bluuebunny/crossref_metadata_2025 huggingface dataset:
An Investigation of Aluminium Neurotoxicity using some In Vitro Systems
Authors: Atterwill Christopher K. , Davies Wendy J. , Kyriakides Michael A.
Published: August 2020

It has been shown that acute exposure in vitro to high concentrations of aluminium chloride does not appear to perturb neural function in terms of the electrophysiological properties of lower vertebrate leech neurones. Longer term exposure in vitro, however, both non-specifically inhibits cellular differentiation and also produces neural cytotoxicity in the rat midbrain micromass, mixed cell culture model. Furthermore, previous studies from this laboratory have demonstrated a reduction of cholinergic neuronal function in brain organotypic reaggregate cultures following long-term, but not short-term, exposure. More-immature neural cells appear to be most sensitive to the effects of aluminium. Relating these data to the tiered in vitro test system for neurotoxicants previously proposed by Atterwill (13), it is apparent that the neurotoxic effects of aluminium are detectable in a first-stage procedure using the micromass culture model, but not following acute exposure in freshly isolated, ex vivo leech neurones. Functional cholinergic toxicity was also detected in the organotypic reaggregate cultures proposed as a second level screen.
Precision-cut Guinea-pig Liver Slices as a Tool for Studying the Toxicity of Volatile Anaesthetics
Authors: Ghantous Hanan N. , Fernando Jeanne , Morgan Scott E. , Gandolfi A. Jay , Brandel Klaus
Published: August 2020

Cultured precision-cut liver slices retain normal liver architecture and physiological biochemical functions. Hartley male guinea-pig liver slices have proven to be a good model for studying the biotransformation and toxicity of halothane. This system was used to evaluate the biotransformation and toxicity of different volatile anaesthetics (halothane, enflurane, isoflurane and sevoflurane), and compare their effects to those of new anaesthetics (desflurane). Liver slices (250–300μm thick) were incubated in sealed roller vials, containing Krebs Henseleit buffer at 37°C under 95% O2:5% CO2 atmosphere. Volatile anaesthetics were delivered by volatilisation after pre-incubation for 1 hour to produce a constant concentration in the medium. Production of the metabolites, trifluroacetic acid and fluoride ion, was measured. Intracellular potassium ion content, protein synthesis and secretion were determined as indicators of viability of the slices. The rank order of biotransformation of anaesthetics by the liver slices was halothane >sevoflurane>isoflurane and enflurane>desflurane. The rank order of hepatotoxicity of these anaesthetics was halothane>isoflurane and enflurane>sevoflurane and desflurane. Halothane is the anaesthetic which is metabolised furthest and has the most toxic effect, while desflurane is the least metabolised anaesthetic and has the least toxicity. This in vitro cultured precision-cut liver slice system appears to be suitable for studying the biotransformation of volatile anaesthetics and correlating its role in the resulting toxicity.
The Fish Pigment Cell: An Alternative Model in Biomedical Research
Authors: Karlsson Jan Olof G. , Grundstrom Nils , Elwing Hans , Andersson Rolf G. G.
Published: August 2020

Receptor-mediated pigment aggregation within pigment cells (chromatophores) of an isolated fish scale is an ideal model system for functional receptor studies. The superficial layer of the scale contains both dermal chromatophores and postganglionic sympathetic nerves. By means of stimulation of the nerves, or by addition of appropriate receptor agonists, it is possible to elicit pigment aggregation within the chromatophores. A single fish can contribute hundreds of scales, various pharmacological and biochemical experiments are easily carried out and the physiological response, i.e. pigment aggregation, is readily evaluated by the aid of a light microscope or a simple scale photometer.

A denervation model, based on isolated scales, permits studies of factors involved in the sensitivity change, which typically takes place after experimental or pathological denervation.

By using isolated fish scales it is quite simple to illustrate many biomedically important concepts, like receptor theory and nerve-effector cell communication. This makes the scale a very useful preparation in biomedical education.
Applications of Human Peripheral Blood Lymphocytes in Genotoxicity and Cytotoxicity
Authors: Celotti Lucia , Bianchi Vera
Published: August 2020

A number of features make peripheral blood lymphocytes an excellent system for studying both genotoxicity and cytotoxicity in humans. They are an abundant and readily accessible source of somatic cells, mostly in a non-proliferative state, but able to be stimulated by mitogens to enter the cell cycle. The blastocyte transformation of lymphocytes is a useful model for investigating the mechanisms which regulate cell-cycle progression in mammalian cells. By stimulating lymphocytes in vitro, it is possible to detect the genetic damages they have sustained in vivo, which become manifest as chromosomal aberrations, sister-chromatid exchanges or gene mutations. The metabolic properties of lymphocytes have been extensively studied, especially with reference to their characteristic collection of enzymes involved in nucleotide turnover, which makes them exquisitely sensitive to changes in intracellular levels of DNA precursors. The data collected on the ability of lymphocytes to metabolise xenobiotics show a marked quantitative difference between resting and proliferating lymphocytes, and minor qualitative differences between lymphocytes and other cell types, e.g. hepatocytes. An indirect approach to detect the metabolism of genotoxic xenobiotics by lymphocytes is the analysis of DNA adducts in their chromatin after in vivo or in vitro exposure. Lymphocytes can be employed to identify the (cyto)genetic consequences of in vivo genotoxic exposure and inter-individual variation in sensitivity to genotoxic agents. The analysis of mutations at the hgprt locus in lymphocytes is a promising approach for the study of somatic-cell mutations in humans and of the possible mechanisms of in vivo selection against mutants.

