bluuebunny/crossref_metadata_2025 huggingface dataset:
Testing Wound-healing Activity in T15 Fibroblast Cultures: A Morphometric Analysis
Authors: Korkmaz Seval , Zeytinoglu Hülya , Zeytinoglu Melih , Aydin Süleyman , Öztürk Yusuf , Baser Hüsnü Can
Published: April 2019

The purpose was to evaluate the use of mouse T15 fibroblast cell cultures for the investigation of wound-healing activity. In order to investigate their mechanisms of action, the effects of drugs with wound-healing activities were compared by using morphometric analyses by microscopy after cell staining. A number of parameters were used to evaluate the effects of titrated extracts from Centella asiatica and dexpanthenol (drugs that have been used in medical practice for their wound-healing activities) on cultured mouse T15 fibroblasts. These parameters were: the total number of cells; the number of T15 cells in mitosis; the percentages of fusiform, polygonal, round and vacuole-containing cells; and the number of intracellular collagen granules. The results indicate that these two drugs exhibit wound-healing activities by activating fibroblast cells, and have cytoprotective effects, although their mechanisms of action on mouse T15 fibroblasts were different. On the basis of our findings, mouse T15 fibroblast cell cultures seem to be useful for the pharmacological screening of compounds with wound-healing activity.
Local Irritation/Corrosion Testing Strategies: Development of a Decision Support System for the Introduction of Alternative Methods
Authors: Zinke Stephan , Gerner Ingrid , Graetschel Gabriele , Schlede Eva
Published: April 2019

The notification procedure for new chemicals of the European Union (EU) requires protocols on physicochemical and toxicological tests for the evaluation of physico-chemical properties and probable toxic effects of each notified substance. In order to reduce the amount of animal testing, alternative methods should be introduced into toxicity testing. Therefore, we have developed a rule-based decision support system (DSS) for the prediction of the local corrosive/irritant properties of new chemicals. To this end, data on more than 1000 substances were examined, which resulted in approximtely 180 “exception-rules” of the kind IF (physicochemical property) A THEN not (toxic) Effect B. In addition, the structural formulae of the chemicals were analysed, which resulted in approximately 160 “structure-rules” of the kind IF Substructure A THEN Effect B. The DSS can predict (based on theoretical structure-activity relationships) whether a chemical produces: a) corrosive effects (i.e. no testing is necessary; b) might have corrosive effects (i.e. no animal testing, in vitro tests are suitable); and c) will produce no effects or only marginal effects (i.e. animal tests are necessary based on current EU legislation for hazard assessment purposes). In addition, the DSS provides reliable data for legal classification and labelling based on a specific result.
Alternatives to Animal Testing in the Safety Evaluation of Products
Authors: Knight Derek J. , Breheny Damien
Published: April 2019

The conventional method for assessing the safety of products, ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals — including cosmetics — involves determining their toxicological properties by using experimental animals. The aim is to identify any possible adverse effects in humans by using these animal models. Providing safe products is undoubtedly of the utmost importance but, over the last decade or so, this aim has come into conflict with strong public opinion, especially in Europe, against animal testing. Industry, academia and the regulators have worked in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals required and the severity of the tests in which they are used. There is a long way to go before products can be evaluated without any animal studies, and it may be that this laudable aim is an impossible dream. Nevertheless, considerable progress has been made by using a combination of in vitro tests and the prediction of properties based on chemical structure. The aim of this review is to describe these important and worthwhile developments in various areas of toxicological testing, with a focus on the European regulatory framework for general industrial and household chemicals.
A Comparative Study of the Toxicity of Mercury Dichloride and Methylmercury, Assayed by the Frog Embryo Teratogenesis Assay-Xenopus (FETAX)
Authors: Prati Mariangela , Gornati Rosalba , Boracchi Patrizia , Biganzoli Elia , Fortaner Salvador , Pietra Romano , Sabbioni Enrico , Bernardini Giovanni
Published: April 2019

The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a powerful and flexible bioassay that makes use of the embryos of the anuran amphibian Xenopus laevis. The FETAX can detect xenobiotics that affect embryonic development, when mortality, teratogenicity and growth inhibition are used as endpoints. The FETAX was used to compare the embryotoxic and teratogenic potentials of two chemical species of mercury: inorganic mercury(II) chloride (HgCl2) and organic methylmercury chloride (MeHgCl). MeHgCl, with an estimated median lethal concentration [LC50] of 0.313μM and a median teratogenic concentration [TC50] of 0.236μM, showed a higher toxicity than HgCl2, with estimated LC50 and TC50 values of 0.601μM and 0.513μM, respectively. On the basis of these results, HgCl2 and MeHgCl can be classified as “slightly teratogenic compounds”, as the ratio of LC50/TC50 is less than 1.5. There was a significant deviation from the commonly described monotonic behaviour of the concentration–response curves, suggesting a hormetic effect of both species of mercury. Uptake experiments, followed by neutron activation analysis, showed a higher incorporation of mercury in embryos exposed to MeHgCl compared with those exposed to HgCl2. Interestingly, Hg-exposed embryos showed a higher content of selenium and zinc than did control embryos.
Cystathionine Pathway-dependent Cytotoxicities of Diethyl Maleate and Diamide in Rat and Human Hepatoma-derived Cell Cultures
Authors: Dierickx Paul J. , De Beer Jacques O. , Scheers Ellen M.
Published: April 2019

