bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Sensitivity of Two Mouse Lymphoid Tumour Cell Lines (P388 and P388D1) to Five Anticancer Drugs In Vitro
Authors: Arnould Roland , Dubois Jacque , Abikhalil Fokri , Libert Anita , Ghanem Ghanem , Atassi Ghanem , Hanocq Michel , Lejeune Ferdy J
Published: August 2020

The sensitivity and the selectivity of two murine lymphoid cell lines (P388 and P388D1) to five chemotherapeutic drugs were investigated in vitro. The cytotoxicities of melphalan, daunorubicin, hexamethylmelamine (HMM), hydroxymethylpentamethylmelamine and dihydroxymethyltetramethylmelamine, two HMM derivatives, were measured in the two cell lines using two different techniques: reduction of a tetrazolium derivative (MTT) and tritiated thymidine uptake into DNA. Cytotoxicity was expressed by the 50% inhibitory concentration (IC50) after one hour and after exposure of cells to each drug for two days. The IC50 results indicate that the P388 cells were generally more sensitive to melphalan, daunorubicin, hydroxymethylpentamethylmelamine and dihydroxymethyltetramethylmelamine than the P388D1 cell line. HMM was found to be inactive in both cell lines. Despite the lower sensitivity of the P388D1 cell line compared with the P388 cell line, because of its greater homogeneity, it could replace the P388 line for the in vitro assays, bearing in mind that the P388D1 cell line sensitivity is 1.21 - 24.63 times lower than that of the P388 cell line, at least as far as the drugs tested are concerned. Moreover, our results emphasise that variations of sensitivity could occur with repeated passage in vitro.
The Use of Liver Cell Cultures Derived from Different Mammalian Species in In Vitro Toxicological Studies: Implementation in Extrapolation Models?
Authors: Blaauboer Bas J. , Wortelboer Heleen M. , Mennes Wim C.
Published: August 2020

In vitro techniques can be applied to obtain insight into the organotoxic potential of compounds and in the underlying mechanisms of action. During the last few decades the applicability of these techniques in toxicology has been demonstrated for an increasing number of cell types. Cultured hepatocytes have been shown to provide convenient in vitro systems for studying the role of biotransformation in the toxicity of a compound. The use of in vitro systems derived from a range of species may lead to a better understanding of species differences in the toxicity of xenobiotics, thus improving interspecies extrapolations. The applicability of this approach will be illustrated with results from experiments on the biotransformation and cytotoxicity of bromobenzene, the in vitro inducibility of biotransformation enzyme systems and the effects of hypolipidemics in primary hepatocyte cultures derived from a numberof animals.
ELISA for Guinea-pig IgG1: An Alternative to the PCA Test for Relative Allergenicity
Authors: Prentø Annette
Published: August 2020

An ELISA method to replace the PCA (passive cutaneous anaphylaxis) test has been developed for use in the evaluation of relative allergenicity in guinea-pigs (Dunkin-Hartley). The method detects antigen-specific IgG1 antibodies formed in the guinea-pig during sensitisation, and correlates well with the corresponding PCA test.

The relative importance of IgG1 and IgE for relative allergenicity in the Dunkin-Hartley guinea-pig strain is discussed.
Effects of 4-Hydroxynonenal on Microtubular System of Normal and Transformed Fibroblasts
Authors: Gadoni Elena , Olivero Antonella , Miglietta Antonella , Gabriel Ludovica
Published: August 2020

Microtubules are known to be important in cell division and proliferation; however, their role in cell transformation is controversial. Cell proliferation can be inhibited by antimicrotubular drugs, and 4-hydroxynonenal, a product of lipid peroxidation with antiproliferative characteristics, is able to interact with tubulin, the main protein of microtubules. In this study, the effects of 4-hydroxynonenal on both normal and transformed cells (3T3 and SV40 transformed 3T3 fibroblasts) were examined, in order to investigate the significance of in vivo alterations of microtubules. An immunofluorescence technique was used to examine any modifications of the microtubule network, and colchicine-binding activity was assayed to determine tubulin content. Morphological observation of the microtubule network did not reveal any significant difference between normal and transformed fibroblasts; the colchicine-binding assay detected a stronger resistance in the latter cells.
ATP and Protein Synthesis Assays to Evaluate the Toxicities of Preservatives in Vitro
Authors: Galli Corrado L. , Viviani Barbara , Casartelli Giampiero , Marinovich Marina
Published: August 2020

Cellular protein content, protein synthesis, ATP level and lactate dehydrogenase (LDH) activity, measured in a murine epidermal cell line (HEL/30), were used as the endpoints for determining the cytotoxicities of 17 antimicrobial chemicals. The relative toxicities of the test compounds were quantified by the determination of the concentrations inducing a 50% inhibition of [3H]-leucine incorporation into proteins (IC50), causing a 50% reduction of ATP level or final cell protein content or producing the maximal effect on LDH leakage (EC50) after 2 hours of treatment. The results indicate a good correlation between both the reduction of ATP level and inhibition of protein synthesis and the minimal inhibitory concentration (MIC) on different microorganisms, suggesting that ATP and protein synthesis assays could be useful as prescreening methods for testing the cytotoxicities of preservatives.
Two Procedures for the Prediction of Acute Toxicity (LD50) from Cytotoxicity Data
Authors: Halle Willi , Spielmann Horst
Published: August 2020

Single linear regression analysis was used to characterise the relationship between cytotoxicity in a variety of mammalian cell culture systems and acute oral toxicity (LD50) in experimental animals. The following results were obtained.

