , Cerrai Piero
Published: August 2020

Level I (neutral red uptake) and level II (cell adhesion and proliferation) in vitro cytotoxicity tests were used to evaluate the biocompatibility of newly-synthesised poly(ester-ether-ester) block copolymers to be used for the construction of vascular prostheses. The synthesis of these copolymers does not require the presence of added catalysts and results in high molecular mass materials with good tensile and mechanical properties. The results of the cytotoxicity tests suggest that the level of cytocompatibility of all the copolymers tested is high and very promising for their future use as biomaterials.
Toxicity of the First Ten MEIC Chemicals to Bacteria
Authors: Kerszman Gustaw
Published: August 2020

The toxicity of the first ten MEIC chemicals to Escherichia coli and Bacillus subtilis was examined. Nine of the chemicals were toxic to the bacteria, with the minimal inhibitory concentration (MIC) ranging from 10-3 to 4.4M. The sensitivities of both organisms were similar, but the effect on E. coli was often bactericidal, while it was bacteriostatic for B. subtilis. Digoxin was not detectably toxic to either bacterial species. Amitriptyline and FeSO4 were relatively less toxic to the bacteria than to human cells. For seven chemicals, a highly significant linear regression was established between log MIC in bacteria and log of blood concentration, giving lethal and moderate/mild toxicity in humans, as well as with toxicity to human lymphocytes.
An Alternative Test Using Algal Cultures for Testing Chemicals for Toxicity
Authors: Nielsen Søren Achim , Hougaard Thomas
Published: August 2020

An alternative test is presented, in which algal cultures are used for testing toxic substances. This test system is based on variations in the size distribution of cells in test cultures as a measurement of growth. Thus, inhibition of mitotic activity is used as a measurement for toxic effects. The test can be performed on a short time-scale and is very sensitive to even weak toxic doses.
Use of the Syrian Hamster Embryo (SHE) Cell Transformation Assay for the In Vitro Testing of Five Pesticides
Authors: Dušinská Maria , Slamen˘ova Darina , Kolman Ada
Published: August 2020

The morphological transformation of Syrian hamster embryo (SHE) cells has been used for the in vitro testing of the transforming activity of five pesticides (Dual, Vucht 524, Piritione, Decemtione and Supercypermethrin). Morphological transformation with two known inducers, 3-methylcholanthrene and N-methyl- N‘-nitro- N-nitrosoguanidine, was used as a positive control. It was found that only one of the tested pesticides, Dual, failed to induce morphological transformation in SHE cells, whereas the remaining four demonstrated varying degrees of positive response and could be regarded as potential carcinogenic chemicals.
Of Bacteria and Men: Toxicity of 30 MEIC Chemicals to Bacteria and Humans
Authors: Kerszman Gustaw
Published: August 2020

The toxicity of chemicals (numbers 11–30 from the multicenter evaluation of in vitro cytotoxicity [MEIC] programme) to Escherichia coli and Bacillus subtilis was examined. Malathion was not detectably toxic to bacteria. Theophylline was toxic to E. coli but not to B. subtilis. All of the other chemicals were toxic to both bacteria, with minimal inhibitory concentrations (MIC) ranging from 10-5M to 1.3M. The sensitivities of both organisms to the chemicals were quite similar. A highly significant correlation could be established for the first 30 MEIC chemicals between MIC in bacteria and acute lethal blood concentration in humans. A significant correlation was also established for sets of MEIC chemicals between MIC in bacteria and the relevant endpoint concentrations in various tests employing human cells.
Frog Skin: A Computer Simulation of Experiments Performed on Frog Skin In Vitro to Investigate the Epithelial Transport of Ions
Authors: Dewhurst David G. , Williams Alan
Published: August 2020

An interactive computer-assisted learning program is described, based on experiments performed on frog skin in vitro, a preparation commonly used to teach the principles of ion transport across tight epithelia. It is aimed at undergraduate students on a variety of biomedical science courses and is designed for use on any IBM-compatible microcomputer. The program uses data derived from theoretical models to allow students to design experiments by altering certain experimental parameters. They can investigate, for example, the effects of changing the concentrations of certain ions on either side of the skin or the actions of certain drugs on either passive or active transport. Such investigations involve taking measurements from a simulated voltmeter, ammeter or radiation (scintillation) counter, as appropriate. The complete learning package includes background information for the student, tutor's notes, and suggested student assignments. The use of the program in teaching physiology and its value as an alternative to animal experiments are discussed.
Effects of Benzalkonium Chloride on Pilocarpine-induced Opacity in Porcine Isolated Corneas
Authors: Igarashi Haruyoshi , Katsuta Yasunaga , Sawa Kayoko , Chiba Yuu , Kozima Michio , Ogawa Katsuhiro , Nakazato Yoshiharu , Kawasaki Tohru
Published: August 2020