Glutathione (GSH) plays a role in many toxicologically important metabolic processes. It was previously established that L-buthionine S,R-sulphoximine (BSO), a specific inhibitor of γ-glutamylcysteine synthetase, reduces the GSH content more efficiently in rat (Fa32) than in human (Hep G2) hepatoma-derived cells. We therefore investigated whether the cystathionase inhibitor propargylglycine (PPG) could further decrease the BSO-induced GSH depletion in Hep G2 cells. The influence of the cystathionine precursors N-acetylmethionine, methionine and homocysteine on the cytotoxicity of diethyl maleate (DEM) and diamide [1,1′-azobis( N,N-dimethylformamide)] was also investigated. PPG reduced the GSH content in both cell lines. A further GSH decrease in Hep G2 was obtained when using a BSO + PPG combination containing relatively high concentrations of PPG. BSO diminished the toxicity of PPG. Homocysteine was the most efficacious of the tested cystathionine precursors in increasing the GSH content and reducing the cytotoxicity of DEM and diamide in Fa32 and Hep G2 cells.
ECVAM's Contributions to the Implementation of the Three Rs in the Production and Quality Control of Biologicals
Authors: Halder Marlies , Hendriksen Coenraad , Cussler Klaus , Balls Michael
Published: April 2019

A summary is presented of the activities initiated, and the progress achieved, between April 1993 and December 2001 in implementing the Three Rs in one of the main priority areas of the European Centre for the Validation of Alternative Methods (ECVAM) — the production and quality control of biologicals. These have included organising eight key workshops, and financial contributions to, and sponsorship of, relevant international workshops, symposia and conferences. Noteworthy activities include financial support and/or participation in a number of prevalidation and validation studies. These involved alternative methods for the batch potency testing of: human tetanus vaccines; human and veterinary tetanus antisera and immunoglobulin; rabies vaccines; Leptospira hardjo vaccines; Clostridium perfringens vaccines; and erysipelas vaccines. They also involved a cell culture test for specific toxicity testing of diphtheria toxoid vaccines. In addition, ECVAM funded a study on the use of humane endpoints for vaccine quality control tests involving severe suffering, such as the potency testing of erysipelas, rabies and pertussis vaccines. ECVAM has also contributed financially to the compilation of manuals and expert reports, and to training in test methods. Following the report of an ECVAM Task Force, ECVAM financially supported the prevalidation of some in vitro methods for the potency testing of a recombinant hormone. A proposal is presented for promotion of regulatory acceptance, and suggestions are made for possible future activities.
Organotypic Brain-slice Cultures from Adult Rats: Approaches for a Prolonged Culture Time
Authors: Wilhelmi Eckbert , Schöder Ulrich H. , Benabdallah Akilah , Sieg Frank , Breder Jörg , Reymann Klaus G.
Published: April 2019

Animal experiments are widely used in neurobiological and neuropharmacological research. Today, juvenile brain organotypic slice cultures have partially replaced in vivo experiments, but there is no adequate in vitro counterpart for the adult brain. The present study was aimed at the long-term culture of physiologically intact hippocampal slices from adult rats, by improving the conditions for preparation and culture, and the development of a new culture medium. A cerebrospinal fluid (CSF)-like medium was used, which was modified with a variety of supplements, including energy precursors, free-radical scavengers, and compounds known to inhibit neurotoxicity. The population spike amplitude (PSA) was used as a measure of viability, and amplitudes larger than 1mV indicated viable cultures. The addition of MK-801 during slice preparation improved PSA values during the first two days in vitro (DIV). Ascorbic acid and insulin prolonged the culture time up to DIV 4. FK-506 and vitamin E, alone or in combination, supported slice culture up to DIV 5. An increase in ATP, unless combined with vitamin E, and/or insulin, increased culture time up to DIV 6. Vitamins B1, B2, B12 and D2 had no effect. The modified CSF-like medium developed in this study permits the culture of adult hippocampal tissue for at least 6 days.
Is it Possible to Replace Stimulus Animals by Scent-filled Cups in the Social Discrimination Test?
Authors: van den Bos Ruud , van der Horst Klaske J. , Baars Annemarie M. , Spruijt Berry M.
Published: April 2019