Firstly, in a cytotoxicity assay using the calf aortic endothelial cell line BKEz-7, IC50 values determined for 44 chemicals in culture showed significant correlation with the oral LD50 values for rat and mouse (computed correlation coefficient r=0.546). After eliminating three chemicals that were characterised by extreme lethality indices (LI = IC50/LD50), the correlation coefficient of the remaining 41 chemicals increased to a value of r=0.728. By using the linear regression model for these 41 chemicals, the oral LD50 for rat and mouse can be predicted correctly from the IC50 values for 83% of substances from a variety of chemical substance classes within a range of approximately one order of magnitude of dosage unit of LD50 for rat and mouse.

Secondly, the mean IC50 values (IC50x¯) determined as the geometrical mean of two or more IC50 values per substance, which were generated in a wide spectrum of mammalian cell lines and collected in a “Registry of Cytotoxicity” (RC), gave similar results (r=0.644). Likewise, with the aid of this method, the oral LD50 for rat and mouse can be predicted for 74% of non-selected chemicals from structurally-different classes in the same dosage range, e.g., 1–25 millimoles per kg body weight.

The prediction of LD50 values from in vitro cytotoxicity data may permit the calculation of a more precise dose range-finding and offers a new way for reducing the number of animals in acute toxicity testing.
In Vitro Cytotoxic Effects of Extracellular ATP
Authors: Murgia Marta , Pizzo Paola , Zanovello Paola , Zambon Annalisa , Di Virgilio Francesco
Published: August 2020

ATP is released in the body from several cells under various physiological and pathological conditions. A number of authors have postulated a role for extracellular ATP (ATPo) as a neurotransmitter, a secretagogue or an inflammatory mediator. Here, we propose an additional role for ATPo, as a cytotoxic factor, and discuss in vitro experiments showing that this nucleotide causes cell death by two mechanisms: colloido-osmotic lysis and apoptosis.
In Vitro Reconstitution of Differentiated Human Epithelia
Authors: Pellegrini Graziella , Gherzi Roberto , Franzi Adriano Tito , D'Anna Fiorella , De Luca Michele , Cancedda Ranieri
Published: August 2020

Human keratinocytes obtained from skin biopsies can be serially cultured in vitro. When plated on lethally irradiated 3T3 fibroblasts, keratinocyte colonies reconstitute a stratified squamous epithelium devoid of stratum corneum. The expression of a mature cornified epidermis, expressing all the morphological and biochemical markers of the in vivo epidermis, can be obtained by the “emerged dermal equivalent” culture system. Melanocytes grown under the same culture conditions, maintain a physiological melanocyte/keratinocyte ratio, are organised in the basal layer of the cultured epidermis, and maintain differentiated functions such as dendritic arborisation, melanin synthesis and melanosome transfer. This allows the reconstitution of an epidermis physiologically populated by functionally active melanocytes. Epithelial cells from different mucosal body sites, namely palate, urethra, conjunctiva, cornea and vagina, can also be cultured and maintain the characteristics of the original donor sites. The in vitro reconstituted human epithelia, permanently transplanted onto patients presenting large epidermal or mucosal defects, retain the characteristics of the original donor site, suggesting an intrinsic site-specific differentiation programme. These three-dimensional human epithelium models could prove useful in standard cytotoxicity assays and could be used as a tool to study the effects of a variety of compounds on normal human epithelia in vitro.
The Micronucleus Test in Cultured Mammalian Cells and in Mytilus Gill Tissue
Authors: Gabriele Marco , Brunetti Riccardo , Majone Franca
Published: August 2020

The kinetochore-containing micronuclei induced in CHO cells by clastogenic and aneuploidogenic substances, such as mitomycin C (MMC) or colchicine, were distinguished by means of a technique based on the use of human CREST antikinetochore antibodies. Both substances induced their expected effects. However, 48 hours after the end of treatment, colchicine and MMC induced a significant increase of CREST– and CREST+ micronuclei, respectively, suggesting an induction of clastogenicity by colchicine and of aneuploidy by MMC. These results were confirmed by the characterisation of the micronuclei induced by the same substances in gill cells of the marine mussel, Mytilus, treated in vivo. A cytofluorimetric analysis on the isolated gill of this animal showed that the cell cycle remained unaltered until almost 24 hours after excision. This suggests the possibility of using the isolated gill of Mytilus as a new biological system for laboratory experiments.
BaP Metabolism and DNA-adduct Formation in Cultured Human Lymphocytes Treated In Vitro with BaP and (-)-BaP-7,8-dihydrodiol
Authors: Pavanello Sofia , Zanesi Nicola , Levis Angelo Gino
Published: August 2020

Nine samples of human lymphocytes from six healthy subjects were treated in vitro for 24 hours with [3H]-benzo[a]pyrene (BaP) and unlabelled (-)-BaP-7,8-dihydrodiol; 1μg/ml and 0.5μg/ml, respectively. The separation by HPLC of BaP-DNA adducts showed a significant formation of syn-BaPDE-dGuo adducts in five out of the nine human lymphocyte samples treated in vitro with [3H]-BaP. BaP-(7,10/8,9) tetrol and BaP-(7/8,9,10) tetrol derived from (+)-anti-BaPDE were the predominant isomers extracted from the culture medium of all the lymphocyte samples treated with (-)-BaP-7,8-dihydrodiol (mean ratio of anti-BaPDE/syn-BaPDE tetrols 5.6 + 1.2), suggesting that the metabolic activation of (-)-BaP-7,8-dihydrodiol occurred by its epoxidation to (+)-anti-BaPDE much more frequently (4–8 times) than by epoxidation to the (-)-syn-BaPDE form. In contrast to the predominant formation of anti-BaPDE tetrols, the analysis by HPLC of DNA adducts in the five subjects examined also revealed the significant formation of syn-BaPDE-DNA adducts (mean adduct ratio of (+)-anti-/(-)-syn-BaPDE-DNA = 2.6 ± 0.7). In six human lymphocyte samples treated in vitro with anti-BaPDE and syn-BaPDE, the anti-BaPDE bound to DNA 2–5 times more efficiently than did the syn-BaPDE. However, after 24 hours, the level of anti-BaPDE-DNA adducts significantly decreased almost to the level of the syn-BaPDE-DNA adducts, suggesting that a stereoselective repair mechanism could have preferentially removed the anti-BaPDE-DNA adducts.
A New In Vitro Model for Predicting the Toxicity of Cosmetic Products
Authors: Carrara Maria , Cima Lorenzo , Cerini Roberto , Carbonare Maurizio Dalle
Published: August 2020