The incubation of isolated porcine corneas (intact, with the epithelium or endothelium plus Descemet's membrane removed, or with both the epithelium and endothelium plus Descemet's membrane removed) with solutions of pilocarpine HCl (5 X 10 4M or 5 x 10-3M) for four hours caused very little increase in opacity when compared with corneas incubated with physiological saline. However, at a higher concentration (5 x 10-2M), the application of pilocarpine to the endothelial surface, or to both the epithelial and endothelial surfaces of intact corneas, caused an obvious increase in opacity. The addition of the preservative benzalkonium chloride (BC; 0.005%) to pilocarpine solutions caused an increase in opacity, but in no circumstances did this appear to be other than an additive effect, since incubation with BC alone had an opacifying effect. This in vitro test confirms that pilocarpine is a safe drug for application as eye-drops. Studies using high performance liquid chromatography showed that BC increased the amount of pilocarpine passing through the cornea from the epithelial to the endothelial surface. A small amount of BC also passed through the cornea over the 4-hour experimental period.
Magnetic Resonance Imaging and Spectroscopy: New Noninvasive In Vivo Approaches in Toxicology Research
Authors: Brauer Manfred
Published: August 2020

Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) give anatomical and biochemical information about a human patient or animal in a non-invasive manner. This unique quality permits the study of toxicological responses of an organ within an intact animal and in a manner in which many fewer animals are needed than by conventional methods of investigation. The use of MRI and MRS in the study of hepatotoxicants, particularly bromobenzene and ethanol, is reviewed. Bromobenzene causes localised hepatic oedema and bioenergetic deterioration; these changes were followed with time by 1H MRI and 31P MRS, respectively. Phosphocholine levels in the liver were found to increase dramatically during bromobenzene-induced damage, possibly related to an intracellular control mechanism in response to tissue damage. The ability of the bromobenzene-challenged liver to metabolise a fructose load was followed by dynamic 31P MRS. Chronic ethanol administration damages the liver. This toxicological process results in the accumulation of fat in the liver, which was followed by fat-selective 1H MRI. When ethanol is no longer administered to the subject, the fatty infiltration subsides, and this process was followed over 16 days in the same animal using fat-selective 1H MRI. Chronic ethanol renders the liver in situ more susceptible to hypoxic injury and less likely to recover afterwards, as shown by 31P MRS.
Skin Organ Culture as an Alternative to In Vivo Dermatotoxicity Testing
Authors: van de Sandt Johannes J.M. , van Schoonhoven Jacqueline , Maas Wilfred J.M. , Rutten Alphons A.J.J.L.
Published: August 2020

Various aspects of acute cutaneous toxicity were studied in a skin organ culture model. Chemicals were applied topically for four hours, after which cytotoxicity was assessed by measuring the conversion of the tetrazolium salt, MTT. The relationship between pKa and cytotoxicity was investigated for a homologous series of benzoic acids. In this series, salicylic acid had the lowest pKa and proved to be the most toxic compound. Furthermore, the pH of the carrier solution was shown to influence the toxicity of chloroacetic acid and acetic acid in a different way. Using skin discs of both human and rabbit origin, we found that human skin was more resistant to toxicity induced by the irritants benzalkonium chloride and formaldehyde. As an additional aspect of dermal toxicology, the percutaneous absorption of testosterone was studied. After topical application to rabbit skin discs, testosterone was absorbed in a dose-dependent manner and concurrent metabolism was demonstrated.
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.
Neutral Red Uptake, Cellular Growth and Lysosomal Function: In Vitro Effects of 24 Metals
Authors: Repetto Guillermo , Sanz Pilar
Published: August 2020