A study in which the rat social discrimination test was refined is described. This test measures social memory by using, in general, juvenile rats as stimulus animals. Rats are offered a first juvenile to investigate (learning trial), and after a specified interval, the rats are offered the same rat and a second juvenile rat to investigate again (retrieval trial). When the rats sniff the second juvenile in the retrieval trial more than the first, social memory for the first juvenile is said to be present. This test is mainly based on scents from the juvenile. Attempts were made to refine the test to reduce the number of animals used, to enhance the scope of the test, and to improve its validity. Firstly, the stimulus animals were replaced by the scent of juveniles, in the form of cups filled with sawdust taken from cages of juvenile rats. Similar results to those in the original test were obtained when using these scents. Furthermore, male and female scents were tested, and showed the same results as for the juvenile scents. Secondly, rats were also given two cups (one scent-filled and one filled with plain sawdust) in the learning trial, to determine which allowed a more-precise delineation of motivational, discriminatory and memory components. Overall, it is possible to replace stimulus animals by scent-filled cups in the social discrimination test, to enhance the scope of the test, and to draw more-valid conclusions with respect to social memory.
In Vitro Toxicity: Mechanisms, Alternatives and Validation — A Report from the 19th Annual Scientific Meeting of the Scandinavian Society for Cell Toxicology
Authors: Forsby Anna
Published: April 2019

The Scandinavian Society for Cell Toxicology (SSCT) has arranged annual scientific meetings since 1983. These workshops were the forum for the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. Along with the MEIC programme, which was completed in 1998, a wide range of topics relating to cytotoxicity have been discussed. The meetings have also given an opportunity for graduate students and young scientists to present their work to an international audience. At the same time, experts in the fields of in vitro toxicity have been invited as speakers. The 19th SSCT scientific meeting, which was held in 2001 at Sørup Manor in Ringsted, Denmark, was no exception. The meeting consisted of four sessions: mechanisms of toxicity; environmental toxicological testing; alternatives to animal experiments; and validation of in vitro tests.
A Two-centre Evaluation of the Human Organotypic Skin Explant Culture Model for Screening Contact Allergens
Authors: Lehé Cynthia L. , Jacobs John J.L. , Elliott Graham R. , Das Pranab K.
Published: April 2019

Animal models are considered to be the “gold standard” for determining the potential contact allergenicity of low molecular weight chemicals. However, governmental regulations and ethical considerations limit the use of animals for such purposes. There is therefore a need for in vitro alternative models. The human organotypic skin explant culture (HOSEC) model is reported to be a promising alternative method for the predictive testing of contact allergens. The accelerated migration of Langerhans cells from the epidermis upon exposure to contact allergens is used to identify chemicals that are potentially capable of inducing a delayed-type hypersensitivity. In the study described in this paper, the model was further refined, and used, in two independent laboratories, to screen 23 low molecular weight compounds of known classification for their allergenicity. Each laboratory was able to accurately detect the contact allergens, despite small variations in the protocols used. However, the classification of dermal irritants, which have often been falsely classified as allergens, varied between the two laboratories. Despite the current limitations of the HOSEC model, the accuracy of the predictions made (sensitiser or non-sensitiser) compare favourably with classifications obtained with commonly used animal models. The HOSEC model has the potential to be developed further as an in vitro alternative to animal models for screening for contact allergens.
Botulinum Toxin Testing in Animals: The Questions Remain Unanswered
Authors: Balls Michael
Published: April 2019

Questions are raised concerning the testing of botulinum toxin in animals, and the British Government's answers to Parliamentary Questions on this issue are reviewed, with an emphasis on the potential for reducing, refining and replacing the animal tests, which can involve substantial severity, and on the responsibility of the Home Office so see that the Three Rs approach is fully applied in this specific case.
In Vitro Pollen Tube Growth Reveals the Cytotoxic Potential of the Flavonols, Quercetin and Rutin
Authors: Antognoni Fabiana , Ovidi Elisa , Taddei Anna Rita , Gambellini Gabriella , Speranza Anna
Published: April 2019

Flavonols are phytochemicals widely found in commonly consumed foods. In spite of their beneficial effects on human health, however, cytotoxicity and even suspected genotoxicity have also been reported for the flavonol, quercetin. This points to the need for preventive studies to identify any cytotoxic effects associated with pure flavonol intake. This work was performed with the aim of verifying whether a plant-based in vitro system, the pollen tube, could be used to evaluate the cytotoxic potential of exogenous flavonols. Increasing concentrations of the aglycone, quercetin, and its glycoside, rutin, were assayed with regard to tube growth of kiwifruit pollen, determined by applying the pollen tube growth test protocol. This test, based on the photometric quantification of pollen tube mass production in suspension cultures, has already been applied in the sensitive and reliable toxicological evaluation of a wide range of chemicals. Whereas 60–800μM rutin promoted kiwifruit pollen tube elongation, 10–50μM quercetin strongly inhibited growth, and also produced irreversible malformations, such as screw-like tube growth, abnormal vacuolation, alteration of organelle streaming, and nuclear positioning. Thus, the cytotoxic potentials of the two flavonols have been confirmed to differ. Pollen tubes seem to afford a promising test system for a preventive, rapid in vitro biosafety assessment of antioxidant nutritional supplements, without using laboratory animals.
The Development of a Standardised Protocol to Measure Squamous Differentiation in Stratified Epithelia, by using the Fluorescein Cadaverine Incorporation Technique
Authors: Gray Alison C. , Malton Joanne , Clothier Richard H.
Published: April 2019