A method has been developed whereby cosmetic products which are not soluble in water or in alcohol can be brought into contact with cell cultures by being placed in a cell culture insert, which is then placed in the cell culture well. Preliminary experiments were carried out with L929 cells, and cytotoxicity was evaluated by measuring neutral red uptake and the total protein content of treated cultured cells. Encouraging results were obtained in comparisons of three cosmetic emulsions and of one emulsion containing a range of concentrations of two preservatives, Kathon CG and Bronopol.
Effects of the First Twenty MEIC Reference Chemicals on Viability, Glucose Consumption and Spontaneous Contractility of Primary Cultured Rat Skeletal Muscle Cells
Authors: Gulden Michael , Finger Jutta
Published: August 2020

Primary cultured rat skeletal muscle cells were used to determine concentration-dependent effects of the first twenty MEIC chemicals on three endpoints, spontaneous contractility and viability after 1 and 24 hours, and glucose consumption during 24 hours of exposure. The contractions of cultured muscle cells depend on spontaneous electrical activity of the excitable cell membranes. The majority of the test compounds inhibited contractility at concentrations which affected neither viability nor glucose consumption. Most of these compounds are known to interact with excitable membranes in a site-specific or non-site-specific manner, thereby causing therapeutically intended or toxic effects. The results indicate that inhibition of spontaneous contractility of cultured skeletal muscle cells may reflect important non-cytotoxic biological activities of test chemicals which might be more relevant for their acute toxicity than cytotoxic action.
Evaluation of Cytotoxicity of the First Twenty MEIC Chemicals by Using Haemolysis of Human Erythrocytes as an Endpoint
Authors: Isomaa Boris , Lilius Henrik , West Anne
Published: August 2020

The cytotoxicity of the first twenty chemicals on the MEIC list was evaluated by determining the haemolytic activity of the chemicals. All the tested chemicals, except malathion, were haemolytic. With digoxin and sodium fluoride, however, the maximum degree of haemolysis achieved remained less than 50%. The EC10 for the chemicals varied from 0.38mM (digoxin) to 4,830mM (ethylene glycol). Comparing our data for the first ten MEIC chemicals with data obtained with other in vitro test systems, they correlated well with data reported for human lymphocytes (r = 0.93), rat hepatocytes (r = 0.98) and 3T3 cells (r = 0.95). However, in comparison with these tests, the haemolysis assay was less sensitive. The EC10 for the first ten MEIC chemicals correlated rather well (r = 0.84) with human lethal blood concentrations, but the haemolysis assay underestimated the toxicity of most of the chemicals and particularly underestimated the toxicity of digoxin.
Modification of the FRAME Kenacid Blue Method for Cytotoxicity Tests on Volatile Materials
Authors: Smith Lesley M. , Clothier Richard H. , Hillidge Sonia , Balls Michael
Published: August 2020

In attempts to overcome the problem for cell culture cytotoxicity tests represented by highly volatile test materials, the wells of 96-well tissue culture plates were sealed individually, either by means of adhesive plastic film spread across the whole plate or by the addition of a small amount of paraffin oil to each well. Use of the adhesive film reduced the growth of control 3T3-L1 murine fibroblast-like cells by nearly 40%, whereas the addition of paraffin oil did not affect cell growth. The latter method was therefore used to compare the cytotoxicities of 27 liquid and 11 water-soluble solid test materials in sealed and unsealed wells, using the FRAME 72-hour kenacid blue method.

Of the 27 liquids tested, acetaldehyde, acetone, acetonitrile, allyl alcohol, ethanol, ethyl acetate, methanol, pyrrole and propanol had significantly greater cytotoxic effects in oil-sealed than in unsealed wells. The cytotoxicities of acetic acid, aniline, butanol, 1,2-dibromomethane, dimethyl formamide, dimethyl sulphoxide, glycerol, hydrochloric acid, 2-methoxyethanol, octanol, pentanol, sulphuric acid, tributyltin chloride, trichlorobenzene, trifluoroacetic acid and Tween 20 were not affected by the layer of paraffin oil. Heptanol and hexanol showed significantly lower cytotoxic effects in oil-sealed than in unsealed wells, possibly because of interactions between these alcohols and the oil, since certain alcohols are more soluble in oil than in culture medium. Only one of the 11 solid materials tested, sodium hydroxide, showed greater cytotoxic effects in oil-sealed wells, possibly because of an effect on the culture medium buffering system.