In the field of cytotoxicity, the applications of lymphocytes are, as yet, still few: the main effect measured is the impairment of the proliferative response to mitogens. But lymphocytes can be employed as primary human cells to be treated in vitro with mutagenic or toxic chemicals in standard genotoxicity and cytotoxicity assays, and offer the advantage of avoiding the problems of inter-species extrapolation of results by testing in a human system. Moreover, the (geno)toxic effects detected in lymphocytes after treatments in vitro may give information on the spontaneous or environmentally-determined susceptibility of the individual donors to xenobiotics.
The Role of Cell Culture in Improving Continuous Ambulatory Peritoneal Dialysis
Authors: Jabar Zara , Dawson Mary
Published: August 2020

This paper describes the application of cell culture to the investigation of adverse effects associated with continuous ambulatory peritoneal dialysis. Detailed description is given of effects caused by reactive oxygen species, such as could be generated in infections, on confluent sheets of MDCK cells. The electrical effect was reduction in transmonolayer resistance. Various morphological abnormalities were concurrently observed. The method has applications in other barrier membrane studies.
Comparative Cytotoxicity Rankings of Four Aminoglycoside Antibiotics in the Chang, SIRC and LLC-PK1 Cell Lines
Authors: Linseman Daniel A. , Raczniak Timothy J. , Aaron C. Sidney , Bacon James A.
Published: August 2020

A series of structurally related aminoglycosides — neomycin, gentamicin, amikacin, and streptomycin — were screened for cytotoxicity in three cell lines, Chang (liver), SIRC (corneal epithelial), and LLC-PK1 (kidney). The main objectives of this study were: firstly, to determine whether the proximal tubule origin of the LLC-PK1 cell line conferred increased sensitivity to this class of xenobiotic when compared to cell lines derived from organs other than the kidney; and secondly, to determine whether any of the cell lines would rank the in vitro cytotoxic potential of the compounds in an order consistent with their in vivo toxicities. LDH leakage and cell proliferative effects (CP) were the endpoints used to measure cytotoxicity.

The proximal tubule derivation of the LLC-PK1 cell line did not appear to confer significantly increased sensitivity to any of the aminoglycosides tested using LDH release and cell proliferation as endpoints of cytotoxicity. The relative cytotoxicity rankings were as follows: Chang — gentamicin>neomycin>amikacin>streptomycin (LDH), neomycin∼gentamicin∼streptomycin >amikacin (CP); SIRC — neomycin∼gentamicin∼streptomycin>amikacin (LDH and CP); and LLC-PK1 — gentamicin∼streptomycin>neomycin>amikacin (LDH), and streptomycin >neomycin>gentamicin∼amikacin (CP). The results suggest that the Chang line provides a cytotoxicity ranking consistent with in vivo nephrotoxicity data. The SIRC line ranks amikacin the least cytotoxic, but fails to discriminate between the cytotoxicities of gentamicin, neomycin and streptomycin. The LLC-PK1 cell line ranks the compounds in an order which is inconsistent with in vivo results. The LLC-PK1 cell line appears to be the most sensitive to streptomycin, which is the only agent tested that is not accumulated in the kidney in vivo. The results may reflect basal cytotoxicity, since relatively non-specific endpoints were used. Perhaps the LLC-PK1 cell line would rank the cytotoxic potential of this class of compounds more accurately if parameters which are more renal-specific were measured as endpoints.
Drosophila Melanogaster Embryo Cultures: An In Vitro Teratogen Assay
Authors: Bournias-Vardiabasis Nicole
Published: August 2020

An in vitro teratogen assay has been developed that uses Drosophila embryo cell cultures. The endpoints selected in assessing the teratogenic potential of any agent (physical or chemical) involves detection of interference with normal muscle and/or neuron differentiation, induction of heat stock (stress) proteins, and inhibition of normal neurotransmitter levels. Current studies involve use of reporter gene technology (protein fusions) to identify teratogenicity. Results so far suggest that the Drosophila assay is capable of accurately establishing if a particular agent tested can act as a teratogen by a variety of appropriate endpoints (morphological, biochemical, molecular). Furthermore, this assay can be used not only as a teratogen screen, but also in mechanistic studies of abnormal development, gene involvement in teratogenic resistance, and the possible role of heat shock proteins in preventing birth defects.
Progestins and Antiprogestins: A Review of Their Role in Medicine and Bioassays Used in Their Development
Authors: Bigsby Robert M.
Published: August 2020

The physiological role of ovarian progesterone in reproduction and the medical uses of synthetic progestins and antiprogestins are briefly reviewed. Although a number of progestins are in wide use, the search continues for better analogues that display fewer of the unwanted effects of the present compounds. The one antiprogestin approved for human use to date, RU486, exhibits considerable antiglucocortoid activity