Fluorescein cadaverine (FC) incorporation into cornified envelopes during squamous differentiation in stratified epithelia acts as a fluorescent substitute for endogenous transglutaminase substrates that can be visualised and quantified. The FC incorporation technique has been used to evaluate squamous differentiation in keratinocytes cultured in a medium that stimulates differentiation and in response to modulation by chemicals. A Standard Operating Procedure for the measurement of squamous differentiation is required as part of the prevalidation procedure for in vitro assays. In the present study, keratinocytes were isolated from the epidermis of 34 human donors. Cellular metabolic activity (resorufin production), total protein (kenacid blue uptake) and transglutaminase activity (FC incorporation) were measured in 87 and 21 independent runs at 6 and 12 days, respectively. Analysis of the control data showed that the cultures had a mean resorufin production that decreased over 12 days. This was inversely related to FC incorporation, which increased over 12 days. Mean protein concentration was reduced over the 12 days, but not in analyses that used donors for whom both 6-day and 12-day data were available. This information was used to define the normal limits within which the data should fall (mean ± 1 SD). Data sets falling outside the normal limits performed statistically no differently from the normal responders, in experiments conducted to investigate the effects of chemicals on the modulation of squamous differentiation in keratinocytes. This was demonstrated by using compounds that modify transglutaminase expression and/or activity. All- trans retinoic acid significantly inhibited FC incorporation, but stimulated resorufin production at 1 × 10–7M and above. Nicotine significantly up-regulated both FC incorporation and resorufin production at 125μg/ml. Hence, it was concluded that this robust assay approach, in which keratinocytes from a range of donors respond predictably to the test chemicals employed, did not justify the limitations that would be imposed by setting criteria that eliminated all data lying outside the normal range.
Peer Review of Validation Studies: An Assessment of the Role of the OECD by Reference to the Validation of the Uterotrophic Assay for Endocrine Disruptors
Authors: Combes Robert D.
Published: April 2019

The involvement of the OECD in managing the validation of the rat uterotrophic assay for endocrine disruptors, and in organising the peer review of the results of this study, has been assessed and compared with the many conclusions and recommendations in several published reports of international workshops on validation, and information in guidance documents, produced by the European Centre for the Validation of Alternative Methods (ECVAM), the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the OECD itself. It is concluded that the OECD has not followed the recommendations for full transparency and independence of the peer-review process. This is based on the fact that it has published a draft guidance document that differs from the report of a recent OECD workshop on validation, in such a way as to give the OECD the flexibility to fully control the peer-review process and, in so doing, to avoid full transparency. Comparison of the timing of the organisation of workshops by the OECD and the progression of the validation study, together with the fact that a draft test guideline for the assay was written before completion of the peer review, suggest that the OECD has given a higher priority to the expedition of the validation and regulatory acceptance of the uterotrophic assay than it has to good scientific and logistical practice. This severely undermines its credibility in the validation process, so, in order for the OECD to be rightly perceived as an honest broker, it is recommended that the OECD should play no role in the validation of new or revised tests, until after they have been successfully validated, peer reviewed, and endorsed by the appropriate authorities, and are ready for test guideline development. With regard to the on-going OECD validation studies of other in vivo assays for endocrine disruptors, the OECD should take immediate steps to ensure full independence and transparency of their peer review.
Neurotoxicology: Principles and Considerations of In Vitro Assessment
Authors: Aschner Michael , Syversen Tore
Published: April 2019

Neurotoxicology is an exciting area of science, not only because of the importance of toxic injury to the nervous system in human disease, but also because specific toxicants have served as invaluable tools for the advancement of our knowledge of “normal” neurobiological processes. In fact, much of our understanding of the organisation and function of the nervous system is based on observations derived from the actions of neurotoxicants. This paper addresses various physiological aspects behind the exquisite sensitivity of the nervous system to toxic agents, including the privileged status of the nervous system vis-à-vis blood-brain barrier function, the extensions of the nervous system over space and the requirements of cells with such a complex geometry, and the transmission of information across extracellular space. In addition, in vitro models and their utility in the assessment of neurotoxicological outcome are discussed, with reference to both their advantages and disadvantages.
Cellular Effects of Electromagnetic Fields
Authors: Naarala Jonne , Höytö Anne , Markkanen Ari
Published: April 2019