The use of paraffin oil as a partial well sealant in 96-well tissue culture plates promises to improve the applicability of in vitro cytotoxicity tests to volatile test materials, by improving confidence that their toxicities have not been underestimated.
Paraquat Alters Growth, DNA and Protein Synthesis in Lung Epithelial Cell and Fibroblast Cultures
Authors: Barile Frank A. , Arjun Seeta , Senechal Jean-Jacques
Published: August 2020

In vivo exposure to paraquat, a bipyridyl herbicide, is associated with selective necrosis of type I and type II alveolar pneumocytes. We examined the influence of paraquat on cell growth, DNA and protein synthesis in human and rat lung epithelial and fibroblast cell lines, to determine the effects and affinity of the chemical in vitro for cells of various origins. Cells were incubated with [3H]-thymidine or [3H]-proline in the absence or presence of 0.05–10mM paraquat for periods of 6 or 24 hours. Cell cultures grown in the presence of 50–200μM paraquat exhibited significantly lower cell numbers than comparable controls after 3 days. The suppression of DNA synthesis by paraquat was dose-dependent, but did not rely on the length of exposure. Paraquat inhibited protein synthesis and selectively decreased net production of low molecular weight proteins. NADPH levels were the same in both control and treated cultures. [14C]-Paraquat was accumulated during a 4-hour exposure period via an active non-competitive process. The results suggest that lung fibroblasts are as sensitive to the herbicide as epithelial cells. In addition, paraquat appears to interfere with transcription and/or translation by its ability to generate superoxide radicals, rather than by depletion of reducing equivalents.
The Possible Use of Isolated Human Platelets in In Vitro Cell Toxicology
Authors: Mäkelä Jan-Henrik , Isomaa Boris
Published: August 2020

Isolated human platelets represent a model system which could be used in in vitro cell toxicology for mechanistic studies on the short-term effects of cytotoxic compounds. We have recorded changes in the concentration of free cytosolic Ca2+ ([Ca2+]i) with Fura 2 in isolated human platelets treated with sphingosine, a compound suggested to be a specific inhibitor of protein kinase C (PKC). We huve shown that sphingosine is able to induce a rapid and transient release of Ca2+ from the intracellular stores in platelets. Furthermore, a massive efflux of released Ca2+ was shown. These effects of sphingosine on the human platelet might leave the cell incapable of performing Ca2+-dependent reactions, including PKC-mediated phosphorylation, upon stimulation subsequent to sphingosine-treatment.
The Use of Artemia Salina in Toxicity Testing
Authors: Lewan Lillemor , Andersson Marianne , Morales-Gomez Paloma
Published: August 2020

This study shows that the Artemia assay, which is usually performed by incubation for a 24-hour period in artificial sea water, can also be performed in phosphate buffered saline (PBS) at pH 7.2, or in a cell culture medium, both of which are used in toxicity assays employing mammalian cells. Thus, by using the equivalent media for incubation, toxic effects in the Artemia assay and toxic mechanisms can more accurately be compared with results obtained in mammalian cell toxicity. Survival of control animals is good for 72 hours, provided that infection can be avoided. A pH greater than 6 is essential for good survival of Artemia Salina, and a pH greater than 10.5 should be avoided. Because of the risk of infection at low saline concentrations, a decreased incubation time of 16 or 12 hours is recommended.

The lethal concentrations (LC10 and LC50) in the 24-hour Artemia assay of the first ten chemicals in the MEIC programme were measured in PBS, and the results compared with those from a previous study of the effects in a 10-minute acute ATP leakage assay, specifically indicating lesions in the cell membrane. The EC10 and EC50 values for the alcohols in the Artemia assay were 35–75% of the corresponding values in the ATP leakage assay. For paracetamol and amitriptyline, the EC10 and EC50 values in the Artemia assay were 2–16% of the corresponding values in the ATP-leakage assay. The greatly increased toxicity of the two compounds in the animals may be related to systemic effects. FeSO4 was very toxic to Artemia salina at a concentration of lug/ml, but it did not cause leakage of ATP from cultured cells, even at a concentration of 8,000μg/ml, showing that widely different mechanisms of interaction with FeSO4 are measured by the two assays. A lower toxicity of polygodial, a sesquiterpenoid unsaturated dialdehyde, in cell culture medium, was obvious in the Artemia assay. Thus, some factors in cell culture medium must, by interacting with the toxic molecule, protect the animals against the toxicity of polygodial, as was previously found in cultured cells.
Prediction of Eye Irritation Potential of Surfactants using the SIRC-NRU Cytotoxicity Test
Authors: Roguet Roland , Dossou Koovi G. , Rougier Andre
Published: August 2020

The SIRC cell line, derived from rabbit cornea was used to assess the cytotoxicity of a wide range of surfactants. The measurement of their effect on the cell membrane, the critical target of surfactants, has been performed using the neutral red uptake (NRU) bioassay. The results obtained for 30 surfactants have been correlated with in vivo data (Draize test) obtained from a data bank. The cytotoxicity of the tested surfactants ranks as follows: cationic > anionic > amphoteric > non-ionic. A good correlation (r = 0.80 and p < 10-4) was obtained. In the case of non-ionic surfactants (n 11) a higher correlation with in vivo data was obtained (r = 0.92 p < 10-4), showing that the SIRC-NRU test is particularly useful for the evaluation of non-irritants or weak irritants currently used in cosmetic formulations. The results demonstrated that this easily performed method leads to few false negatives (6%) and thus merits its inclusion in a battery of screening tests for predicting eye irritancy without the use of animals.
Contributions of Physicochemical Properties to the Evaluation of Ocular Irritation
Authors: Régnier Jean-François , Imbert Christophe
Published: August 2020

International guidelines for the evaluation of ocular irritation advise that all the available information, structural and physicochemical, should be taken into account in order to minimise the testing of substances under conditions that are likely to produce severe reactions in laboratory animals.

In order to determine whether the pH and the acidic/alkaline reserve would be suitable parameters for the prediction of ocular irritation, we have measured, under standard conditions, the pH and the acidic/alkaline reserve of 166 chemicals (90 non-irritant, 22 irritant, and 54 severely irritant for the eye, according to the EEC classification and labelling system).