Specific toxic interference in lysosomal activity was evaluated by the relative uptake of neutral red by lysosomes. In this adaptation of the standard neutral red cytotoxicity assay, the results are expressed relative to cell culture protein content to avoid misinterpretation due to the influence on cell proliferation of the chemicals tested. Neuro-2a mouse neuroblastoma cells were exposed in vitro to 24 metal compounds and the lysosomal activity was quantified. Five different patterns of alterations were identified. Group A (ZnCl2, NaAsO2, Na2HAsO4, CdCl2, SnCl2, HgCl2, HgCH3Cl and TlCOOCH3) produced an inhibition of the relative uptake of the dye, in marked contrast to the greater inhibition shown by the neutral red uptake assay. Group B (MgCl2, A1C13, KMnO4) NiCl2, BaCl2 and Pb[NO3]2) showed less inhibition with strong parallelism with the neutral red assay. In Group C (CrCl3, Na2Cr2O7, FeCl3, CoCl2, CuCl2 and Sn[C2H5]4), low-dose stimulation, and inhibition at higher concentrations were found. Group D (LiCl and CrCl2) stimulated uptake, and Group E (MnCl2 and Sr[NO3]2) produced no significant modifications. The relative uptake of neutral red can be a convenient tool for the study of specific toxic alterations of lysosomes.
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.
Comparative Cell Toxicology: The Basis for In Vitro Toxicity Testing
Authors: Seibert Hasso , Gulden Michael , Voss Jens-Uwe
Published: August 2020

If “cell toxicology” is defined as the discipline aimed at studying the general principles of chemical interference with cellular structures and/or functions, then “comparative cell toxicology” may be defined as the study of the variety of responses to xenobiotics using: (a) different endpoints within one cell type; (b) cell types from different tissues from one species; and (c) homologous cell types from different species. If the full potential of in vitro models for toxicity testing is to be realised and the scientific basis for hazard assessment improved, then comparative cell toxicological approaches have to be developed further. In the present paper, an approach using different in vitro systems is described. The approach is aimed at the assessment of the basic toxicological characteristics of chemicals.
Inclusion of Physicochemical Data in Quantitative Comparisons of In Vitro and In Vivo Toxic Potencies
Authors: Gulden Michael , Seibert Hasso , Voss Jens-Uwe
Published: August 2020

In order to evaluate the relevance of in vitro test systems for acute toxicity assessment, quantitative comparisons of in vitro and in vivo potency data have to be performed. The potency of chemicals to cells in vitro is usually characterised by nominal effective concentrations (e.g. EC50 values). Often, the only available in vivo data are acute lethal body doses (e.g. LD50 values). To enable a reasonable quantitative in vitro–in vivo comparison to be made, a formula has been developed to permit the conversion of EC50 values into “effective model body doses, ED50 values”. This formula takes into account the lipophilicity of the compounds and the very different relationships between the volumes of the lipid and water compartments in vitro and in vivo. The suitability of this approach is evaluated with results obtained for the first 30 MEIC chemicals.
The Effect of Non-ionic X-Ray Contrast Media on Glial Cells In Vitro
Authors: Aardal Nils-Petter , Andersen Knut-Jan , Christensen Erik I. , Vik Hogne
Published: August 2020

The rat glial cell line C6 has been used to assess the toxicity of two different iodine-based X-ray contrast media: the non-ionic dimer iodixanol and the non-ionic monomer iohexol. The cells were exposed to increasing concentrations (0-75mg iodine/ml) during exponential growth (up to eight days). A dose-dependent inhibition of cell growth was observed when iohexol was added to the culture medium, but not when iodixanol was added. While iohexol had a profound, long lasting effect on cell viability, iodixanol, after an initial adverse impact, had no effect. Iohexol and iodixanol both caused increased lysosomal peptidase activity, as shown for dipeptidyl peptidase, but had no effect on other lysosomal enzymes, such as acid β-galactosidase. Electron microscopy revealed no changes in the cellular ultrastructure after exposure to iodixanol. In contrast, there was extensive vacuolisation in the cells exposed to iohexol, including a significantly increased number of autophagocytic vacuoles. It would appear that iodixanol has only minor effects on glial cells in culture, compared to those induced by iohexol.
A Mathematical Model, Describing the Kinetic Behaviour of the Thrombopoietic System in Mice and Rats
Authors: Kodym Reinhard
Published: August 2020

A mathematical model of thrombopoiesis in small rodents, principally rats, is presented. It is a compartmental model, consisting of seven compartments, which contain the following cell populations: early megakaryocyte progenitor cells, late megakaryocyte progenitor cells, megakaryocytes of ploidy classes 8N, 16N, 32N and 64N, and platelets. Signals for feedback regulation are derived from the platelet compartment and from all megakaryocyte compartments. The main characteristic of the model is its ability to simulate megakaryocyte ploidy and volume distribution. Platelet number and the ploidy distribution of megakaryocytes after acute and chronic disturbances in the platelet compartment were simulated and were found to approximate experimental data published in the literature. The kinetic parameters for the progenitor cell compartment, which were derived from published in vitro data, were sufficient to stimulate the platelet number after administration of hydroxyurea and 5-fluorouracil. The proposed model is capable of reproducing the reactions of the system to several experimental disturbances in the thromobopoietic lineage, but the action of agents such as radiation, which cause damage to the stem cells or to other lineages, cannot be simulated.
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.
The Intestinal Mucosa of Coeliacs in Remission is Unable to Abolish the Agglutinating Activity of Gliadin Peptides on K562(S) Cells
Authors: de Vincenzi Massimo , Giovannini Claudio , Cornell Hughes J.
Published: August 2020

