bluuebunny/crossref_metadata_2025 huggingface dataset:
Intelligent Testing Strategies for Chemicals Testing — A Case of More Haste, Less Speed?
Authors: Combes Robert , Balls Michael
Published: April 2019

The prospects for using (Q)SAR modelling, read-across (chemical) and other non-animal approaches as part of integrated testing strategies for chemical risk assessment, within the framework of the EU REACH legislation, are considered. The potential advantages and limitations of (Q)SAR modelling and read-across methods for chemical regulatory risk assessment are reviewed. It is concluded that it would be premature to base a testing strategy on chemical-based computational modelling approaches, until such time as criteria to validate them for their reliability and relevance by using independent and transparent procedures, have been agreed. This is mainly because of inherent problems in validating and accepting (Q)SARs for regulatory use in ways that are analogous to those that have been developed and applied for in vitro tests. Until this issue has been resolved, it is recommended that testing strategies should be developed which comprise the integrated use of computational and read-across approaches. These should be applied in a cautious and judicious way, in association with available tissue culture methods, and in conjunction with metabolism and biokinetic studies. Such strategies should be intelligently applied by being driven by exposure information (based on bioavailability, not merely on production volume) and hazard information needs, in preference to a tick-box approach. In the meantime, there should be increased efforts to develop improved (Q)SARs, expert systems and new in vitro methods, and, in particular, ways to expedite their validation and acceptance must be found and prospectively agreed with all major stakeholders.
Prediction of Human Acute Toxicity by the Hep G2/24-hour/Total Protein Assay, with Protein Measurement by the CBQCA Method
Authors: Dierickx Paul J.
Published: April 2019

In our previously described Hep G2/24-hour/total protein assay, protein levels were measured by using the Lowry method. This assay was the best acute in vitro assay for the prediction of human toxicity within the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) study. In order to increase the MEIC data-base with a wider range of chemicals, we were interested in introducing the more practical 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde (CBQCA) method for the quantification of the total protein content. Therefore, we investigated whether the same good results for the prediction of acute human toxicity would be obtained with the CBQCA method. The cells were treated for 24 hours, then cytotoxicity was determined by measuring the total protein content with CBQCA. The results were quantified by using the PI50c: the concentration (in mM) of test compound required to reduce the total protein content measured with the CBQCA-method by 50% as compared to the control cells. The results were compared with the PI50, the corresponding value when the Lowry method was used. A relatively low correlation was observed between PI50 and PI50c, reflecting the large and unexpected, differences when using the two protein assays. However, when comparing the log PI50c with the human toxicity, a correlation coefficient of r2 = 0.761 ( n = 44) was obtained for exactly the same series of MEIC chemicals. This value is clearly higher than that for the Lowry method ( r2 = 0.695). Compared to the Lowry method originally used, the Hep G2/24-hour/CBQCA total protein assay has the additional important advantage that it can be very easily adapted for large-scale analyses with robotic systems, including the on-line calculation of the results.
The Role of Biomembranes in Chromium (III)-induced Toxicity In Vitro
Authors: Rudolf Emil , Červinka Miroslav
Published: April 2019

The role of biomembranes in the chronic toxicity of environmentally occurring chromium acetate hydroxide was investigated by using primary human fibroblasts. Transport of chromium acetate hydroxide across the plasma membrane of the cell, and the effects of chromium (III) ions on the plasma membrane as well as other intracellular membranes, were determined during six weeks of continuous exposure by using atomic absorption spectrometry, observation of cell morphology, membrane integrity assays (for lactate dehydrogenase leakage and lysosomal membrane disruption), and mitochondrial assays (for mitochondrial dehydrogenase activity and mitochondrial transmembrane potential analysis). The type of cell death induced by long-term exposure was determined in terms of phosphatidylserine externalisation, caspase-3 activation, and chromatin fragmentation. Chromium acetate hydroxide, at a concentration of 100μmol/l, accumulated in exposed cells, inflicting plasma membrane damage and suppressing mitochondrial function. Antioxidant co-enzyme Q, at a concentration of 10μmol/l, partially prevented plasma membrane damage and mitochondrial dysfunction. Exposure to chromium acetate hydroxide produced apoptosis, necrosis and an intermediate type of cell death in primary human fibroblasts. These results show that the plasma membrane and mitochondrial membrane are important targets for chronic chromium acetate hydroxide toxicity, and that this in vitro system holds promise for studying the toxicity resulting from long-term exposure to metal ions.
Recommendations for Improving the Home Office Project Licence Abstracts
Authors: Hudson Michelle
Published: April 2019

