bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Carbon Tetrachloride on Perfused Cultures of Hepatic and Neuronal Cells
Authors: Wallin Stefan , Walum Erik
Published: August 2020

Cultured hepatocytes and hemisphere neurons from chick embryos and mouse neuroblastoma cells were exposed to carbon tetrachloride (CC14; 0, 1, 2, 3 and 4mM) for 1 hour, using a perfusion system developed for studying the effects of volatile substances. In the perfused cultures, three parameters were compared: lipid peroxidation, membrane integrity and cellular respiration. In addition, cytochrome C oxidase activity was determined after incubation of cell homogenates with CC14. A concentration-dependent increase in lipid peroxidation and membrane permeability was found in the neuroblastoma cells. The hepatocytes responded to a lesser extent with respect to membrane permeability and their lipid peroxidation did not differ from that of controls. The hepatocytes responded with a 35% decrease in respiration when exposed to 3mM CC14, and a 20% decrease in cytochrome C oxidase activity after treatment with 1.5mM CCl4. In the neuronal cells, much smaller decreases in respiration were found and their cytochrome C oxidase activity remained unaffected. These results are very similar to those obtained after incubation in a closed chamber system. However, the perfused cells were found to be less sensitive to CCl4 than cells exposed under static conditions.
Changes in Astroglial Cell Volume after Exposure to HgCl2 or CH3HgCl
Authors: Rönnbäck Lars , Hansson Elisabeth
Published: August 2020

Cell volume was determined by measuring [14C]-3- O-methyl glucose uptake in astroglial-enriched primary cultures. Control cell volume was 3.20μl/mg protein. After incubation in 10 5M HgCl2 for 60 minutes, there was a 71% increase in cell volume. This increase was partially inhibited in the presence of the α1 receptor agonist, phenylephrine, or by the α2 receptor agonist clonidine, and was completely reversible by their respective antagonists, prazosine and yohimbine. The β receptor agonist, isoproterenol, which in itself increased cell volume, and 5-hydroxytryptamine (5HT) did not affect the HgCl2-induced changes in cell volume. 10 5M CH3HgCl increased cell volume by 26% after 30 minutes of incubation. This increase was not significantly influenced by adrenoceptor agonists or 5HT.

It therefore seems that mercurial-induced changes in cell volume can be regulated by astroglial receptor stimulation.
Use of External Metabolising Systems in the Neutral Red Assay
Authors: Rasmussen Eva
Published: August 2020

BALB/C 3T3 mouse fibroblasts or CHO cells grown in 96-well microtitre plates were used for the neutral red viability assay. The cytotoxicity and ability of microsomes or an S9 mixed function oxygenase to activate cyclophosphamide were compared. A pronounced cytotoxic effect of both activation systems was found using CHO cells, but not using 3T3 cells. 3T3 cells were more sensitive than CHO cells to the active metabolites of cyclophosphamide. Cyclophosphamide was more efficiently converted to toxic metabolites by microsomes than by the S9 mix. A concentration of microsomes corresponding to 0.032mg protein per ml of medium was shown to be optimal for the activation of cyclophosphamide in both cell lines.
The Use of Basal Cytotoxicity and Target Organ Toxicity Tests in Hazard Identification and Risk Assessment
Authors: Balls Michael , Fentem Julia H.
Published: August 2020

Non-animal procedures, including in vitro test systems and test strategies, can already make a significant contribution to the background to risk assessment — in predicting both the toxic potential and toxic potency of chemicals, as well as, in some circumstances, the toxic hazard they may represent under specified conditions of exposure. They can be particularly useful for investigating molecular and cellular mechanisms of chemical-induced toxicity, and for identifying species-specific effects, which greatly limit the value of data from laboratory animal studies in the human risk assessment process. Attention is focused on the need for greater effort to be invested in the development of non-animal procedures for evaluating the biokinetic factors which will determine the ultimate form and concentration of a particular chemical at possible sites of toxic action. The relative merits of correlative and mechanistic approaches to test development and test validation are discussed. The need for realism is emphasised, not only in relation to our expectations of the validation process, but also in terms of the current and future status of regulatory toxicology, in vitro or in vivo, as a scientific discipline. Finally, it is concluded that the intelligent and strategic use of in vitro test systems, in conjunction with predictive computer modelling, could markedly improve the scientific basis of human risk assessment.
The HET-CAM Test: A Study of the Irritation Potential of Chemicals and Formulations
Authors: de Silva Odile , Rougier André , Dossou Koovi G.
Published: August 2020

The HET-CAM (hen's egg test-chorio-allantoic membrane), described by Luepke in 1985, permits the study of immediate effects following the administration of test substances to the chorioallantoic membrane of 10-day-incubated White Leghorn chicken eggs. The results of a study of 60 chemicals and 41 cosmetic formulations are presented here.

Intralaboratory reproducibility was established with a double-blind study of 20 surfactants at two concentrations. The results show a high rank correlation between the scores given by both experimenters: the Spearman's rho is greater than 0.9 (p < 10-8).

The 60 chemicals were studied at a concentration equivalent to 10% of the concentration tested in vivo. They were classified according to the three EEC categories of ocular irritancy, and when a correlation between the HET-CAM scores and historical Draize in vivo data was determined, the corresponding Spearman's rho value was 0.72 (p < 10-4).