Studies at the cellular level are needed to reveal the cellular and molecular biological mechanisms underlying the biological effects and possible health implications of non-ionising radiation, such as extremely low frequency (ELF) magnetic fields (MFs) and radiofrequency (RF) fields. Our research group has studied the effects of 50Hz ELF MFs (caused by power lines and electric devices) and 872MHz or 900MHz RFs (emitted by mobile phones and their base stations) on cellular ornithine decarboxylase activity, cell cycle kinetics, cell proliferation, and necrotic or apoptotic cell death. For RFs, pulse-modulated (217Hz modulation frequency corresponding a global system for mobile communication-type signal) or continuous wave (unmodulated) signals were used. To expose the cell cultures to MFs or RFs, specially developed exposure systems were used, where levels of electromagnetic field exposure and the conditions of cell culture could be precisely controlled. A co-exposure approach was used in many studies, i.e. the cell cultures were exposed to other stressors in addition to MFs or RFs. Ultraviolet radiation, serum deprivation, or fresh medium addition, were used as co-exposures. The results presented in this short review show that the effects of mere MFs or RF on cell culture models are quite minor, but that various co-exposure approaches warrant additional study.
Effects of Aluminium and Lead on ATPase Activity of Knockout GDNF+/– Mouse Cerebral Synaptosomes In Vitro
Authors: Kohila T. , Tähti H.
Published: April 2019

In this in vitro study, changes in the activity of the neural membrane integral protein, ATPase, were recorded after the exposure of isolated synaptosomes to different concentrations of aluminium and lead. Both total ATPase activity and Mg2+-ATPase activity were studied. A specific mouse strain, heterozygous for a glial cell line-derived neurotrophic factor (GDNF), and the corresponding wild-type mouse cerebral tissue, were used for the synaptosome isolations. The ATPase activities of the GDNF+/– mouse synaptosomes were compared with those of wild-type synaptosomes. The decrease in total ATPase activity was similar in both types of synaptosomes, but after exposure to aluminium, the decrease of Mg2+-ATPase activity in the GDNF+/– synaptosomes was smaller than that in the wild-type synaptosomes. After exposure to lead, the protective effect of GDNF was not so clear. The synaptosomal effects of lead were already found at concentrations lower than those where cell toxicity appeared in SHSY5Y cell cultures. Thus, synaptosomal ATPase activity was considered to be a sensitive marker for the detection of lead-induced neurotoxicity.
Cytotoxicity of the Dicarboximide Fungicides, Vinclozolin and Iprodione, in Rat Hepatoma-derived Fa32 cells
Authors: Dierickx Paul J.
Published: April 2019

Dicarboximide fungicides are widely used to control various fungal species. Their primary action is not known, due to a lack of knowledge concerning the mechanism of action of the dicarboximide group. The cytotoxicities of vinclozolin and iprodione in rat hepatoma-derived Fa32 cells were investigated. Cytotoxicity was measured by neutral red uptake inhibition after treatment for 24 hours. Iprodione was more toxic than vinclozolin. Vinclozolin was less toxic in glutathione-depleted cells than in control cells. This was also true for iprodione at lower concentrations, but iprodione became more toxic at higher concentrations. Both the fungicides increased the endogenous glutathione content by 20% after 1 hour. After 24 hours, the glutathione content was doubled by vinclozolin, but was not affected by iprodione. No effect on glutathione S-transferase activity or reactive oxygen species formation could be observed. Cytochrome P450-dependent ethoxyresorufin- O-deethylase and pentoxyresorufin- O-depentylase activities were moderately activated by iprodione and strongly activated by vinclozolin. A glutathione-related cytochrome P450-dependent metabolic attack of vinclozolin and iprodione could be responsible for their cytotoxicity in Fa32 cells. Further research is needed to fully elucidate these (or other) mechanisms.
Cytotoxicity Assays with Fish Cells as an Alternative to the Acute Lethality Test with Fish
Authors: Segner Helmut
Published: April 2019

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro–in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro–in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.
Modelling of Normal and Premalignant Oral Tissue by using the Immortalised Cell Line, SVpgC2a: A Review of the Value of the Model
Authors: Staab Claudia A. , Vondracek Martin , Custodio Hipolito , Johansson Katarina , Nilsson Jan Anders , Morgan Peter , Höög Jan-Olov , Cotgreave Ian , Grafström Roland C.
Published: April 2019