The Freedom of Information Act 2000 prompted the Government to evaluate how public understanding about the use of animals in scientific research can be improved and how animal experimentation is regulated within the UK. This resulted in the publication of anonymous project licence abstracts. Here, the abstracts published between December 2004 and October 2005 have been evaluated, by using a scoring system which takes into account their content and format, in order to assess whether they provide information that will facilitate the increased implementation of reduction, refinement and replacement strategies. The potential impact of freely-available Home Office project licence abstracts on public attitudes to animal testing and on the quality of animal-based research, is also discussed. Although some abstracts scored well, in general, the abstracts were found to be severely lacking in detail about the welfare of experimental animals. Thus, the abstracts tend to present a distorted picture of animal-based research. Many abstracts lack the details which would reveal how the licensing criteria had been met. It is concluded that, in their current form, the project abstracts are not sufficiently informative. A number of recommendations are made, which address this problem.
Reconstructed Human Epidermis for Skin Absorption Testing: Results of the German Prevalidation Study
Authors: Schäfer-Korting Monika , Bock Udo , Gamer Armin , Haberland Annekathrin , Haltner-Ukomadu Eleonore , Kaca Monika , Kamp Hennicke , Kietzmann Manfred , Korting Hans Christian , Krächter Hans-Udo , Lehr Claus-Michael , Liebsch Manfred , Mehling Annette , Netzlaff Frank , Niedorf Frank , Rübbelke Maria K. , Schäfer Ulrich , Schmidt Elisabeth , Schreiber Sylvia , Schröder Klaus-Rudolf , Spielmann Horst , Vuia Alexander
Published: April 2019

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.
Induction of Apoptosis and Activation of JNK and p38 MAPK Pathways in Deoxynivalenol-Treated Cell Lines
Authors: Baltriukiene Daiva , Kalvelyte Audrone , Bukelskiene Virginija
Published: April 2019

Deoxynivalenol (DON) is a mycotoxin produced by what are thought to be the most prevalent toxin-producing fungi of the Fusarium genus. Here, we present the results of apoptosis induction, phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs), and expression of the c-Jun protein after DON treatment, in a pre-B lymphocyte REH cell line. In addition, human pre-T lymphocyte Jurkat, hamster kidney-derived BHK21 and mouse hepatoma MH-22a cells were used in comparative experiments in vitro. We found that the DON effect was cell origin-dependent and dose-dependent, with a significant slow-down of cell proliferation and increase of apoptotic cells in blood cell lines. BHK21 and MH-22a cells were less sensitive to the DON effect. In blood-derived REH and Jurkat cells, DON-induced apoptotic changes were preceded by an increase in JNK and p38 MAPKs phosphorylation, as well as in c-Jun expression. However, the activation of JNK phosphorylation and c-Jun expression were transient, but did not coincide with each other. An inhibitor of JNK1/2, SP600125, had a negligible negative effect on REH cell viability after DON treatment, demonstrating that JNK does not contribute to DON-induced apoptosis. In contrast, studies on the role of p38 MAPK revealed that p38 signalling is required for DON-induced apoptosis in REH cells.
Comments on the Sub-group Reports of the EU Technical Expert Working Group on the Revision of Directive 86/609/EEC on the Protection of Animals Used for Experimental and Other Scientific Purposes
Authors: Combes Robert , Balls Michael
Published: April 2019