The combination of these parameters permitted the correct identification of 74% of the severely irritant chemicals and 97% of the non-severely irritant (non-irritant or irritant) chemicals. The predictive value for a severely irritant potential was 93%, and 88% for non-severely irritant chemicals.

This physicochemical approach, used as a first line screen for ocular irritation, is very simple and inexpensive.
Evaluation of the EYTEX™ System as a Screening Method for Ocular Tolerance: Application to Raw Materials and Finished Products
Authors: Courtellemont Pascal , Hebert Patrice , Redziniak Gerard
Published: August 2020

In vitro evaluation of ocular irritation requires the use of a battery of tests, for example cell culture assays and the HET-CAM test. In order to check the field applications of the different methods and their complementation, we have studied the EYTEX™ physicochemical model in an internal validation study.

In this study, we compared the EYTEX™ results with the in vitro ocular irritation potencies of 52 formulations and 49 chemicals. The in vivo/in vitro correlation was highly significant, p < 0.01%, K = 0.8 (Kappa method). On the other hand, the concordance, sensitivity and specificity of this method were, 85%, 89% and 83% respectively. The compatability of 98% obtained showed the kit to be applicable to cosmetic formulations.

The EYTEX™ system has also been evaluated during a comparative study of EYTEX™ human clinical tests, performed on 12 finished products; we have observed a concordance equal to 92%, indicating an excellent correlation between the two methodologies.

These results suggest that the EYTEX™ test could be a reliable in vitro prescreening system for determining the eye irritation potential of cosmetic formulations, and that it should be accepted as a component of our battery of alternative methods.
K562. A Human Cellular System Capable of Undergoing In Vitro Differentiation: Measurement of Genotoxic Parameters Useful for Cytotoxicity Evaluation
Authors: Sapora Orazio , Maggi Antonella , Maione Barbara , Pazzaglia Simonetta , Tabocchini Maria-Antonella
Published: August 2020

The initial damage produced by ionising radiation and its subsequent repair have been studied in a cellular system. K562 cells are used which are capable of undergoing in vitro pseudoerythroid differentiation in the presence of 2mM butyric acid. The level of initial damage is similar in actively growing and 48-hour differentiated cells, while it is lower by a factor of two in cells irradiated after exposure to the inducer for 72 hours. In differentiated cells, the kinetics of repair measured up to 60 minutes is slower than that in actively growing cells. These findings suggest that the genome of actively proliferating cells is not only more susceptible to radiation-induced damage, but also more accessible to repair enzymes than the more compact genome of differentiated cells.

Repair after a longer time interval has also been investigated. After 24 hours of repair, the amount of residual damage is higher in actively proliferating cells than in differentiated ones. However, in proliferating cells, DNA synthesis can interfere with repair of the lesions or vice versa, while in differentiated cells, due to the lack of proliferation, damage occurring in non-transcribing genes can presumably be sustained for longer periods without biological consequences.

A method to evaluate the relative sensitivity of non-duplicating differentiated cells is proposed. It is based on measurement of the residual damage, detected after 24 hours of repair, and on the assumption that a relationship exists between unrepaired DNA damage (residual damage) and the ability of the cell to survive.
Evaluation of the Cytotoxicity of the First 20 MEIC Chemicals in Two Hepatoma Cell Lines with Different Xenobiotic Metabolism Capacities
Authors: Bassi Anna Maria , Bosco Ornella , Brenci Sabrina , Adamo Daniela , Penco Susanna , Piana Sandra , Ferro Margherita , Nanni Giorgio
Published: August 2020

The cytotoxicity of the first 20 chemicals on the MEIC list was evaluated using two hepatoma cell lines having different xenobiotic metabolic capacities, one derived from the rat (HTC), and one of human origin (Hep G2). Two endpoints were measured to evaluate cytotoxicity: colony-forming ability (CF), and cell viability of the attached monolayer (CV), evaluated as total macromolecular content. The CF test provided the most sensitive endpoint, due to the lower number of exposed cells in comparison with the CV test. Using the CF assay, the Hep G2 cell line showed higher sensitivity than the HTC cell line to some chemicals known to be metabolised in vivo. The IC50s obtained under the different experimental conditions varied as a consequence of phenotypic differences between the cell lines, and of the nature of the endpoints. The most toxic chemicals were malathion, diazepam and amitriptyline (IC50: 0.001-0.1mM). The solvents tested produced the lowest toxic effects (IC50: > 10mM).

These findings suggest that hepatoma cell lines possessing various specific enzyme activities could be usefully employed in a battery of tests designed to reproduce in vitro the wide range of biochemical properties expressed by the cells in the whole organism.
A European Interlaboratory Evaluation Study of an In Vitro Ocular Irritation Model (Skin2™ Model ZK1100) using 15 Chemicals and 3 Shampoos
Authors: Pirovano Raffaella , Zaninelli Pietro , Noben Jos , Logemann Pamela , Southee Jacqueline , Joller Peter , Coquette Alain
Published: August 2020

The aim of this interlaboratory study was to ascertain the feasibility of the use of the in vitro Skin2™ Model ZK1100 kit as a tissue substrate for cytotoxicity studies.

Eighteen compounds — 15 raw chemicals and 3 finished products (commercial shampoos) — were tested in four different laboratories on two separate occasions, and the data obtained were compared with the data available in the literature on eye irritancy potential.