The 41 formulations (make-up removers, shower gels, shampoos, creams and body milks) were tested by two protocols: rinsed and non-rinsed. The correlation between the HET-CAM scores and the historical Draize in vivo maximum average scores was studied, and the rhos obtained were 0.77 and 0.76 (p < 10 -8), respectively.

The advantages of the HET-CAM method lie in its sensitivity, rapidity and moderate cost. Both chemicals and cosmetics formulations can be tested, and specific families can be assessed using modified and more-appropriate protocols. However, to ensure that a good reproducibility and sensitivity is provided, the use of reference materials is strongly recommended.

The satisfactory performances of the HET-CAM test in many previous evaluation studies (e.g. those of the BGA, CTFA, EEC and OPAL) show that it can be a useful assay as part of a battery of in vitro tests for the screening of new ingredients and formulations.
The Agarose Overlay Method as a Screening Approach for Ocular Irritancy: Application to Cosmetic Products
Authors: Combrier Emmanuel , Castelli Dominique
Published: August 2020

The in vitro agarose overlay method was investigated as an alternative to the Draize eye irritation test for the evaluation of the ocular irritancy of cosmetic products.

Modifications to the original protocol include the definition of an area of lysis (expressed as a weighty)/toxicity relationship for each product by a planimetric method and the creation of an agarose classification.

56 different cosmetic formulations, including emulsions, gels, lotions and tonics, were evaluated using this modified test, and the results were correlated with in vivo Draize data. Four different agarose classes were delimited: non-irritant; minimally irritant; mildly irritant; and irritant; corresponding respectively to the following scoring scales (x 10 4g): 0; 0 <x<200; 200≤ x <450 and ≥ 450.

A high correlation between the agarose overlay method and the Draize test was observed when taking into account two classes: non-irritant (non-irritant, minimally irritant) and irritant (mildly irritant, moderately irritant [Draize] or irritant [agarose]) as shown by an 86% concordance value. In this study, the sensitivity was 92% (11–12 irritants were predicted by the agarose diffusion method) and the specificity was 84%. The overestimation error of 16% suggests that the agarose overlay method might be slightly more sensitive than the Draize eye test.

When applying the kappa test, the agarose overlay method reached a 64% coefficient, attesting that the good correlation with the Draize test was not due to chance. In relation to the Landis & Koch classification, this percentage places the agarose overlay method in the “good concordance” class.

Based on these results, the use of the agarose overlay method for screening the ocular irritation potential for the cosmetics industry can be considered to be a very interesting alternative to be included in a battery of tests.
The Effects on Porcine Isolated Corneas of the Addition of Various Preservatives to Befunolol-HCl Solutions
Authors: Igarashi Haruyoshi , Katsuta Yasunaga , Sawa Kayoko , Chiba Yuu , Kojima Michio , Ogawa Katsuhiro , Nakazato Yoshiharu , Kawasaki Tohru
Published: August 2020

The opacity which develops in intact porcine isolated corneas during incubation for a period of four hours at 35°C with solutions of befunolol-HCl (5 x 10-4M, 5 x 10 -3M, 5 x 10-2M) is increased by the presence of benzalkonium chloride (BC; 0.005%) or a mixture of chlorobutanol (0.2%), methyl p-hydroxybenzoate (MPB; 0.026%) and propyl- p-hydroxybenzoate (PPB; 0.014%). The addition of BC had a greater effect than the addition of the chlorobutanol/ MPB/PPB mixture, when solutions were applied only to the epithelial surface, only to the endothelial surface, or to both surfaces simultaneously. However, when the individual constituents of the mixture were added separately to more-concentrated befunolol solutions (5 x 10-2M), chlorobutanol, MPB and PPB each increased opacity when solutions were applied only to the epithelial surface; MPB and PPB increased, but chlorobutanol decreased, opacity when solutions were applied only to the endothelial surface; and chlorobutanol increased, but MPB and PPB each decreased, opacity when solutions were applied to both surfaces. Assuming that BC and the mixture of chlorobutanol/MPB/PPB are equally effective as preservatives, the results suggest that the latter mixture may be preferable to BC in eye-drops intended for long-term use.
In Vitro Mechanistic Toxicology Evaluation of the Antibiotic, Paldimycin Sodium, in Chang Liver Cells
Authors: Linseman Daniel A. , Raczniak Timothy J. , Bacon James A. , Linseman Kelly L. , Shilliday F. Barclay , Branstetter Daniel G.
Published: August 2020

The basal in vitro cytotoxic potential of the antibiotic, paldimycin sodium, was investigated in Chang liver cells. The potential roles of lipid peroxidation, protein binding, and proteolytic enzyme activation in the cytotoxic mechanism of paldimycin were also examined. Cytotoxicity was assessed by measuring decreases in cell proliferation, increases in medium LDH activity due to cytoplasmic leakage, and morphological changes. Lipid peroxidation, protein binding, and proteolytic enzyme activation were investigated by co-treating cells with paldimycin and compounds which act as modifiers of the above pathways, such as antioxidants, sulphydryl compounds, and protease inhibitors, respectively.