The classical bioassays currently in use for the development of these steroid analogues depend upon in vivo progestin effects. However, progestins induce measurable responses in several cell culture systems that could be used as alternative bioassays. Rabbit uterine stromal cells in primary culture are especially suitable for such use. Addition of progesterone to these cultures stimulates synthesis of a 42 kilodalton (42kD) protein that appears in the medium. The 42kD protein response is specific for a progesterone receptor-mediated event and it can be blocked by known progestins. Using this culture system as a bioassay, one rabbit yields enough cells to measure the effect of 30 test doses in triplicate. Thus, a culture system is described that could substitute for current in vivo bioassays. This culture bioassay would allow large scale screening for potential progestin or antiprogestin activity.
An International Reference Chemical Data Bank would Accelerate the Development, Validation and Regulatory Acceptance of Alternative Toxicology Tests
Authors: Purchase Iain F.H.
Published: August 2020

Toxicologists involved in safety evaluation have a primary responsibility for ensuring that their results are the best possible for predicting human health effects. Currently, the scientific and regulatory consensus is that animal-based studies provide the best possible evidence. Thus, toxicologists are faced with ethical questions about the use of animals for experimental research. In vitro tests which equal or supersede animal studies, in terms of provision of data for human health assessment, are thus a high priority for toxicologists. There is no shortage of ideas for alternative methods and this emphasises the importance of adequate validation.

One critical component of validation studies is the selection, evaluation and provision of appropriate chemicals for assessing the performance of alternative tests. Problems are encountered with poor published toxicology data, the difficulty of ensuring the selection of chemicals to represent a sufficiently wide range of chemical classes, disagreement as to the toxicological classification of chemicals and many other issues in the selection and supply of validation chemicals.

Validation studies will be much easier to carry out and will be better received by those involved in human hazard assessment if this aspect of the studies is well planned and controlled. It is proposed, therefore, that an international reference collection of chemicals is assembled which is carefully selected for validation of specific toxicological endpoints, carefully reviewed and classified as to toxicity and made available to research workers interested in validating alternative methods. Additionally, the usefulness of the database could be enhanced if the results of validation studies were to be recorded in a central repository.
Induction of Cytoplasmic Vacuolation by Two Alkyl-diamine Compounds
Authors: Valtolina Marinella , Forster Roy
Published: August 2020

Tetramethyl-1,4-butanediamine (TMBD) and tetramethyl-1,6-hexanediamine (TMHD) have been used as model compounds to study cytoplasmic vacuolation. These chemicals rapidly induced clear intracellular vacuoles in an intestinal cell line (I-407 human embryo-derived cells), in primary human smooth muscle cells and in Chinese hamster V79 cells. As has previously been reported, the induction of vacuolation was pH-dependent and dose-related. Vacuole formation was also found to be slowly reversible, and the degree of vacuole formation appeared to be determined not simply by the concentration of test material, but also by the amount of test material available per cell. V79 cells were markedly more sensitive to vacuole induction than the intestinal cells. Image analysis permitted the quantitation of vacuole induction. The vacuolation response is believed to result from lysosomal entrapment of the chemicals.
Computerised Image Analysis of Nuclear Enlargement Assay in HeLa Cells
Authors: Rizzi Renato , Re Francesco , Chiesara Enzo
Published: August 2020

It has been observed that cells often respond to carcinogens by nuclear enlargement. For this reason, new morphometric approaches have been developed to evaluate cell modifications in pre-carcinogenesis assays. Morphometric computerised automatic analysis, with original software, was performed on HeLa cells treated with various compounds (hydroxyurea, dimethylnitrosamine, N-methyl- N’-nitro-nitrosoguanidine and cyclophosphamide) to evaluate nuclear size changes.
Bioactivation and Toxicity of Two Anthraquinones in V79 Chinese Hamster Cells
Authors: Mian Maurizio , Fratta Duccio , Rainaldi Giuseppe , Benetti Dino , Gervasi Pier Giovanni
Published: August 2020

The role of the flavoprotein NADPH cytochrome P450 reductase, NADH-dehydrogenase and DT-diaphorase in the biotransformation of the anthraquinones rhein and danthron was investigated using purified enzymes, subcellular fractions and cell cultures. The results demonstrated that rhein was more able than danthron to produce O2 by P450 reductase, NADH-dehydrogenase and V79 homogenate and microsomes, and this may explain its higher toxicity to V79 cells. In addition, unlike other quinones, DT-diaphorase appeared not to play a detoxication role in rhein and danthron biotransformation. On the contrary, by inhibiting DT-diaphorase with dicoumarol, the rhein- and danthron-derived cytotoxicity in V79 cells was reduced. This finding suggests that, for these quinones, other coumarol-sensitive biotransformation routes are operative in V79 cells.
Exogenous Histidine as a Co-factor which Exacerbates the Toxicity of Hydrogen Peroxide in Cultured Mammalian and Bacterial Cells
Authors: Brandi Giorgio , Sestili Piero , Guidarelli Andrea , Schiavano Giuditta Fiorella , Albano Amedeo , Cattabeni Flaminio , Cantoni Orazio
Published: August 2020

The killing of Escherichia coli cells by H2O2 is higher when exposure to the oxidant is performed in a complete culture medium, as compared to saline. Whereas MgSO4, CaCl2, thiamine or glucose, added separately or in combination with the saline, had no effect on the cytotoxic response to H2O2, the cytotoxicity appeared highly dependent upon the presence of the casamino acids in the incubation medium. One of these amino acids, histidine, was found to greatly augment the toxicity of H2O2 in E. coli. This effect of histidine was also observed in mammalian cells. In fact, both the cytoxicity and the DNA damage produced by H2O2 in Chinese hamster ovary (CHO) cells were significantly increased by this amino acid.
In Vitro Models for Biochemical Investigations on Programmed Cell Death
Authors: Perotti Mariarosa , Taddei Filippo , Mirabelli Francesca , Bellomo Giorgio
Published: August 2020

Isolated rat thymocytes treated with methylprednisolone (MPS), cultured human synovial (McCoy's) cells exposed to cold shock and human mammary adenocarcinoma (BT-20) cells treated with Tumour Necrosis Factor (TNF), all showed DNA fragmentation and the morphological and biochemical features of cell death characteristic of a process known as apoptosis.