Normal oral keratinocytes (NOKs), and a Simian virus 40 T-antigen-immortalised oral keratinocyte line termed SVpgC2a, were cultured in an effort to model the human oral epithelium in vitro, including normal and dysplastic tissue. Monolayer and organotypic cultures of NOKs and SVpgC2a were successfully established in a standardised serum-free medium with high levels of amino acids, by using regular tissue culture plastic for monolayers and collagen gels containing oral fibroblasts as the base for generating tissue equivalents. NOKs express many characteristics of normal tissue, including those associated with terminal squamous differentiation. After > 150 passages, SVpgC2a cells retained an immortal, non-tumourigenic phenotype that, relative to NOKs, was associated with aberrant morphology, enhanced proliferation, deficiency in terminal differentiation, proneness to apoptosis, and variably altered expression of structural epithelial markers. Transcript and protein profiling, as well as activity assays, demonstrated the expression of multiple xenobiotic-metabolising enzymes in SVpgC2a cells, some of which were higher in comparison to NOKs. A generally preserved, or even activated, ability for xenobiotic metabolism in long-term cultures of SVpgC2a cells indicated that this cell line could be useful in safety testing protocols — for example, in the development of consumer products in the oral health care field. However, SVpgC2a cells displayed some features reminiscent of a severe oral dysplasia, implying that this cell line could also to some extent serve as a model of a premalignant oral epithelium.
Mitochondrial Toxicity Detected in a Health Product with a Boar Spermatozoan Bioassay
Authors: Hoornstra Douwe , Andersson Maria A. , Johansson Tuula , Pirhonen Tuula , Hatakka Maija , Salkinoja-Salonen Mirja S.
Published: April 2019

Seaweed and organic alfalfa capsules sold as “health promoting” products had repeatedly caused emesis in a consumer. Using the boar spermatozoan bioassay, the capsule contents were found to contain a toxic substance that inhibited boar sperm motility and depolarised mitochondria at low exposure concentrations of 10μg/ml. The capsule also contained high amounts (105–107cfu/g), of endospore-forming bacteria and Streptomyces-like bacteria. Bacteria from the capsule produced toxic substances when cultured in the laboratory. Three different toxic responses were provoked in the spermatozoa exposed to extracts from the Streptomyces-like isolates: a) hyperpolarisation of the plasma membrane and depolarisation of the mitochondria; b) depolarisation of mitochondria similar to that caused by the capsule content extract; and c) motility inhibition, with no observed change of any cytosolic transmembrane potential. Membrane potential changes in the sperm cells exposed to the bacterial extracts were similar to those provoked by exposure to valinomycin and bafilomycin A1, to nigericin, and to oligomycin and ionomycin, respectively. Extracts prepared from Bacillus isolated from the capsule non-specifically depolarised all the cellular transmembrane potentials. The results demonstrate the potential value of a cell toxicity assay with boar spermatozoa for detecting hazardous substances in products intended for human consumption, without whole-animal exposure or using fetal calf serum for cell cultures.
In Vitro Methods in the Prediction of Kinetics of Drugs: Focus on Drug Metabolism
Authors: Raunio Hannu , Taavitsainen Päivi , Honkakoski Paavo , Juvonen Risto , Pelkonen Olavi
Published: April 2019

The absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of a candidate drug influence its final clinical success. These properties have traditionally been evaluated by using various in vivo animal approaches, but recently, a number of in vitro and in silico methods have been introduced to determine key ADMET features. Basic events, such as absorption through the gut wall, binding to plasma proteins, active and passive transfer through the blood–brain barrier, and various metabolic parameters, can now be screened with rapid in vitro and computer modelling methods. The focus in this short review is on the basic in vitro and in silico methods that are used for studying the metabolism properties of new drug molecules.
Status and Prospects of In Vitro Tests in Risk Assessment
Authors: Louekari Kimmo
Published: April 2019

According to the new chemicals policy of the European Union (EU), most chemicals, i.e. the 20,000 chemicals manufactured or imported at 1–10 tons annually, should be tested primarily by using in vitro methods. Also, for other chemicals, the use of in vitro methods is encouraged in the testing strategies given in the draft EU legislation. However, the validation and international acceptance of in vitro tests has been slow. Only recently has the OECD approved four new in vitro test methods, validated by the European Centre for the Validation of Alternative Methods. An analysis of ten randomly selected risk assessment reports of the EU Existing Chemicals Risk Assessment Programme showed that in vitro studies, for example, on cytotoxicity to different cell cultures, cell transformation, metabolism and skin penetration (a total of 115 studies) were used for the assessments. Key metabolic pathways and mechanisms of toxicity have been elucidated, for some chemicals, by using in vitro methods. On the other hand, the results of in vitro studies were regarded as secondary or unreliable in some cases. For several toxic endpoints, in vitro methods will probably serve as screening tools and for mechanistic studies, while target organ toxicity or physiologically regulated adverse effects caused by long-term exposure are difficult to observe without the use of animal models.
Expanding the Three Rs to Meet New Challenges in Humane Animal Experimentation
Authors: Schuppli Catherine A. , Fraser David , McDonald Michael
Published: April 2019