Paldimycin demonstrated a greater degree of basal cytotoxicity than was previously shown for several aminoglycoside and tetracycline antibiotics in Chang liver cells. Lipid peroxidation and proteolytic enzyme activation had no significant involvement in the observed cytotoxicity, whereas protein binding (more specifically, sulphydryl binding) was a principal mechanism of paldimycin-induced cytotoxicity in Chang liver cells. In addition, this sulphydryl binding was shown to be mediated via an exposed isothiocyanate moiety on the paldimycin molecule. The results of the co-treatment experiments described above are in agreement with data obtained in several other in vitro systems, including cell-free systems, isolated proteins, tissue homogenates, and primary isolates of cardiac myocytes.
The Scientific Basis of the EYTEX™ System
Authors: Gordon Virginia C.
Published: August 2020

The EYTEX™ system is an in vitro method for predicting in vivo ocular irritation via a target biomacromolecular approach. EYTEX™ uses changes of relevant macromolecules upon exposure to chemicals and formulations to predict in vivo irritancy and toxicity endpoints.

The EYTEX™ system is based on alterations in the conformation and hydration of an ordered macromolecular matrix. An oligomeric protein with a molar mass of approximately 300,000g/mol is the major active component of the reagent. This oligomer, characterised by 12 subunits, associates itself into strands which are then incorporated into a gel network. This network also includes smaller proteins, peptides, amino acids, aminoglycans and mucopolysaccharides, which contribute to the overall response of the macromolecular matrix to a diverse array of test compounds, such as acids, bases, salts, solvents, surfactants, lubricants, preservatives, emulsions, dyes, amines, amides and many others. The method has been extensively evaluated using chemicals and formulations from diverse classes and with different ranges of toxicity, representing various mechanisms of ocular toxicity. Results using the in vitro test correlate broadly with the Draize in vivo eye irritancy test results. Quantification of the EYTEX™ method is based on measurement of changes in optical density of the matrix, which are sensitive to the conformation and hydration of protein, as well as the order in the matrix. These changes, which occur when the matrix is exposed to chemicals and formulations, can be used to predict in vivo ocular irritancy. Calibrators and controls provide assay standardisation with a 4–8% coefficient of variation. Multiple dose and time variables relating to irritancy potential can be assessed and compared with Draize in vivo results. The EYTEX™ system provides a comprehensive set of four protocols suitable for analysing chemicals and formulations with varying degrees and mechanisms of ocular irritancy.
Comparison of Xenobiotic-mediated Cytotoxicity in Rat Cultured Hepatocytes and the V79 Chinese Hamster Lung Fibroblast Cell Line: Can Metabolically-activated Hepatotoxins be Identified by Selective Cytotoxicity to Hepatocytes?
Authors: Fry Jeffrey R. , Hammond Alison H. , Garle Michael J. , Lal Kishan
Published: August 2020

The effects of 17 xenobiotics on rat hepatocytes and V79 cells were evaluated under identical exposure conditions (confluent monolayer for 24 hours) and endpoint measurement (MTT reduction). The data indicated that the majority of metabolically-activated rat hepatotoxins could be identified by greater cytotoxicity to rat hepatocytes relative to V79 cells, but that direct-acting hepatotoxins (galactosamine and ethionine) and a group of eight compounds likely to act through interference of basal functions in non-dividing and dividing cells produced similar toxicity in each cell type. It is possible that the greater water-solubility of the direct-acting hepatotoxins relative to the indirect-acting hepatotoxins may contribute to the lack of effect seen with the former group under the conditions of the assay (top concentration of 1mM).
Multiple Computer Automated Structure Evaluation Methodology as an Alternative to In Vivo Eye Irritation Testing
Authors: Klopman Gilles , Ptchelintsev Dmitri , Frierson Manton , Pennisi Stephen , Renskers Kevin , Dickens Michael
Published: August 2020

The Multiple Computer Automated Structure Evaluation (Multi-CASE) methodology was applied to the semi-quantitative analysis of eye irritation potential of 186 organic chemicals. The resulting expert dictionary of substructural attributes indicates that the following functionalities are linked to eye irritation potential: carboxylate and sulphonate groups in anionic surfactants, phenolic moieties in non-ionic surfactants, amino groups, halogenated and unsaturated structures, anhydrides and epoxides. Ester groups were recognised as having a low irritation potential. The Multi-CASE program successfully predicted a priori the eye irritation potential of: 1) 21 individual chemicals; 2) chemical mixtures such as coconut oil, hydrogenated castor oil, rice bran, eucalyptus oils and potassium alginate; and 3) polymers,’ including polyvinyl alcohols, polyvinyl butyral, polyvinyl pyrrolidone, corn starch and nylon. It was found that the structure of the repeat units together, and not that of the individual monomers, is the best predictor of the eye irritation potential of the corresponding polymers. It appears that the repeat units of polymers dilute out the irritation potential of terminal groups normally expected to contribute significant irritation potential to chemicals of lower molecular weight.