The kit was easy to manage, and no technical difficulties were encountered in the estimation of MTT reduction, LDH and PGE2 release in the medium. Similar MTT50 values were recorded in two independent experiments in each laboratory, and the rankings of irritation potency within the same chemical class were comparable. Both LDH and PGE2 were important parameters to be taken into account along with MTT50 values in establishing the irritation potential of some categories of compounds.
Evaluation of the First Ten MEIC Chemicals in the Allium Test
Authors: Fiskesjö Geirid , Levan Albert
Published: August 2020

After joining the MEIC (multicentre evaluation of in vitro cytotoxicity) programme, and submitting the first ten compounds of the programme to the Allium test, we found that this test correlates well with several other tests (e.g. MIT-24 cell test, and tests with mice, rats or humans in vivo).

The compounds tested were paracetamol, acetylsalicylic acid, iron sulphate, diazepam, amitriptyline, digoxin, ethylene glycol, methanol, ethanol and isopropanol. Among them, paracetamol turned Out to induce chromosome breakage in the meristems of Allium cepa root tips. This is the same qualitative response to paracetamol as that obtained previously in human lymphocytes in vivo and in vitro. The present results indicate that the eukaryotic higher plants may serve as highly useful test systems for biological risk evaluation.
Evaluation of the Cytotoxicity of the First Ten MEIC Chemicals in 3T3 Mouse Fibroblasts with and without Microsomal Activation, Using the Neutral Red Assay
Authors: Rasmussen Eva Selzer
Published: August 2020

The cytotoxicity of the first ten MEIC chemicals was evaluated by determination of neutral red uptake in BALB/C 3T3 mouse fibroblasts both with and without the addition of a microsomal activation system. The IC20 values of all the chemicals tested without microsomes were significantly correlated to in vivo data on acute systemic toxicity. The use of microsomes weakened the overall correlation with the in vivo data. Useful information regarding the activation of acetylsalicylic acid, iron sulphate, methanol and ethanol was obtained using microsomes, while misleading information was obtained regarding the metabolism of paracetamol, diazepam, digoxin and ethylene glycol. In the case of alcohol metabolism, the Aroclor 1254 induction of rat liver enzymes seems to parallel the induction of cytochrome P450 IIE1, which is known to metabolise a number of alcohols to free radicals which might exert a variety of cytotoxic effects. For this reason, the use of Aroclor 1254 induction in cytotoxicity and genotoxicity testing is questioned.
Use of Toxicokinetics in Risk Assessment Based on In Vitro Data
Authors: Johanson Gunnar
Published: August 2020

This presentation addresses some aspects of the methodology, advantages and problems associated with toxicokinetic modelling based on in vitro data. By using toxicokinetic models, particularly physiologically-based ones, it is possible, in principle, to describe whole body toxicokinetics, target doses and toxic effects from in vitro data. Modelling can be divided into three major steps: 1) to relate external exposure (applied dose) of xenobiotic to target dose; 2) to establish the relationship between target dose and effect (in vitro data, e.g. metabolism in microsomes, partitioning in tissue homogenates, and toxicity in cell cultures, are useful in both steps); and 3) to relate external exposure to toxic effect by combining the first two steps. Extrapolations from in vitro to in vivo, between animal and man, and between high and low doses, can easily be carried out by toxicokinetic simulations. In addition, several factors that may affect the toxic response by changing the target dose, such as route of exposure and physical activity, can be studied. New insights concerning the processes involved in toxicity often emerge during the design, refinement and validation of the model. The modelling approach is illustrated by two examples: 1) the carcinogenicity of 1,3-butadiene; and 2) the haematotoxicity of 2-butoxyethanol. Toxicokinetic modelling is an important tool in toxicological risk assessment based on in vitro data. Many factors, some of which can, and should be, studied in vitro, are involved in the expression of toxicity. Successful modelling depends on the identification and quantification of these factors.
The Use of Several Leakage Markers which Enable Platelet Membrane Damage and Platelet Activation to be Distinguished
Authors: Mäkelä Jan-Henrik , Isomaa Boris
Published: August 2020

We have studied the effects of four differently charged detergents and sphingosine on platelet membrane integrity and platelet activation, using the leakage of pre-loaded 43K+, pre-loaded [14C]-serotonin ([14C]-5-HT) and lactate dehydrogenase (LDH) as markers.

Sphingosine, at a concentration of 20μM, induced a release of [14C]-5-HT as well as 43K+, without releasing LDH. The [14C]-5-HT743K+ ratio was about 1.5. A similar leakage pattern and the same [14C]-5-HT/43K+ ratio was also induced by thrombin, a potent platelet agonist, indicating that the release induced by sphingosine is due to platelet activation and not to membrane damage. The detergents, on the other hand, induced a marked release of 43K+ followed by an increased release of LDH. No pronounced leakage of [14C]-5-HT occured with the detergents; the [14C]-5-HT/43K+ ratio was ≤ 0.2. In this manner, we can distinguish between marker-release caused by membrane-damage and that caused by platelet-activation, by using the combination of leakage markers presented here.

Our study also showed that fixation prior to centrifugation is crucial in order to avoid leakage of markers, which are released upon activation of the platelets. About 35% of the [14C]-5-HT content of the platelets was released when unfixed platelets were centrifuged, and, interestingly, the detergents, as well as ethanol, inhibited the centrifugation-induced release of [14C]-5-HT.
In Vitro Toxicity Testing Using Marine Luminescent Bacteria (Photobacterium phosphoreum): the Biotox™ test
Authors: Kahru Anne
Published: August 2020