DNA fragmentation in MPS-treated thymocytes and in cold shock-exposed McCoy's cells was preceded by a marked increase in cytosolic free Ca2+ concentration resulting either from a sustained influx of Ca2+ from the extracellular medium or from the release of Ca2+ from intracellular pools. On the other hand, in TNF-treated BT-20 cells, DNA fragmentation was not associated with any early, marked increase in the cytosolic free Ca2+ level. In all three models, however, DNA fragmentation was efficiently prevented by intracellular Ca2+ chelators, calmodulin inhibitors and activators of protein kinase C (PKC). These results suggest that the activation of common mechanisms involving Ca2+ and PKC may be essential for the development of apoptosis.
A Preliminary Investigation into the Opacifying Effects In Vitro of Compounds with Varying Numbers of Nitrogen-containing Groups using Isolated Porcine Corneas
Authors: Igarashi Haruyoshi , Katsuta Yasunaga , Nakazato Yoshiharu , Kawasaki Tohru
Published: August 2020

Isolated intact porcine corneas were incubated for 4 hours with solutions of various compounds, bathing either the epithelial or endothelial surface or both surfaces, and the resulting opacities were measured. All compounds were tested at 5 x 10-4 M, 5 x 10-3 M, 5 x 10 2M and 1 x 10 1M. Solutions of pilocarpine HC1, carteolol HC1 and timolol maleate (which contain 2 or 4 nitrogens) caused a greater opacity when applied to the endothelial surface than when applied to both the endothelial and epithelial surfaces. By contrast, solutions of sodium lauryl sulphate, befunolol HC1 and dibucaine HC1 (which contain 0, 1 or 3 nitrogens) caused a greater opacity when applied to both surfaces than when applied to the endothelial surface alone. In both groups, there was a good correlation between the greatest opacities and the osmolalities of the various solutions.

The difference in responses to the two groups of compounds may be related to the number of nitrogen atoms in the molecules. It is suggested that there is something in the porcine corneal epithelium which inhibits the uptake of compounds containing 2 or 4 nitrogens. Alternatively, in the presence of these compounds, the epithelium may be able to actively pump out water that has entered by diffusion.

We wish to thank Mrs A.M. Northover for her linguistic assistance, and the local slaughterhouse at Koriyama-shi for the supply of fresh porcine eyes.
Improved Housing of Mice, Rats and Guinea-pigs: A Contribution to the Refinement of Animal Experiments
Authors: Scharmann Wolfgang
Published: August 2020

The keeping of experimental animals requires housing systems appropriate to the needs and behaviour of each species, as demanded by various supranational and national guidelines. It is questionable whether conventional housing systems for rodents such as mice, rats and guinea-pigs meet this demand. It is suggested that the housing of laboratory rodents should be improved by the use of larger and more appropriate cage types, as well as by reducing the monotony of conventional housing systems.
Can the In Vivo Maximum Tolerated Dose be Predicted Using In Vitro Techniques? A Working Hypothesis
Authors: Shrivastava Ravi , John Gareth W. , Rispat Ginette , Chevalier Annick , Massingham Roy
Published: August 2020

All new chemical entities synthesised in our laboratories have routinely been subjected to in vitro toxicity tests. Out of curiosity, we established a working hypothesis in which the in vitro data could be empirically transformed to predict the in vivo four-week standard maximum tolerated dose (MTD) studies in rats and dogs.

As a first step to verifying this hypothesis, we report here the findings of an in vitro cytotoxicity study of 25 compounds randomly selected from our files, possessing a wide range of pharmacological activities and for which data from standard four-week MTD studies were available. Single blind in vitro toxicity studies in three carefully selected types of primary and cell line cultures were carried out. In vitro CT50 (concentration inducing 50% cell death) and CT100 (concentration inducing 100% cell death) values were obtained for each of the three cell types and, using empirical assumptions, these results were used to predict the MTD in vivo in the rat and dog. The actual in vivo threshold and toxic doses were obtained from the MTD study reports. The in vivo toxicity values predicted from the in vitro toxicity results with this series of 25 compounds showed a better than 80% correlation with the actual in vivo results obtained in the MTD studies.