The Multi-CASE eye irritation model can be used for: 1) in vitro prescreening for eye irritation potential; and 2) design and modification of chemicals when low eye irritation potential is important.
An In Vivo/In Vitro Study of Allyl Alcohol Toxicity Using Enzyme Inhibitors
Authors: Pulci Romana , Moneta Donatella , Dostert Philippe , Brughera Marco , Scampini Giovanna , Castellino Silvana , Carminati Paolo , Mazué Guy
Published: August 2020

The aim of this study was to verify an in vitro model of hepatotoxicity, designed to assess the production of reactive species from biologically-inert chemicals through their metabolic transformation. One example is allyl alcohol, which produces acrolein through the action of the enzyme alcohol dehydrogenase. Acrolein is a highly hepatotoxic aldehyde which is detoxified to acrylic acid by aldehyde dehydrogenase (ALDH). A deficiency of this enzyme, common in some Asian populations, can give rise to pathological conditions of hepatotoxicity.

Isolated rat hepatocytes were incubated with allyl alcohol with and without cyanamide, a known inhibitor of ALDH. The toxicity of allyl alcohol, assessed on the basis of release of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) into the culture medium, was dramatically increased by the addition of cyanamide. In vivo, the same treatment scheme was used in rats treated with allyl alcohol with or without cyanamide pretreatment. It was also demonstrated that allyl alcohol toxicity is dramatically enhanced by the addition of an aldehyde dehydrogenase inhibitor, as shown by plasma levels of hepatic enzymes (GOT, GPT and LDH) and by histological findings. We believe that this in vitro model, involving the use of enzyme inhibitors, could be useful for verification of the hypothesis that hepatotoxins, such as acrolein, are produced from some pharmaceutical and other chemical compounds.
The Use of the Vitality and Chemotaxis of Human Polymorphonuclear Leukocytes for the In Vitro Estimation of the Acute Toxicity of the First Ten Chemicals from the MEIC List
Authors: Valentino Matteo , Monaco Francesca , Pizzichini Maria Antonietta , Governa Mario
Published: August 2020

The acute cytotoxicity of the first ten MEIC chemicals has been estimated by others in various cell lines. In the present investigation, isolated human polymorphonuclear leukocytes (PMN) from ten healthy non-smoking laboratory personnel were used to assess in vitro toxicity of the same chemicals.

The cells were treated with different concentrations of the respective chemicals for three hours and their vitality and chemotaxis were tested. Vitality was measured by fluorescence microscopy after the addition of fluorescein diacetate and ethidium bromide. Living cells which took up and hydrolysed fluorescein diacetate, and dead cells, stained by ethidium bromide, were counted and the percentage of live cells was calculated.

Locomotion stimulated by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (F-MLP), was measured in blind-well Boyden chambers and a chemotactic index was calculated.

The results were mathematically transformed to produce a linear curve, and then fitted by the linear least squares procedure, from which LC50 and IC50 values were obtained by interpolation. All the chemicals decreased the vitality and inhibited the chemotaxis of the PMN. Obviously the chemotactic test was more sensitive than the vitality one. A correlation (r = 0.933) was found between vitality and chemotaxis inhibition. Spearman rank correlation analysis revealed significant correlations between our results and those from in vitro experiments conducted in other laboratories, as well as with data concerning mouse, rat and human lethal doses.
The Influence of Animal Discomfort, Human Interest and Scientific Quality on the Ethical Acceptability of a Projected Animal Experiment as Assessed with Questionnaires
Authors: Stafleu Frans R. , Baarda Ben D. , Heeger F. Robert , Beynen Anton C.
Published: August 2020

This study attempts to assess to what extent three selected variables (animal discomfort, scientific quality and human interest) determine the ethical acceptability of a projected animal experiment, as judged by animal experimenters. Two levels of each of the three variables were incorporated into otherwise identical protocols of a hypothetical animal experiment. Thus, there were eight different protocols with various combinations of the variables. In a postal survey, animal experimenters were asked to assign an acceptability score to the projected animal experiment described and to give a short written justification of their score. Human interest had the greatest influence on acceptability scores, followed by animal discomfort and scientific quality. Arguments concerning scientific quality played a major role in determining acceptability scores. At high levels of animal discomfort, the projected experiment was considered acceptable when both human interest and scientific quality were high. Thus, it remains questionable whether, in practice, a well-designed experiment with significant, expected human interest would be dismissed because of a high or moderate degree of anticipated animal discomfort.
In Vitro Cytotoxicity Testing: 24-hour and 72-hour Studies with Cultured Lung Cells
Authors: Hopkinson Desirée , Bourne Rae , Barile Frank A.
Published: August 2020

This study was designed to evaluate the potential of an in vitro cell culture method for its ability to determine cytotoxicity and to compare the cytotoxic concentrations with established LD50 values for the same chemicals. Rat lung epithelial cells (L2) were incubated in the absence or presence of increasing concentrations of the test chemical for 24 hours, and the inhibition of incorporation of radio-labelled amino acids into newly synthesised proteins was used as a marker for toxicity. In addition, cultured cells were exposed to the test chemicals for 72 hours, and cell proliferation experiments were performed as parallel measures of toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. The biological significance of the results of testing 20 chemicals shows that the experimental IC50 values are as accurate as predictors of human toxicity as are equivalent toxic blood concentrations derived from rodent LD50s. Results obtained from 72-hour growth studies reveal a greater sensitivity to cytotoxicity than from the 24-hour protein synthesis experiments. Statistically, however, the differences between the two protocols are inconclusive. It is anticipated that these procedures, together with a related battery of tests, may supplement or replace animal protocols currently used for human risk assessment.
Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing
Authors: Andersen Knut-Jan , Christensen Erik Ilsø , Vik Hogne
Published: August 2020

The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro.