Marine luminescent bacteria emit light naturally when they are in an optimal environment. If harmful substances are present, their bioluminescence decreases, and the amount of light loss is proportional to the toxicity. In this paper, a new short-term toxicity test system, Biotox™, based on freeze-dried Photobacterium phosphoreum, is assessed. Concentration-effect curves for 25 laboratory chemicals were measured, and EC50 values (the concentrations of chemical, μg/ml, which reduce the light output of bacteria by 50% after being in contact with bacteria over a certain time period) were calculated. The sensitivity of the Biotox™ test to hydrophilic compounds was approximately two times greater than the sensitivity of the Microtox™ test. However, the sensitivity of the Biotox™ test to hydrophobic compounds was shown to be 2–4 times lower than the sensitivity of the Microtox™ test. The Biotox™ test was especially sensitive to As5+, giving an EC50 value of 0.04μg/ml, compared with 1.5μg/ml in the Microtox™ assay.
A Multicentre Study of Acute In Vitro Cytotoxicity in Rat Hepatocytes: Tentative Correlation Between In Vitro Toxicities and In Vivo Data
Authors: Fautrel Alain , Chesné Christophe , Guillouzo André , Sousa Georges De , Placidi Michel , Rahmani Roger , Braut Françoise , Pichon Jacqueline , Hoellinger Henri , Vintèzou Pascale , Melcion Celine , Cordier André , Lorenzon Giocondo , Benicourt Marc , Fournex Robert , Bichet Nicole , Gouy Daniel
Published: August 2020

A multicentre validation study of the acute in vitro cytotoxicities of 31 liquid or solid chemicals was carried out by six laboratories, using primary rat hepatocyte cultures as a model system. We report here a comparison of neutral red uptake IC50 and LD50 values. Oral, i.p. and i.v. LD50 values were available for 27, 24 and 18 chemicals, respectively, and an IC50 value was obtained for 15, 14 and 11 of these compounds, respectively. A significant correlation was found only between IC50 and i.v. LD50 values.
Research and Development of In Vitro Pharmacotoxicology: A European Perspective
Authors: Rodríguez-Farré Eduardo , Roberfroid Marcel , Fracchia Giovanni N.
Published: August 2020

The experts taking part in the Workshop were: E. Rodríguez-Farré ( Coordinator); G.N. Fracchia, (Secretary); M. Adolphe, École des Hautes Études, Paris, France); P.H. Bach (University of East London, UK); M. Baeder (Hoechst Ltd, Hattersheira, Germany); R. Bass (BGA, Berlin, Germany); H.G. Baumgarten (Frei Universität, Berlin, Germany); H. Bazin (DGXII, CEC, Brussels, Belgium); P. Bentley (Ciba-Geigy, Basle, Switzerland); A. Boobis (Royal Postgraduate Medical School, London, UK); J. Castell (Hospital La Fé, Valencia, Spain); J.P. Contzen (DGXII, CEC, Brussels, Belgium); A. Cordier (Sandoz Pharma Ltd, Basle, Switzerland); J. Diezi (Université de Lausanne, Switzerland); L. Dubertret (INSERM U-312, Creteil, France); P.M. Fasella (DGXII, CEC, Brussels, Belgium); J.H. Fentem (FRAME, Nottingham, UK); A. Guillouzo (INSERM U-49, Rennes, France); I. Kimber (Zeneca, Macclesfield, UK); T. Krieg (Universität zu Koln, Germany); A. Mantovani (Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy); K. Miller (BIBRA, Carshalton, UK); J.P. Morin (INSERM U-295, Rouen, France); D. Paul (Fraunhofer Institut für Toxikologie und Aerosolforschung, Hannover, Germany); P.W.J. Peters (Riijkinstituut voor Volksgezondheid en Milieuhygiene, Bilthoven, The Netherlands); J. Picard (Faculté des Sciences, Louvain la Neuve, Belgium); D. Poggiolini (Ministry of Health, Rome, Italy); C.M. Regan (University College, Dublin, Ireland); C.A. Reinhardt (SIAT, Zurich, Switzerland); B. Robaire (McGill University, Montreal, Canada); M. Roberfroid (Université Catholique de Louvain, Brussels, Belgium); V. Rogiers (Vrije Universiteit Brussels, Belgium); J. Rueff (Istituto de Higiene e Medicina Tropical, Lisbon, Portugal); H. Spielmann (ZEBET, Berlin, Germany); H. Stolte (Medizinische Hochschule, Hannover, Germany); J. van Noordwijk (European Pharmacopeia Commission, Bosch en Duin, The Netherlands); E. Walum (University of Stockholm, Sweden); D.C. Williams (Trinity College, Dublin, Ireland); and M. Yaniv (Institut Pasteur, Paris, France), and their contributions are gratefully acknowledged.
The Predictive Potential of a Battery of Ecotoxicological Tests for Human Acute Toxicity, as Evaluated with the First 50 MEIC Chemicals
Authors: Calleja Mabel C. , Persoone Guido , Geladi Paul
Published: August 2020

The acute toxicities of the first 50 chemicals (dextropropoxyphene hydrochloride excluded) of the multicentre evaluation of in vitro cytotoxicity (MEIC) programme were determined on four aquatic invertebrates and a bacterial strain (Photobacterium phosphoreum for the Microtox™ test) commonly used in ecotoxicology testing. Three of the aquatic invertebrate tests consisted of cyst-based toxicity tests (Artoxkit M with Artemia salina, Streptoxkit F with Streptocephalus proboscideus, and Rotoxkit F with Brachionus calyciflorus), and the Daphnia magna test. Results of simple linear regression analyses indicated that the rodent tests (rat and/or mouse) were better than the ecotoxicological tests for predicting acute oral lethal doses in man. However, it appears that the batteries of ecotoxicological tests resulting from the partial least squares method appear to be better than the rodent tests for predicting human oral lethal doses.
Bovine Pulmonary, Hepatic and Renal Tissues: Models for the Study of Mammalian C-S Lyase Enzymes
Authors: Buckberry Lorraine D. , Blagbrough Ian S. , Bycroft Barrie W. , Shaw P. Nicholas
Published: August 2020