Whether or not in vitro cytotoxicity predictions are ultimately found to be directly and consistently related to the MTD in vivo for all pharmacological classes of compounds will require many additional studies, but it is hoped that these results will stimulate the necessary research effort required to answer this question.
Toxicity of Sodium Dodecyl Sulphate and Other Detergents in Cultures of Human Oral Mucosa Epithelium
Authors: Arenholt-Bindslev Dorthe , Bleeg Henry S. , Richards Alan
Published: August 2020

The aim of the present study was to investigate the relative cytotoxic effects of the detergents sodium dodecyl sulphate, Triton X-100, sodium oleate, stearyl etoxylate, sodium cholate and sodium taurocholate. Sodium dodecyl sulphate and stearyl etoxylate are used as dentifrice ingredients. Cultures of human oral epithelial cells were exposed to dilutions of the detergents for five minutes. Acute cytomorphological changes and membrane damage (leakage of lactate dehydrogenase, acid phosphatase and UV-absorbing material [280nm]) were monitored. Cytomorphological changes and cell proliferation were subsequently observed during the following week of recovery. Sodium dodecyl sulphate and Triton X-100 caused the most pronounced cytotoxic reactions, and it is concluded that more attention should be focused on the use of non-toxic detergents in oral hygiene products.
Cell Culture Methods to Evaluate the Biocompatibility of Implant Materials
Authors: Ciapetti Gabriela , Cenni Elisabetta , Cavedagna Daniela , Pratelli Loredana , Pizzoferrato Arturo
Published: August 2020

Cell culture techniques are usually used in the field of biomaterials research and development in order to detect toxic components. Morphological assays are the most widely used methods and give the very first information about the biological compatibility of materials. Cell function assays give more quantitative data, but the comparison of data between different laboratories is difficult. Some of the cell culture methods that are used for biocompatibility studies are described briefly here, and results from our laboratory are reported.

Despite some inherent limitations of the cell culture techniques, they are an accurate and reliable method of predicting the biological compatibility of materials to be implanted in vivo.
Inhibition of Growth, Morphological and Morphometrical Changes Induced by Amido-anthraquinone Derivatives in NCTC 2544 and HeLa Cells
Authors: Conconi Maria Teresa , Parnigotto Pier Paolo , Bonali Antonella , Zagotto Giuseppe
Published: August 2020

In this work, we have studied cytotoxicity induced in NCTC 2544 and HeLa cell lines by two newly-synthesised potential anticancer drugs, the amido-anthraquinone derivatives 8-diethylaminopropionamido-1,4,5-trihydroxy-9,10-anthracenedione bromide (A888) and 5,8-bisdiethylaminopropionamido-1,4-dihydroxy-9,10-anthracenedione dibromide (A890). The results have been compared to the results obtained with mitoxantrone, a well-known anticancer agent. Using the neutral red uptake assay, A888 showed less cytotoxic action compared to mitoxantrone and A890. According to the results of morphological analysis after exposure for five hours, anthraquinone derivatives induced the formation of cytoplasmic vacuoles and changes in the arrangement of nucleus in both cell lines. On the basis of morphometrical analysis, we have observed an increase in total cellular area with a decrease in the nuclear/cytoplasmic ratio. We have found that the increase in total cellular area was higher in NCTC 2544 cells than in HeLa cells. From these data, we can conclude that NCTC cells are more sensitive to the compounds tested than HeLa cells. However, the growth inhibition assay and morphological analysis did not confirm these results.
Photoprotection Exerted by Sunscreens in a Human Cell Line after UV Damage
Authors: Viviani Barbara , Marinovich Marina , Galli Corrado L.
Published: August 2020

In recent years, attention has been devoted to the biological effects of solar radiation. Exaggerated exposure to sunlight has underlined the importance of evaluating the ability of different products to protect skin from all the adverse effects of sunlight. In preliminary experiments, we used human keratinocytes in vitro to study the possible influence of sun irradiation on different biological events, in order to identify specific markers of photodamage. Unscheduled DNA synthesis was clearly observed in all the samples exposed to irradiation, in a dose-dependent manner. The best response was obtained when the UVA/UVB irradiation dose reached 44/7.2mJ/cm2 respectively. Under these conditions, the ability of the different sunscreens, mainly benzophenones, to protect from UV damage was assessed. The results seem to confirm the ability of such an in vitro model to evaluate the photoprotective action of the tested sunscreens.
Application of the EYTEX™ System to the Evaluation of Cosmetic Products and their Ingredients
Authors: Kruszewski Francis H. , Hearn Laura H. , Smith Kyle T. , Teal Janice J. , Gordon Virginia C. , Dickens Michael S.
Published: August 2020

465 cosmetic product formulations and raw ingredients were evaluated with the EYTEX™ system to determine the potential of this in vitro alternative for identifying eye irritation potential. The EYTEX™ system is a non-animal, biochemical procedure developed by Ropak Laboratories, Irvine, CA, that was designed to approximate the Draize rabbit eye irritation assay for the evaluation of ocular irritation.

Avon Products Inc. provided all the test samples, which included over 30 different product types and represented a wide range of eye irritancy. All the EYTEX™ protocols available at the time of this study were used. Samples were evaluated double-blind with both the membrane partition assay (MPA) and the rapid membrane assay (RMA). When appropriate, the standard assay (STD) and the alkaline membrane assay (AMA) were used, as well as specific, documented protocol modifications.

EYTEX™ results were correlated with rabbit eye irritation data which was obtained from the historical records of Avon Products Inc. A positive agreement of EYTEX™ results with the in vivo assay was demonstrated by an overall concordance of 80%. The assay error was 20%, of which 18% was due to an overestimation of sample irritancy (false positives) and 2% was attributed to underestimation (false negatives). Overestimation error in this study was due in part to the inability of the protocols to accurately classify test samples with very low irritation potential. Underestimation of sample irritancy was generally associated with ethoxylated materials and high concentrations of specific types of surfactants.