In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.
Survival and Function of Fibroblasts Stored as Stock Cultures at a Non-freezing Temperature
Authors: Andrén Anders H. G. , Wieslander Anders P.
Published: August 2020

Cytotoxicity, measured as inhibition of cell growth of cultured cell lines, is a widely used method for testing the safety of biomaterials and chemicals. One major technical disadvantage with this method is the continuous routine maintenance of the cell lines. We decided to investigate the possibility of storing stock cultures of fibroblasts (L-929) in an ordinary refrigerator as a means of reducing the routine workload. Stock cultures of the mouse fibroblast cell line L-929 were prepared in plastic vials with Eagle's minimum essential medium. The vials were stored in a refrigerator at 4–10°C for periods of 7–31 days. The condition of the cells after storage was determined as cell viability, cell growth and the toxic response to acrylamide, measured as cell growth inhibition. We found that the L-929 cell line can be stored for 2–3, weeks with a viabilty > 90% and a cell growth of about 95%, compared to L-929 cells grown and subcultured in the normal manner. The results also show that the toxic response to acrylamide, using refrigerator stored L-929 cells, corresponds to that of control L-929 cells. We concluded that it is possible to store L-929 cells in a refrigerator for periods of up to 3 weeks and still use the cells for in vitro cytotoxic assays.
Cytotoxicity of 28 MEIC Chemicals to Rat Hepatocytes using Two Viability Endpoints: Correlation with Acute Toxicity Data in Rat and Man
Authors: Roguet Roland , Cotovio José , Gaetani Quintino , Dossou Koovi G. , Rougier André
Published: August 2020

28 of the chemicals on the MEIC list were tested on rat hepatocytes in culture. Firstly, the metabolic capacities of the cell model were characterised, i.e. cytochrome P450 content and conjugation activities. Two independent endpoints, MTT (tetrazolium salt) reduction (for mitochondrial integrity) and neutral red uptake (NRU, for lysosomal integrity), were then used to measure the cytotoxicities of the test compounds after incubation with the cells for 24 hours. The relative toxicities of the test chemicals were measured by the determination of IC50 values for each parameter. Statistical analysis revealed a correlation between the results obtained in the two assays. The NRU assay, however, proved to be more sensitive than the MTT reduction assay. When the in vitro results were compared with those obtained from an in vivo data bank, good agreement was found with the acute toxicities of the test products in rats, with the exception of thioridazine, malathion, copper sulphate (all overestimated) and potassium cyanide (underestimated). In addition, a good correlation between basal cytotoxicity values for rat hepatocytes and LD50 for rats or lethal doses for humans was observed. These results suggest that the MTT reduction and/or NRU assays could be useful indicators of the cytotoxic potential of chemicals in rat hepatocyte cultures and thus provide information on the intrinsic lethal toxicity of compounds and their metabolites to rats and humans.
Anticholinesterase Activity of Aldicarb and Carbofuran in Neuron Cultures
Authors: Sawyer Thomas W. , Weiss M. Tracy , Bianco Angela
Published: August 2020

The anticholinesterase activity of the carbamate insecticides, carbofuran and aldicarb, was examined in primary cultures of chick embryo forebrain neurons. Both compounds inhibited acetylcholinesterase, with carbofuran being more potent than aldicarb by more than two orders of magnitude. Preincubation of carbofuran with hepatic S9, derived from rat or chick, decreased its anticholinesterase activity, with chick S9 exerting the greater deactivating effect. Conversely, preincubation of aldicarb with rat control S9 increased its anticholinesterase activity, while chick S9 had only a slight effect. Preincubation of the test compounds with S9 obtained from animals induced with phenobarbitone or 3-methylcholanthrene generally had a slightly deactivating effect, with the exception of aldicarb, where preincubation with induced rat hepatic S9 fractions slightly increased its potency as an anticholinesterase agent when compared to the effects of control S9. Preincubation of aldicarb and carbofuran with hepatic S9 fractions from both rat and chick altered their anticholinesterase activities, so that they were more predictive of their relative potential species toxicities in vivo. The relative toxicities of aldicarb and carbofuran, within a given species, however, were not as well predicted. This model may be of use in assessing the potential toxicities of carbamate pesticides with respect to species differences.
Assessment of the Functional Integrity of Hepatocytes: A Brief Review
Authors: Fry Jeffrey R. , Hammond Alison H.
Published: August 2020

A variety of approaches to assessment of cellular integrity exist, based on tests of integrity of the plasma membrane, tests of metabolic competence, and asessment of morphology. By definition, such approaches address different aspects of cellular integrity and hence are not interchangeable indices of cellular integrity. Accordingly, it would be most appropriate to characterise hepatocyte preparations on the basis of more than just trypan blue dye exclusion (a test of plasma membrane integrity) as is customary. A scheme for the choice of the most appropriate mix of tests of cellular integrity is presented.
The Galileo Data Bank on Toxicity Testing with In Vitro Alternative Methods. I. General Structure
Authors: Loprieno Nicola , Boncristiani Guido , Bosco Elena , Nieri Maria , Loprieno Gregorio
Published: August 2020

Toxicity testing of chemicals by means of in vitro alternative methods to the use of animals has been extensively developed, as documented by a variety of studies. The interpretation of results and the comparative evaluation of data derived from various cell toxicity studies require organisation by a computerised data system, capable of handling the large number of variables included in different assays, such as cell lines, methods of treatment with the chemical, methods used to evaluate the biological effect, endpoints considered, etc.