The peptic-tryptic-cotazym digest of a wheat gliadin was fractionated into ten primary fractions. Subfraction 2R of fraction 9 is known to be toxic to patients with coeliac disease. Fraction 9 and subfraction 2R also agglutinate K562(S) cells, previously shown to be a good indication of toxicity to in vitro intestinal bioptic specimens from coeliac patients. Subfraction 2R was still able to agglutinate K562(S) cells after digestion by morphologically normal small intestinal mucosa of coeliacs in remission, but was inactivated after digestion by normal mucosa. These results are consistent with the hypothesis that there is a mucosal defect in handling gliadin peptides in coeliac patients, and suggest that there is either a primary (or secondary) enzyme deficiency, or some other mechanism, operating in the intestinal mucosa of coeliac patients in remission.
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.
Education and Training: A Basis for the Introduction of the Three Rs Alternatives into Animal Research
Authors: van Zutphen Bert F.M. , van der Valk Jan B.F.
Published: August 2020

Education is a highly effective way of promoting the introduction of alternatives into the everyday practice of biomedical research and testing. In some countries, specific requirements for the education of persons involved in animal experimentation have been made compulsory by law. In The Netherlands, young scientists must take a course on laboratory animal science as part of, or in addition to, their biomedical graduate programme. This course provides information on the proper design of animal experiments, but also covers alternatives, animal welfare issues and ethical aspects of animal experimentation. The Three Rs of Russell & Burch are the guiding principles of the course, during which participants are challenged to seek methods or techniques that can replace, reduce or refine the use of animals. Since 1985, more than 2500 people in The Netherlands have taken the course, and evaluations have indicated that a large majority of the participants appreciated this education as a contribution to both the quality of experiments and the welfare of the animals, and considered the course to be indispensable for those who are responsible for the design and performance of animal experiments.
A Multicompartmental Dynamic Computer-controlled Model Simulating the Stomach and Small Intestine
Authors: Minekus Mans , Marteau Phillipe , Havenaar Robert , Veld Jos H.J. Huis in't
Published: August 2020

A multicompartmental in vitro model has been described, which simulates the dynamic events occurring within the lumen of the gastrointestinal tract of man and monogastric animals. The accuracy of the model for reproducing in vivo data on gastrointestinal transit, pH, bile salt concentrations and the absorption of glucose was tested. The in vivo conditions simulated in the model were based on studies in healthy human volunteers. Mathematical modelling of gastric and ileal delivery with power exponential equations was used for the computer control of meal transit. The model appeared to reproduce accurately the pre-set data on meal transit, pH and bile salt concentrations in the different gastrointestinal compartments. Glucose absorption from the small intestine was almost complete. This model reproduces very closely the dynamic conditions based on the in vivo situation in monogastric animals and man. Therefore, the model can be an important tool in studying the fate of ingested components (for example, food, microorganisms and medicines) during gastrointestinal transit and, consequently, may contribute to the replacement of studies using laboratory animals.
The Role of Prevalidation in the Development, Validation and Acceptance of Alternative Methods
Authors: Curren Rodger D. , Southee Jacqueline A. , Spielmann Horst , Liebsch Manfred , Fentem Julia H. , Balls Michael
Published: August 2020

Experience has shown that the outcome of large and expensive validation studies on alternative methods can be compromised if their managers do not insist that optimised test protocols and proof of their performance are submitted before the start of the formal validation study. One way for the sponsors of validation studies to confirm both the likely relevance of a method for its stated purpose and its readiness for validation would be to require a prevalidation study before formal validation was contemplated. This process would involve the developers (or other proponents of the method) and selected independent laboratories in protocol refinement (Phase I) and protocol transfer (Phase II). The optimised protocol would then be assessed in a protocol performance phase (Phase III), which would involve the testing of a relevant set of coded test materials and an evaluation of a proposed prediction model. In certain circumstances, a successful outcome of Phase III might be sufficient for promotion of the regulatory acceptance of the method. Normally, however, the method would proceed to a formal validation study. The European Centre for the Validation of Alternative Methods, a recognised validation authority, now proposes to introduce this prevalidation scheme into its validation strategy.
Responsible Animal Research: A Riff of Rs
Authors: Rowan Andrew , Goldberg Alan
Published: August 2020