100% sensitivity and 85% predictability were described by the data, indicating the efficiency of EYTEX™ in identifying known irritants. A specificity rate of 39% showed the EYTEX™ assay to be weak in discerning non-irritants. However, the EYTEX™ protocols used in this study were not designed to identify non-irritants. A compatibility rate of 99% proved the effectiveness of the EYTEX™ assay in accommodating a diversity of product types. The EYTEX™ system protocols, when used appropriately, can provide a conservative means of assessing the irritant potential of most cosmetic formulations and their ingredients.
Heavy Metal Ions, Cytotoxicity and Gap Junctional Intercellular Communication in Syrian Hamster Embryo Cells
Authors: Mikalsen Svein-Ole , Rivedal Edgar , Sanner Tore
Published: August 2020

The effects of NiSO4, K2CrO4, PbAc2, CrCl3 and CdAc2 on gap junctional intercellular communication (GJIC) were studied. Two exposure procedures were used — A: an established monolayer of cells was exposed to the compound, and B: the compound was mixed with the cells before seeding. The apparent inhibition of GJIC by CrCl3 and CdAc2 could be accounted for by cytotoxicity, i.e. disruption of physical contacts between cells. PbAc2 had no effect on GJIC. The effects of NiSO4 and K2CrO4 depended on the exposure procedure and cell type studied. Specifically, K2CrO4 induced a significant increase in GJIC in BPNi cells by procedure A, and a decrease by procedure B. The cytotoxic effects of NiSO4 were examined in more detail. The toxic concentrations varied widely between the methods used. The results indicate that the effect of compounds on GJIC may vary with different exposure procedures, and that inhibition of GJIC is not due to general cytotoxic effects.
Evaluation of the Relevance of Selected In Vitro Toxicity Test Systems for Acute Systemic Toxicity
Authors: Seibert Hasso , Gulden Michael , Kolossa Marike , Schepers Gabi
Published: August 2020

Progress in the implementation of in vitro methods in testing procedures for non-genetic toxicity is limited by poor or missing validation of the various approaches. Many efforts have focused on determinations of general cytotoxicity in vitro using proliferating, non-differentiated cell lines. The aim of our present studies is to evaluate the relevance to acute systemic toxicity of three in vitro systems using differentiated mammalian cells (bovine spermatozoa, primary cultured rat hepatocytes, primary cultured rat skeletal muscle cells) and also one in vitro system which uses a continuous cell line (BALB/C 3T3 cells). This combination of in vitro systems covers various aspects of cellular toxicity and permits the determination of the intrinsic activity of chemicals with respect to general cytotoxicity, selective cytotoxicity and interference with selected cell-specific functions. In order to contribute to the establishment of a basis for the selection of methods to be included in test batteries in the future, we are contributing to the MEIC programme. In the present communication, results for the first ten chemicals of the MEIC list are presented and discussed.
Toxicity of Glycols and Allyl Alcohol Evaluated by Means of Co-cultures of Microcarrier-attached Rat Hepatocytes and BALB/C 3T3 Mouse Fibroblasts
Authors: Voss Jens-Uwe , Seibert Hasso
Published: August 2020

The toxicity of allyl alcohol and several glycols (ethylene glycol, 1,2-propanediol, 1,3-propanediol, methoxyethanol, and the glycol ether dioxane) was studied in cultures of 3T3 cells and in co-cultures of 3T3 cells with microcarrier-attached hepatocytes. Metabolism-mediated effects on the cytotoxicity to 3T3 cells were recorded by differences in the growth of the cultures exposed in the presence or absence of hepatocytes. Hepatocyte viability was determined by depletion of intracellular lactate dehydrogenase and effects on the biotransformation ability of hepatocytes were assessed by determination of O-deethylation of 7-ethoxycoumarin (EOD activity). Allyl alcohol was the only substance more toxic to the hepatocytes than to 3T3 cells cultured in the absence of hepatocytes. Toxicity to 3T3 cells of allyl alcohol, ethylene glycol, and 1,3-propanediol, but not of 1,2-propanediol, methoxyethanol and dioxane, was markedly enhanced when the cells were co-cultured with hepatocytes. The results indicate that the toxicity of allyl alcohol, ethylene glycol, and 1,3-propanediol, to 3T3 cells depends on the formation of active metabolites. For ethylene glycol and 1,3-propanediol, growth of 3T3 cells in co-cultures was reduced at concentrations without effects on hepatocyte viability. Co-culture of 3T3 cells with microcarrier-attached rat hepatocytes represents a suitable approach for the in vitro evaluation of metabolism-mediated cytotoxicity.
The Use of Microglia Activation in the Evaluation of Neurotoxicity
Authors: Grundt Inger K. , Nyland Harald
Published: August 2020

The toxicities of the therapeutic drugs amitriptyline and diazepam, the metal compounds methyl mercury and triethyllead, and the essential fatty acids linoleic acid and α-linolenic acid, were determined in a system of rat glial cells in culture with microglia activation as the parameter for evaluation of toxicity. The markers of activated microglia — increased expression of complement receptor 3 and transformation of microglia to phagocytes — were evaluated by immunocytochemical staining with the monoclonal antibody OX-42 and after incubation with fluorescent latex beads. Concentrations which activated microglial cells were compared to EC50 values from acute toxicity tests, and to concentrations which in previous studies activated oligodendroglial cells. The results show that the drugs amitriptyline and diazepam, and fatty acids linoleic acid and α-linolenic acid, activated microglia at 10–25 times lower levels when compared to the EC50 values. Microglia activation was induced by α-linolenic acid and amitriptyline at about 10 times lower levels when compared to linoleic acid and diazepam. The microglia activating concentrations of the metal compounds were, 1,000 times lower, when compared to acute toxicity values. The activation of microglia was initiated by the same concentrations of the compounds that in previous studies were found to activate oligodendroglial cells.
Cytotoxicity of Continuous Ambulatory Peritoneal Dialysis (CAPD) Solutions. A Biocompatibility Study Involving Human Polymorphonuclear Granulocytes Rather than Laboratory Animals
Authors: Schambye Hans T. , Pedersen Fritz B. , Wang Palle
Published: August 2020