The Galileo Data Bank has been developed by the Laboratory of Genetic Toxicology at the University of Pisa, as a scientific instrument to be used in the analysis and organisation of results obtained in the toxicity testing of chemicals by means of in vitro alternative methods.
Evaluation of the EYTEX® System as a Screening Method for the Ocular Irritancy of Chemical Products
Authors: Régnier Jean-François , Imbert Christophe , Boutonnet Jean-Charles
Published: August 2020

The EYTEX® method is an in vitro test used to predict ocular irritation, based on alterations in a protein matrix. We have evaluated this method with the aim of using it to screen chemicals. One hundred and forty-two products (commodities and specialities), having a wide range of chemical properties and ocular irritation potential in the rabbit, were tested with either the standard, MPA, AMA, KMA or UMA protocols. The results were compared with in vivo data obtained previously for each chemical and with the EEC labelling of eye irritation for dangerous substances. Intralaboratory repeatability and interlaboratory reproducibility were evaluated with seven other laboratories.

Ninety-three per cent of the chemicals tested were qualified with the EYTEX method. The coefficients of the linear correlation between the EYTEX score and the maximal Draize score on the one hand and the maximal corneal score on the other hand, were 0.69 and 0.65, respectively. Compared to the EEC classification, the labelling of dangerous substances and for the prediction of severe irritants, sensitivity, specificity and equivalence were 94%, 89% and 78%, respectively. We observed nine false positives (22%) and two false negatives (2%) for the identification of R41-, R34- and R35-labelled products. The predictive values, for identifying R41, R34 and R35 products and non-irritants or R36 products were 78% and 97%, respectively. Repeatability (6.3) and reproducibility (8.9) were quite satisfactory.

The EYTEX system exhibits the characteristics of a good screening method: compatibility with a large range of chemicals; a simple and rapid procedure; good intralaboratory and interlaboratory reproducibility; cost effectiveness; high sensitivity, specificity and predictive value; and a low incidence of false negative and false positive results. Based on these results, we consider the EYTEX method to be a valuable tool for the screening of eye irritancy.
Hydrogen Peroxide Stimulation of Active Sodium Transport in Isolated Frog Skin: Indicative of a Possible Prostaglandin Interaction
Authors: Bjerregaard Henning F.
Published: August 2020

The influence of reactive oxygen metabolites on ion transport across the plasma membrane was investigated by measuring the effects of hydrogen peroxide (H2O2) on short-circuit current (SCO in isolated frog skin. Addition of H2O2 to the basolateral (inner) membranes of the polarised epithelial cells induced a dose-dependent stimulation of SCC with a half maximal stimulating concentration of 41üM H2O2. This stimulation could be blocked by 100μM amiloride in the apical (outer) solution, showing that H2O2 induced a specific activation of the active transport of sodium (Na+). The effect of H2O2 was inhibited in skins pretreated with 5μM indomethacin, implying the involvement of prostaglandins in the response. Furthermore, the effect of H2O2 was inhibited in the presence of 0.1mM quinacrine, indicating that the activation of the cyclo-oxygenase pathway is dependent on phospholipase A2 activity. The present data demonstrate that H2O2 in low concentrations (1μM-1mM) induced a specific stimulation of active Na+ transport due to activation of prostaglandin synthesis. An acute toxic effect on the electrophysiological parameters was noted after the addition of higher H2O2 concentrations (10–100mM). This effect resulted in a non-specific increase in the ion permeability of the epithelium, probably as a result of membrane damage due to lipid peroxidation.
Effects of Polyunsaturated Fatty Acids on Glial Cell Activation. A Study Using Primary Cultures
Authors: Grundt Inger K. , Nyland Harald
Published: August 2020

The aim of this study was to examine the effects of essential fatty acids (gammalinolenic acid [18:3 n-6; GLA] and alpha-linolenic acid [18:3 n-3; Lin]) on the activation of glial cells, using lipopolysaccharides as the activating agent. Primary cultures of mixed glial cells from rat brain were used as the model. The morphological activation of microglia was the most significant response to the exposure. This activation was followed by an increase in 5’-nucleotidase (5’-NT) activity. The 5’-NT activity was increased by GLA or Lin alone to 250–350% of the control value and further increased by co-incubation with lipoteichoic acid (a lipopolysaccharide) to 500–600% of the control value.

The lipopolysaccharide-induced activation of glial cells was also followed by an augmented release of prostaglandin E2. GLA increased the release of prostaglandin E2 in a dose-dependent manner, whereas Lin had no effect on its release.