The Three Rs are the main principles used by Animal Ethics Committees in the governance of animal experimentation, but they appear not to cover some ethical issues that arise today. These include: a) claims that certain species should be exempted on principle from harmful research; b) increased emphasis on enhancing quality of life of research animals; c) research involving genetically modified (GM) animals; and d) animals bred as models of disease. In some cases, the Three Rs can be extended to cover these developments. The burgeoning use of GM animals in science calls for new forms of reduction through improved genetic modification technology, plus continued attention to alternative approaches and cost–benefit analyses that include the large numbers of animals involved indirectly. The adoption of more expanded definitions of refinement that go beyond minimising distress will capture concerns for enhancing the quality of life of animals through improved husbandry and handling. Targeting refinement to the unpredictable effects of gene modification may be difficult; in these cases, careful attention to monitoring and endpoints are the obvious options. Refinement can also include sharing data about the welfare impacts of gene modifications, and modelling earlier stages of disease, in order to reduce the potential suffering caused to disease models. Other issues may require a move beyond the Three Rs. Certain levels of harm, or numbers and use of certain species, may be unacceptable, regardless of potential benefits. This can be addressed by supplementing the utilitarian basis of the Three Rs with principles based on deontological and relational ethics. The Three Rs remain very useful, but they require thoughtful interpretation and expansion in order for Animal Ethics Committees to address the full range of issues in animal-based research.
Growth and Characterisation of Primary Bovine Colon Epithelial Cells In Vitro
Authors: Birkner Sascha , Weber Susanne , Dohle Angelika , Schmahl Günter , Föllmann Wolfram
Published: April 2019

Epithelial crypts from the bovine colon were obtained by using a combined mechanical and enzymatic isolation method, followed by differential D-sorbitol gradient centrifugation. By using this isolation technique, a pure fraction of epithelial crypts with minimal mesenchymal contamination was obtained. The crypts were seeded in collagen-coated plastic flasks. The attached epithelial cells proliferated and formed a confluent monolayer after 6 days in culture. Under low-serum culture conditions (1% fetal calf serum), the cells had a population doubling time of 21–22 hours. During the culture period, the colonocytes were characterised morphologically and enzymatically. The morphology of the cultured cells was confirmed by scanning electron microscopy and transmission electron microscopy. The presence of microvilli, tight junctions and desmosomes demonstrated the ability of the cultured cells to restore an epithelial-like cell monolayer. The epithelial origin of the cells was demonstrated by labelling the cells with antibodies against epithelial-specific cytokeratins 7 and 13. The functional integrity of the cells was evaluated by measuring various marker enzymes (γ-glutamyltranspeptidase, acid phosphatase, alkaline phosphatase, NADH-dehydrogenase) and membrane-associated Na+–K+-ATPase activity. Membrane integrity was determined by measuring the leakage of lactate dehydrogenase into the culture medium. This new culture system for bovine colon epithelial cells could be used as an in vitro model of the colon epithelium in physiological and toxicological studies.
The Mandatory CAM Testing of Cells and Scaffolds for Tissue Engineering: Benefits for the Three Rs of Cooperation with the Vaccine Industry
Authors: Falkner Erwin , Eder Claudia , Kapeller Barbara , Fröschl Walter , Schmatz Christine , Macfelda Karin , Losert Udo M.
Published: April 2019

In cooperation with BAXTER Vaccine AG, which supplies incubated special pathogen-free chicken eggs (including a full veterinary record), a permanent hen's egg chorio-allantoic membrane test (HET-CAM) unit has been established, where angiogenesis testing, cell culture, and digital and histological analyses are performed. At the Core Unit for Biomedical Research, the location of the animal testing facility of the Medical University Vienna, cell–scaffold constructs must be evaluated in vitro and in ovo prior to eventual in vivo tissue engineering experiments. The animal testing advisory committee requires that new test proposals are first evaluated by using cell culture and HET-CAM models. Approvals for in vivo experiments are postponed and not issued prior to in vitro/ in ovo evaluation. Examples are presented of protocols planned for in vivo studies on cell seeded scaffolds, which were refined after in vitro/in ovo evaluations.
Development of a Size-dependent Aerosol Deposition Model Utilising Human Airway Epithelial Cells for Evaluating Aerosol Drug Delivery
Authors: Cooney Daniel , Kazantseva Masha , Hickey Anthony J.
Published: April 2019

Aerosol delivery to the airways of the human respiratory tract, followed by absorption, constitutes an alternative route of administration for compounds unsuitable for delivery by conventional oral and parenteral routes. The target for aerosol drug delivery is the airways epithelium, i.e. tracheal, bronchial, bronchiolar and alveolar cells, which become the site of drug deposition. These epithelial layers also serve as a barrier to the penetration of inhaled material. An in vitro model for aerosol deposition and transport across epithelia in the human airways may be a good predictor of in vivo disposition. The present preliminary studies begin an investigation that blends the dynamics of aerosol delivery and the basis of an in vitro simulated lung model to evaluate the transport properties of a series of molecular weight marker compounds across human-derived bronchiolar epithelial cell monolayers. An Andersen viable cascade impactor was used as a delivery apparatus for the deposition of size-segregated particles onto monolayers of small airway epithelial cells and Calu-3 cells. It was shown that these cell layers can withstand placement in the impactor, and that permeability can be tested subsequent to removal from the impactor.
Trends in Animal Use and Animal Alternatives
Authors: De Greeve Paul , De Leeuw Wim , van Zutphen Bert
Published: April 2019