Continuous amulatory peritoneal dialysis (CAPD) is a well-established treatment for renal failure. Peritonitis is the most important and serious complication to CAPD. The predisposition of patients to contract peritonitis has been attributed to functional impairment of immunoactive cells in the peritoneal cavity due to cytotoxicity of the dialysis solutions used.

A model for biocompatibility studies of CAPD solutions is presented, in which the migration and phagocytotic capacity of normal isolated human polymorphonuclear granulocytes (PMN) were examined after exposure to the test solutions. The methods proved to be highly reproducible.

The commercially-available lactate-based CAPD solutions tested reduced PMN function, mostly due to their low pH. Cell function was improved by elevation of pH, but not to the control level, when the cells were exposed to RPMI — a standard cell culture medium. A new bicarbonate-based CAPD solution proved to be less cytotoxic than the lactate-based ones, and is at present undergoing clinical evaluation.
Estimation of Toxicity of Chemicals from the MEIC List by Colony Radial Growth Rate Measurements in the Fungus Geotrichum Candidum
Authors: Jacobsen Tomas
Published: August 2020

The cytotoxicities of the first ten MEIC chemicals were assayed by estimation of IC50 values on cultures of the fungus Geotrichum candidum. Colony radial growth rates were measured between 24 and 48 hours after inoculation on solid media containing different concentrations of the chemicals. The IC50 values obtained in this study were higher than the human lethal blood concentrations obtained for the chemicals, but good correlation (r = 0.958) was found between human lethal blood concentrations and IC50 values obtained from growth rate measurements of G. candidum. Ferrous sulphate, methanol, ethanol and diazepam gave linear relationships between decrease in growth rates and concentration of the chemical. With ethylene glycol, the growth rate decreased linearly with the logarithm of ethylene glycol concentration.
The Neurotoxicity of Organic Solvents, Studied with in Vitro Models
Authors: Tähti Hanna
Published: August 2020

The present paper deals with the general anaesthetic effect of organic solvents. The emphasis is on cell membranes as models for predicting the adverse reactions of the human nervous system to organic solvents, and for studying the mechanisms of their anaesthetic action. Human and rat erythrocyte and platelet membranes, rat synaptosomal membranes and cultured neural cell membranes were exposed in vitro to various concentrations (3–30mM) of benzene, toluene, o-xylene and styrene in the incubation mixtures. After exposure to a solvent for 30 minutes, the membrane integral protein integrity was determined by measuring ATPase and acetylcholinesterase activities. All measurements were performed in triplicate from 3 or 4 independent experiments with each exposure of solvent and each membrane isolation. The enzyme inhibition was dose-dependent, and differed after solvent treatment in all membrane preparations tested in increasing order, as follows: benzene, toluene, styrene, o-xylene. The results support the protein theories on the mechanism of the anaesthetic action of various industrial organic solvents. The integral membrane proteins contain hydrophobic pockets which may bind anaesthetics, e.g. solvents which change the properties of integral enzymes. The effects of solvents in peripheral models are quite similar to those in neural membranes in vitro. Therefore, these membrane models can serve as test models when the narcotic potencies of the solvents are being evaluated, or at least for screening purposes and for studies of the mechanisms of solvent neurotoxicity.
Time-lapse Phase-contrast Microphotography of Cell Populations as a Basis for Improvement of In Vitro Toxicity Assessment
Authors: Červinka Miroslav
Published: August 2020

Recent trends in the field of in vitro toxicology have centred around the validation of in vitro methods. The ultimate goal is to obtain pertinent data with the minimum of effort. In our laboratory, we have used toxicological methods based on the evaluation of cell morphology and cell proliferation. A method suitable for this purpose is time-lapse microcinematographic (or video) recording of cellular changes, which we used for many years. For practical in vitro toxicity testing, however, this method is far too complicated. Therefore, we have tried to develop a simple modification for the evaluation of cell morphology and cell proliferation, which would still allow for a basic time-dependent analysis. Comparison of detailed microcinematographic analysis with analysis according to our new proliferation assay is demonstrated with cisplatin as the toxicant. We believe that a time-dependent approach could improve the in vitro assessment of toxicity.
A Simple Method for Cytotoxicity Studies of Non-hydrosoluble Substances. Possible Application as an Alternative to the Draize Test for Cosmetics and Toiletries
Authors: Boue-Grabot M. , Halaviat B. , Pinon J. François
Published: August 2020

The use of a microporous membrane as an interface between a cell culture and a substance dissolved into a non-aqueous, non-cytotoxic medium made it possible to study the cytotoxicity of non-hydrosoluble substances, without addition of any adjuvant for dispersion or solubilisation. The LC50s of raw materials widely used in cosmetic and pharmaceutical products were established on skin human fibroblasts.