The results show that this model system is useful for studies on factors affecting the activation of glial cells. GLA and Lin did not reverse glial activation induced by lipopolysaccharides under these experimental conditions.
Induction of Sister-chromatid Exchanges, Micronuclei and Gene Mutations by Indirectly Acting Promutagens Using Human Hepatoma Cells as an Activation System
Authors: Darroudi Firouz , Natarajan Adayapalam T.
Published: August 2020

An established human hepatoma cell strain (designated Hep G2) was used in sister-chromatid exchange (SCE) and micronuclei (MN) assays to study, in vitro, the genotoxic potentials of indirectly acting promutagenic carcinogens, such as 2-acetylaminofluorene (2-AAF) and hexamethylphosphoramide (HMPA), and a non-carcinogen, 4-acetylaminofluorene (4-AAF). In addition, Hep G2 S9-fractions were isolated and used to activate the same chemicals using Chinese hamster ovary (CHO) cells as target cells in vitro. The percentage survival and the frequencies of MN, SCEs and point mutations (at the HPRT locus) were used as biological endpoints.

A dose-dependent increase in the frequencies of SCEs, MN, cytotoxicity, and/or point mutations was found in Hep G2 cells, and in CHO cells in the presence of Hep G2 S9-fractions, with 2-AAF and HMPA, but with 4-AAF no increase was found. The results obtained demonstrate that the Hep G2 cells and their S9-fractions are capable of activating different classes of mutagens to form biologically active metabolites.
The Use of Porcine Proximal Tubular Cells for Studying Nephrotoxicity In Vitro: Validation of the Effects of Culturing and Cryopreservation
Authors: Kruidering Marieke , Maasdam Diedka H. , Leeman Winfried R. , de Heer Emile , Nagelkerke J. Fred
Published: August 2020

The susceptibility to nephrotoxins of freshly isolated porcine proximal tubular cells (PPTC) and cultured PPTC in suspension were compared, with a view to using PPTC as in vitro models for studying nephrotoxicity. In a previous paper, we described how, in freshly isolated PPTC, well-known nephrotoxins such as mercury (II) chloride, cisplatin, p-aminophenol and halogenated hydrocarbons caused a dose-dependent decrease in the viability and mitochondrial membrane potential of the PPTC. In this paper, we show that suspensions of cultured PPTC, harvested by trypsinisation, are less susceptible to nephrotoxins, possibly due to the synthesis of extracellular matrix components. PPTC in primary culture are suitable for relatively long-term nephrotoxicity studies. This was demonstrated by incubation with mercury (II) chloride for 24 hours, resulting in a dose-dependent loss of viability.

Freshly isolated PPTC can be cryopreserved by computer-controlled freezing. The cryopreserved PPTC displayed an increased susceptibility to mercury (II) chloride and a decreased susceptibility to cisplatin and 1,1-dichloro-2,2-difluoroethylene-cysteine, the toxicity of the latter indicating that the renal enzyme β-lyase remains active during cryopreservation.
The Finkleman Preparation: A Computer Simulation for Teaching Undergraduate Students of Pharmacology
Authors: Dewhurst David , Hughes Ian , Ullyott Richard
Published: August 2020

An interactive computer-assisted learning program is described, which simulates a number of experiments which can be performed on the isolated, innervated duodenum of the rabbit (the Finkleman preparation). This preparation is one of the classical pharmacological preparations used to demonstrate to undergraduate students the effects of selected drugs: those acting on adrenoceptors or intestinal smooth muscle, or those affecting responses to sympathetic nerve stimulation. The program runs 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. A screen display which emulates a chart recorder presents simulated results (spontaneous or evoked contractions of the gut), derived from actual data, in response to the selection by students of predetermined experimental protocols from a menu. The program is designed to enhance or replace the traditional laboratory-based practical using this preparation, whilst achieving the majority of the same teaching and learning objectives.
A Comparison between COMPACT and Hazardexpert Evaluations for 80 Chemicals Tested by the NTP/NCI Rodent Bioassay
Authors: Brown Susanna J. , Raja Asad A. , Lewis David F.V.
Published: August 2020

A total of 80 structurally diverse chemicals for which rodent carcinogenicity data are known, have been evaluated by two predictive computer-based systems, COMPACT and Hazardexpert. The results demonstrate that these two predictive test procedures are, to some extent, complementary, in that COMPACT can identify carcinogens which require metabolic activation, whereas Hazardexpert is able to provide an alert for potentially direct-acting agents. In this study, COMPACT was able to identify correctly most rodent carcinogens, and the Hazardexpert system showed a high concordance with non-carcinogens, in particular. It is suggested that some form of combined approach to the use of predictive methods in chemical safety evaluation may represent a way forward for the future.
The Development and Evaluation of In Vitro Tests by the FRAME Alternatives Laboratory
Authors: Clothier Richard H. , Atkinson Karen A. , Garle Michael J. , Ward Rachel K. , Willshaw Angela
Published: August 2020