The Three Rs, reduction, refinement and replacement, were introduced in 1959 by Russell & Burch in their important publication The Principles of Humane Experimental Technique. These Three Rs are now collectively referred to as alternatives. Using the letter R, we have developed an expanded list of Rs not only to stimulate our own thinking, but to encourage others to examine the issue of alternatives in animal research.
Regulatory Genotoxicity Testing: A Critical Appraisal
Authors: Combes Robert D.
Published: August 2020

This review considers current approaches to regulatory genotoxicity testing, focusing on how the use of animals can be further replaced, reduced and refined. The complementary roles of in vitro and in vivo testing, and the justification for using animals, are discussed in detail. Recommendations are made for improvements and further work, in the light of the considerable current controversy surrounding the composition and deployment of testing strategies, and the interpretation of the data generated, particularly for carcinogenicity prediction. The major problems are the oversensitivity of in vitro tests and the insensitivity of in vivo assays. On the basis of an analysis of some published databases, it is concluded that there is insufficient support for using in vivo genotoxicity assays for screening. Also, it is questionable whether the scientific benefits of using such assays always outweigh the costs to the animals involved. The considerable efforts being made to harmonise in vivo protocols and to develop improved methods for detecting genotoxicity are discussed. It is recommended that more emphasis be placed on characterising genotoxins in vitro, especially for mechanisms of activity, to optimise the benefits of any confirmatory animal tests.. Also, regulatory agencies are urged to require better-designed and more-scientifically sound protocols, in which animal numbers are minimised and data interpretation, particularly that of negative results, is facilitated. Lastly, in the development and validation of transgenic rodent systems, emphasis should be placed on developing protocols in which other acute toxicity and metabolism endpoints can be measured simultaneously with in vivo mutagenesis, while minimising animal numbers.
Difficulties with Animal Models: Chimpanzee Research
Authors: Kaufman Stephen
Published: August 2020

Animal models are generally used to elucidate human physiology or pathology. However, attempts to extrapolate animal model findings to humans are undermined by differences in the aetiology and natural history between any animal model condition and the analogous human condition, and by unavoidable interspecies differences in anatomy and physiology. Even when working with species “closely related” to humans, such as chimpanzees, the animal model paradigm is fundamentally unsound. Unfortunately, few animal researchers seriously question the utility of animal models, and consequently they rarely consider other, perhaps more efficient and more reliable, means of conducting biomedical research.
Chimpanzees in AIDS Research
Authors: Ichinose Lester Y.
Published: August 2020

Although the chimpanzee displays an immunological response to an HIV-1 challenge, it is a deficient animal model in AIDS research since it never develops the progressive symptoms of the disease. The continued use of the chimpanzee is also precluded because of its endangered status in the wild, the high cost per animal, and its failure to exhibit a CD4 decline (apoptosis) while infected. However, it is likely that the chimpanzee model will continue to be used in investigations relating to the epidemiology and mucosal transmission of the human AIDS virus.
The Work of Pro Primates in Laboratories
Authors: Spruit Ignaas
Published: August 2020

Entering laboratories and negotiating with scientists promotes the interests of animals more effectively than attacking science verbally, philosophically or by organising demonstrations. This was proven by Pro Primates in a two-year battle against the former TNO Primate Centre.
Present and Future Prospects for Using Alternatives to Chimpanzees in Research
Authors: Langley Gill
Published: August 2020

Three chimpanzee studies of hepatitis C infection were found to be duplicative of each other and of research with human volunteers. Research into taste neurophysiology also used chimpanzees, although data which are highly relevant (but less specific) can be obtained from human studies. The use of chimps to study benzene metabolism was found to be poorly planned and unjustifiable. Scientifically, chimps are not always the best “models” for humans, and their use raises insurmountable ethical concerns. Many alternatives are already available, but researchers will need to adjust their perspectives on the usefulness of different types of data.
HIV Infection: Options for Control
Authors: Moore Eddie
Published: August 2020

Current efforts to control the global spread of HIV infection include drug and vaccine research on animals and human beings, as well as measures to reduce the risk of person-person transmission. By using a simple, five-step problem-solving approach, it can be shown that none of these methods of control can succeed and that only the avoidance of risk will stop the spread of infection. Politicians, teachers, doctors and others in positions of responsibility should therefore be persuaded of the importance of making health education the first priority for funding in programmes to control HIV infection.
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.