This method was used for the study of cosmetics and toiletries which could be tested directly in their actual galenic forms. A relationship between exposure time and cytotoxicity was established for 50 products and a cytotoxicity index was calculated. The results were compared with those of the Draize irritation test on the rabbit eye. This comparison showed a very good correlation between the calculated cytotoxicity index and the ocular irritation index (p < 0.001).

From the results of this preliminary study, it appears that the method could provide an alternative to animal studies on the ocular tolerance of cosmetics and toiletries.
Inhibition of Protein Synthesis after Exposure of Caco2 Cells to Heavy Metals
Authors: Scarino Maria Laura , Poverini Roberta , Di Lullo Giuseppe , Bises Giovanna
Published: August 2020

The toxicity of two essential trace elements, the heavy metals zinc and copper, has been studied in Caco2 cells, a human colon carcinoma cell line able to differentiate in culture into mature enterocytes.

The ID50 for zinc and copper in Caco2 cells was determined from dose-effect curves. The ID50 is the same (500μM) for zinc and copper in undifferentiated cells, but in differentiated cells the ID50 for zinc (1mM) is higher than the ID50 for copper (600μM). Total protein synthesis is more severely affected by both metals in undifferentiated Caco2 cells than in differentiated cells. This difference could be explained by the different intracellular accumulation of the two metals at the two stages of differentiation. Copper inhibits total protein synthesis more than zinc, at both stages of differentiation. A protective role of zinc-induced metallothionein is discussed in relation to these results.
The Haemocompatibility of Biomaterials In Vitro: Investigations on the Mechanism of the Whole-blood Clot Formation Test
Authors: Groth Thomas , Derdau Katrin , Strietzel Frank , Foerster Frank , Wolf Hartmut
Published: August 2020

Twenty years ago Imai & Nose introduced a whole-blood clotting test for the estimation of haemocompatibility of biomaterials in vitro In our paper a modification of this assay is described and the mechanism of clot formation further elucidated. It was found that neither the inhibition of platelet function nor the removal of platelets from blood significantly changed the clot formation rate on glass and polyvinyl chloride in comparison to the rate tor whole blood. Scanning electron microscopy demonstrated that platelets were not involved in clot formation near the blood/biomaterial interface. Thus, it was concluded that the system of contact activation of the coagulation cascade dominates during clot formation under static conditions. The latter conclusion was supported by the fact that preadsorption of human serum albumin or human fibrinogen onto the glass plates used, decreased the clot formation rate in the same manner.
Cyst-Based Toxicity Tests. IV. The Potential of Ecotoxicological Tests for the Prediction of Acute Toxicity in Man as Evaluated on the First Ten Chemicals of the MEIC Programme
Authors: Calleja Mabel C. , Persoone Guido
Published: August 2020

In the framework of the multicentre evaluation of in vitro cytotoxicity (MEIC) programme, the first ten chemicals of the prescribed list were tested for acute toxicity in four standardised cyst-based aquatic invertebrate tests, consisting of two rotifer species (the estuarine Brachionus plicatilis and the freshwater Brachionus calyciflorus) and two crustacean species (the halophilic anostracan Artemia salina and the freshwater anostracan Streptocephalus proboscideus). Mortality was the test criterion and toxic effects, expressed as 24-hour LC50 values, were correlated with rodent and human acute oral toxicity data. Generally, a good correlation was obtained between any of the invertebrate tests and the rodent data. Likewise, the predictive screening potential of the aquatic invertebrate tests for acute oral toxicity in man was slightly better than the rat test for eight (excluding diazepam and digoxin) and nine (including diazepam, excluding digoxin) of the ten substances. The aquatic test systems, however, appear to be more suitable for compounds soluble in water.
ECITTS: An Integrated Approach to the Application of In Vitro Test Systems to the Hazard Assessment of Chemicals,
Authors: Walum Erik , Balls Michael , Bianchi Vera , Blaauboer Bas , Bolcsfoldi George , Guillouzo Andre , Moore Gregory A. , Odland Lena , Reinhardt Christoph , Spielmann Horst
Published: August 2020

As a result of a workshop held at Täljöviken, Åkersberga, Sweden, on 27–29 May 1991, a multicentre collaborative research project was established, with the purpose of developing the concept of integrated in vitro toxicity testing. The first priority was the selection of tests within eight appropriate areas: basal cytotoxicity, irritancy, developmental toxicity, hepatotoxicity, nephrotoxicity, immunotoxicity, neurotoxicity and biokinetics. An ideal battery of tests for each area was identified. Since it was realised that it would not be feasible to include the full ideal list of tests in the project, a minimum test list was also agreed. For each area, ten calibration chemicals were selected. From these 80 compounds, 30 were selected for inclusion, together with 20 of the OECD test programme chemicals, in a first test set of chemicals. The toxicity of these 50 test set chemicals will be investigated in the minimum integrated test scheme.

The aim of the project is to employ non-animal methods to assess the toxicological properties of chemicals, and to improve this assessment through the use of knowledge about mechanisms of toxic action. The information produced will contribute to the establishment of a more-scientific and more-efficient toxicological procedure for hazard assessment. Questions concerning which parameters need to be investigated and combined to make hazard assessments, and which parameters relevant to in vivo toxicity can be determined in non-whole animal test systems, will also be addressed.