This review outlines the work which has been conducted in the FRAME Alternatives Laboratory during the first ten years of the FRAME Research Programme. A number of in vitro tests, including the kenacid blue, neutral red release and fluorescein leakage assay methods, have been evaluated and have subsequently been included in validation schemes organised by the US Soap and Detergent Association, the US Cosmetic, Toiletry and Fragrance Association, the European Commission and the European Cosmetic, Toiletry and Perfumery Association, as well as in the Scandinavian multicentre evaluation of in vitro cytotoxicity testing scheme. More recently, research has been undertaken in the areas of phototoxicity, immunotoxicity, dermal toxicity and intercellular communication, in addition to investigations into fundamental mechanisms of toxicity.
Toxicity Testing with Hepatocytes: Some Methodological Aspects
Authors: Fry Jeffrey R. , Hammond Alison H. , Atmaca Mukadder , Dhanjal Perminder , Wilkinson David J.
Published: August 2020

Two conventional systems — freshly-isolated hepatocytes and hepatocytes in culture — are now widely used for toxicity testing, each possessing advantages and disadvantages. More-recently. developed alternative strategies, which try to minimise the disadvantages apparent in the conventional systems, are described. Results obtained from these alternative strategies are presented, and the importance of culture as a determinant of toxic response is emphasised. Evidence is presented that reduction of the tetrazolium dye, MTT, is a measure of nucleotide redox balance in hepatocytes, rather than of mitochondrial performance as previously believed.
The Role of Structure-activity Relationships and Expert Systems in Alternative Strategies for the Determination of Skin Sensitisation, Skin Corrosivity and Eye Irritation
Authors: Barratt Martin D.
Published: August 2020

A historical database containing the results of 294 defined single substances tested in the guinea-pig maximisation test, carried out according to a single protocol, was used to derive a set of structural alerts for skin sensitisation, which have been incorporated into the expert system, DEREK. Together with an assessment of percutaneous absorption, this system forms an integral part of a strategic approach to the identification of contact allergens.

Quantitative structure-activity relationships (QSARs) were derived for the skin corrosivity of organic acids and bases, and for the eye irritation potential of neutral organic chemicals. The independent variables used for these analyses were selected on the basis of the putative mechanisms for skin irritation or corrosivity and for eye irritation, respectively. Data sets were analysed using principal components analysis; plots of the first two principal components for each data set showed that the analyses were able to discriminate well between chemicals with different classifications of toxicological activity. The derived QSARs are expected to give useful predictions of skin corrosivity and eye irritancy for new or untested chemicals in these classes.

Although the development of these techniques is still at a very early stage, they are already able to play an important part in proposed strategies for the reduction of experimental animal usage. In the long term, it should be possible to conduct safety evaluations using fewer experimental animals or no animals at all. However, acceptance by regulatory authorities will be a key factor in realising the full benefits of the approach.
Appendix A: Quantitative Structure-activity Relationships for Skin Corrosivity
Authors: Barratt Martin D.
Published: August 2020

Quantitative structure-activity relationships (QSARs) have been derived which relate skin corrosivity data on organic chemicals (acids, bases, phenols and neutral chemicals) to their log P (log [octanol/water partition coefficient]) values, molecular volumes, melting points and pKa/ pKb values. Data sets were analysed using principal components analysis. For each group of chemicals, plots of the first two principal components of the above parameters, which broadly model skin permeability and cytotoxicity, showed that the analysis was able to discriminate well between corrosive and non-corrosive chemicals. The QSARs derived should be useful for predicting the skin corrosivity potentials of new or untested chemicals within these categories.
The Development of the Three Rs Concept
Authors: Russell W.M.S.
Published: August 2020

In 1955–57, in the course of a study initiated by Charles Hume and the Universities Federation for Animal Welfare, Rex Burch and I conceived of the Three Rs of humane experimental technique — replacement, reduction and refinement. Except for the foundation, in 1969, and the early work, of FRAME, the next two decades saw little development of the Three Rs, and widespread scepticism about the possibilities of replacement, especially in activity and toxicity testing. In the 1980s and early 1990s, advances in tissue culture technique led to spectacular progress in fundamental pharmacology and toxicology. There have also been important recent advances in aspects of refinement discussed in our book published in 1959, notably in anaesthesia and analgesia, and in the application of ethology. However, there has been less progress in reduction, except for one ingenious new statistical technique for reducing numbers of animals used for testing vaccines. Developments we scarcely touched on in 1959 include the application of the Three Rs to the production and testing of vaccines, the education of experimenters in laboratory animal science (both of these developments pioneered in The Netherlands), and the replacement of animals in teaching by the use of computers and audiovisual displays. A priority for the future is the application of the Three Rs to work with primates. New uses for experimental animals continue to arise, but, after recent successes, we can now have considerable confidence in the progress of humane experimental technique.
Chimpanzees in Laboratories: Distribution and Types of Research
Authors: Stephens Martin L.
Published: August 2020

This review presents the results of an informal 1993 survey of the distribution of chimpanzees (Pan troglodytes) in the world's laboratories, and the types of research conducted on them. Based on the available information, there are over 2200 chimpanzees in. laboratories, most of which are located in several US facilities. Europe apparently has less than 200 chimpanzees housed in two facilities. Worldwide, an estimated 80% of the chimpanzees in laboratories are used in studies on AIDS and hepatitis. It is concluded that, if Europe terminated its use of chimpanzees in research, for either financial, moral or political reasons, the impact on experimental research would be relatively minor.