C-S lyase (CSL) enzymes are responsible for the generation of toxicity via the cleavage of cysteine conjugates to generate reactive thiol species. In order to explore and characterise CSL activity in mammalian organs, cysteine conjugate CSL enzymes were isolated from bovine pulmonary, hepatic and renal tissues. Bovine tissue”, obtained from the abbatoir, affords a readily available source of viable CSL enzymes, without the necessity of sacrificing large numbers of laboratory animals simply to provide tissue. We have demonstrated that significant CSL activity exists in bovine tissues, and that the level of this activity is comparable with that found in human tissues. These enzymes provide an explanation for the previously reported episodes of bovine toxicity, and may provide a reasonable model for other mammalian CSL enzymes.
Critical Evaluation of an Agarose Diffusion Method for the Assessment of Eye Irritancy
Authors: Cottin Martine , Zanvit Alain , Faquet Brigitte , Rougier André , Dossou Koovi G.
Published: August 2020

The agarose overlay method is often used to study the cytotoxicity of non-liquid products. However, diffusion through agarose is a limiting factor in some cases, and this reduces contact between the cells and the test material.

We have developed a new model aimed at reducing this phenomenon. In our technique, rather than being cultured in a monolayer, V79 cells are suspended in 1% agarose medium. We obtained a good linear correlation between this method and the usual L-929 cell monolayer technique (r = 0.95; n = 31). In addition, the new technique is more sensitive. In particular, the cytotoxicity of surfactants is apparent at lower concentrations.

Using this technique, we studied 115 miscellaneous raw materials and cosmetic products, for which in vivo historical ocular irritancy data were available. As expected, our method was unsuitable for the study of alkaline and acidic products, as well as water-alcohol-based lotions. We also adapted the method for testing mascaras and obtained a good ranking for eye irritancy.

For the other 93 products (surfactants, shampoos, lotions, gels, emulsions and miscellaneous products), the mean diameter of the lysis zone after contact for 18 hours was used as the endpoint for comparison with in vivo maximum average scores. There was a strong linear/rank correlation between the two parameters (r = 0.83/rho = 0.79). Furthermore, the concordance between the in vivo and in vitro data in a three-class ranking system was good (weighed kappa index = 0.81). These results suggest that the agarose diffusion method is a potentially useful screening tool for the ocular safety of the categories of products described above.
Use of a Cloned G-Protein-coupled Receptor in the In Vitro Screening of Psychotropic Compounds
Authors: Konings Pierre N.M. , Vanderheyden Patrick M.L. , Stam Nico J. , van Delft Anton M.L. , Ruigt Gé S.F.
Published: August 2020

In the initial preclinical phase of the development of new psychotropic agents, potential drugs are generally characterised by in vitro receptor binding studies using rat brain preparations. We have recently, at this stage, incorporated assays using cell lines transfected with cloned receptors (1). One of the important 5-hydroxytryptamine (5-HT) receptor subtypes used for screening of these compounds is the 5-HT1A receptor, characterised by binding of the selective agonist [3H]-8-OH-DPAT. Results from these binding studies in rat hippocampus membranes appear to correlate very well with results obtained from membranes of fibroblasts expressing cloned human 5-HT1A receptors. This indicates that cloned receptors can be used to characterise psychotropic agents, thereby significantly reducing the number of experimental animals required. Moreover, the information obtained is likely to be of greater relevance for the evaluation of the potential therapeutic action of a drug in man.
Uptake of Organic Chemicals across Fish Gills: Alternatives to the Use of Fish Populations
Authors: Sijm Dick T.H.M.
Published: August 2020

In this study, the isolated perfused gills of rainbow trout (Oncorhynchus mykiss) provided a tool for fundamental research on the rate-limiting step in the uptake of organic hydrophobic chemicals. Data obtained using the isolated gills were comparable to those determined in vivo. Whereas several tens of fish need to be used to obtain statistically sound information on uptake rates in vivo, the isolated perfused gills method requires only three or four fish. A significant reduction in animal use is thus obtained.
Isolation and Culture of Proximal Tubular Cells from Porcine Kidney
Authors: Kruidering Marieke , Prins Frans A. , de Heer Emile , Mulder Gerard J. , Nagelkerke J. Fred
Published: August 2020

Porcine proximal tubular cells (PPTC) were isolated from kidneys obtained from slaughterhouse pigs. After disruption of the connective tissue by collagenase, purification was achieved by filtration and centrifugation on a discontinuous density gradient. Single cells and clusters of 10–40 cells were obtained, having a viability of 93–99%. More than 81% of the single cells showed γ-glutamyltranspeptidase (GGT) activity and more than 95% showed non-specific esterase (NE) activity, marker enzymes for proximal tubule cells. One kidney yielded 1 x 107 single cells and 3x107 cells in clusters. Cells were kept in primary culture on plastic or collagen-coated dishes. In the presence of 10% serum, confluency was reached within four days. The monolayers could be kept in culture for four days after confluency, in serum-free conditions. When seeded in serum-free conditions, PPTC did not reach confluency, but the cells could be kept in culture for at least 16 days. The cells displayed epithelial morphology, i.e. cobblestone shape, dome formation, microvilli, basal infoldings and abundant mitochondria. PPTC in primary culture still displayed NE activity, while 80% of the cells showed GGT activity. In conclusion, the isolated cells are of proximal tubular origin, reach confluency in 3–4 days in the presence of 10% serum, and can be kept as monolayers in serum-free conditions for four additional days and may provide a suitable in vitro model for long-term nephrotoxicity studies.