The Third World Congress (1999, Bologna) celebrated the fortieth anniversary of the publication of Russell & Burch's The Principles of Humane Experimental Technique. There was the general notion that the Three Rs offer a unifying concept that contributes to a progressive reduction and refinement in animal use without compromising the quality of research, human health or the protection of the environment. The Bologna Three Rs Declaration was accepted unanimously, calling upon all parties involved to incorporate the Three Rs into animal-based research. The question is raised, what progress has been made and, in particular, what are the developments in animal use and in the implementation of validated alternative methods. For the present contribution, we requested colleagues from European countries, Canada and the United States to provide information on the numbers of animals currently used for scientific purposes, on the development and implementation of alternative methods and on future perspectives about the issues. Based on the results of this survey, the conclusion is reached that legislative regulations are widely implemented and have become rather strict during the last decade. An exception here is the legislative regulation for rats, mice and birds in the USA. These species are not (yet) protected by the US Animal Welfare Act. The number of animals used has decreased considerably, and the review of protocols by animal ethics committees has become a significant trend. In all countries, there is growing support for the Three Rs concept.
The Use of Human Cells in Biomedical Research and Testing
Authors: Combes Robert D.
Published: April 2019

The ability to use human cells in biomedical research and testing has the obvious advantage over the use of laboratory animals that the need for species extrapolation is obviated, due to the presence of more-relevant morphological, physiological and biochemical properties, including receptors. Moreover, human cells exhibit the same advantages as animal cells in culture in that different cell types can be used, from different tissues, with a wide range of techniques, to investigate a wide variety of biological phenomena in tissue culture. Human cells can also be grown as organotypic cultures to facilitate the extrapolation from cells to whole organisms. Human cell lines have been available for many years on an ad hoc basis from individual researchers, and also from recognised sources, such as the European Collection of Animal Cell Cultures (ECACC) and, in the USA, the Human Cell Culture Centre (HCCC). Such cells have usually been derived from tumours and this has restricted the variety of types of cells available. This problem has been addressed by using primary human cells that can be obtained from a variety of sources, such as cadavers, diseased tissue, skin strips, peripheral blood, buccal cavity smears, hair follicles and surgical waste from biopsy material that is unsuitable for transplantation purposes. However, primary human cells need to be obtained, processed, distributed and handled in a safe and ethical manner. They also have to be made available at the correct time to researchers very shortly after they become available. It is only comparatively recently that the safe and controlled acquisition of surgical waste and non-transplantable human tissues has become feasible with the establishment of several human tissue banks. Recently, the formation of a UK and European centralised network for human tissue supply has been initiated. The problems of short longevity and loss of specialisation in culture are being approached by: a) cell immortalisation to generate a cell type possessing the properties of both primary cells and cell lines; b) the inhibition of intracellular activities resulting in oxidative stress; and c) the use of stem cells, both of embryonic and adult origin.
Cell Transformation Assay Using Balb/c 3T3 Cells or Bhas 42 Cells for the Efficient Detection of Tumour Promoters
Authors: Umeda Makoto
Published: April 2019

In this paper, we report three interlaboratory validation studies with special interest in detecting tumour promoters. The former two validation studies were on the improved two-stage in vitro transformation assay using Balb/c 3T3 cells. The third one was on the Bhas 42 cell assay. From the results, we could confirm that, on the whole, the improved two-stage transformation assay was sensitive, reproducible and reliable, while the Bhas 42 cell assay was a very sensitive and reproducible method for detecting tumour-promoting potential. However, there was some variation in the sensitivity among the laboratories. In order to cope with this sensitivity variation, further refinement is necessary, which will increase the applicability of the methods.
The Research Programme of SCAAT
Authors: de Silva Odile , Basketter David
Published: April 2019

The European Federation of the Cosmetics Industry (COLIPA's) Steering Committee on Alternatives to Animal Testing (SCAAT) is currently focusing its research activity on two areas: skin tolerance and eye irritation. SCAAT has set up two three-year projects on skin sensitisation, one three-year project on skin irritation and one three-year project on eye irritation. The sensitisation projects aim at the standardisation and optimisation of a method involving human dendritic cells and the identification of up-regulated genes following exposure of these cells to chemicals. The skin irritation project aims to identify new markers of irritation with the help of genomic and proteomic technologies. These three projects will be carried out by industry. The scope of the eye project is to identify new in vitro endpoints, predictive of the in vivo response to chemical injury. It involves collaboration with ophthalmologists from the Universities of Aachen and Bristol. The team in Aachen will try to identify whether the kinetics and patterns of changes in physiological function and signals of injury released from the cornea in vitro can predict a chemical's potential to damage the eye, with a focus on recovery. All these projects aim at developing and optimising alternative methods for further prevalidation.