A critical analysis is presented of the reports produced by four Technical Expert Working Group Sub-groups (SGs) on Ethical Review, Cost–Benefit, Authorisation and Scope, which were published on the EC website ( http://ec.europa.eu/environment/chemicals/lab_animals/ia_info_en.htm ), as part of the European Commission (EC)'s review of EU Directive 86/609 EEC. This is in addition to our official response to the internet consultation questionnaire, submitted to the Commission on behalf of FRAME. Whilst the respective SG reports were extensive and detailed, we have identified several shortcomings, and in particular, a frequent lack of consensus among the SG members, resulting in a lack of clear guidance for the EC on what the revised Directive should contain, with reference to a number of crucial issues. Such indecisiveness could lead to wide discrepancies in the approaches of the EC, the European Parliament and the EU Member States concerning many issues of importance to animal welfare and the implementation of alternatives to animal experiments. These concerns range from logistical issues, such as requirements for named officers in authorised establishments, and the recording and publishing of statistics on animal use, to ethical and scientific problems, including the use of non-human primates, local ethical review, and education and training on the essential link between the Three Rs concept and best scientific practice. In each case, the basis for our concerns is explained, and suggestions for improvements to be incorporated into the revised Directive are made, in the hope that the harmonisation of approaches to laboratory animal experimentation and the use of alternative methods in the Member States can be maximised.
Rapid Screening for Soil Ecotoxicity with a Battery of Luminescent Bacteria Tests
Authors: Heinlaan Margit , Kahru Anne , Kasemets Kaja , Kurvet Imbi , Waterlot Cristophe , Sepp Kalev , Dubourguier Henri-Charles , Douay Francis
Published: April 2019

A bacterial test battery, involving i) Microdot™, an aquatic test, ii) the Flash assay, a soil-suspension test (with Vibrio fischeri as the test organism), and iii) the Metal Detector assay, a semi-specific aquatic test for heavy metals (with recombinant luminescent Escherichia coli), was used in a combined toxicological and chemical hazard assessment of Estonian soils sampled from a former Soviet military airfield (13 samples) and from traffic-influenced roadsides (5 samples). The soils showed slightly elevated levels of total petroleum hydrocarbons (TPH), but not of heavy metals. In most of the samples, the levels of TPH did not exceed the Estonian permitted limit values set for residential areas. Toxicity testing was performed on both fresh and dried soils, after aqueous extraction for 1 hour and 24 hours. The toxicity results obtained with the Microtox™ test did not significantly differ in all of the sample treatment schemes; however, it appeared that the drying and sieving of the soils increased the bioavailability of toxicants, probably due to an enlarged reactive soil surface area. According to chemical analysis of the soils and the data from the Microtox™ test and the Metal Detector assay (performed on aqueous elutriates of the soils), these soils would not be considered to be hazardous. In contrast, the Flash assay performed on soil–water suspensions of dried soils, showed that most of the soils were toxic and thus probably contained undetermined particle-bound bioavailable toxicants. The photobacterial toxicity test (the Flash assay) can be recommended for the rapid screening of soils, as it is sensitive, cheap and inexpensive, and provides valuable information on particle-bound bioavailable toxicants, useful for complementing a chemical analysis and for assessing the risks originating from polluted soils.
The New EU REACH Regulation Has Finally Been Adopted: Is this the End of the Campaign Trail… or Just the Beginning?
Authors: Grindon Christina
Published: April 2019

The final EU REACH legislation has recently been adopted. This article considers the progress that has been made toward reducing the numbers of animals likely to be required to fulfil the testing requirements, and also considers the benefits to animal welfare and science that have arisen since the original REACH system proposals were published in 2003. Several positive changes have been made, including: the use of exposure-based testing; the requirement for scientific justification of any proposed animal testing; mandatory data sharing; and the fact that the EU is to take responsibility for the development and validation of alternative methods. While these changes are to be commended, there is still much room for improvement, particularly with respect to the adoption of integrated testing strategies that make maximum use of non-animal approaches to expedite the risk assessment process of existing chemicals, with the use of refined and updated animal tests only as a last resort.
In Vitro Effects of Certain Membrane-acting Agents on Superoxide and Hydrogen Peroxide Production, Protein Synthesis and Membrane ATPase Activity in Buffalo PMN Cells
Authors: Das Hemen , Reddy Golla Ramalinga , More Tukaram , Singh Vineet Kumar
Published: April 2019

Polymorphonuclear (PMN) cells play a key role in innate immunity, due to their ability to produce reactive oxidants such as superoxide (O2–) and hydrogen peroxide (H2O2), and to release antimicrobial proteins and peptides stored in their lysosomal granules. In the present study, the effects of the activation of buffalo PMN cells with various membrane-acting agents were evaluated in terms of O2– and H2O2 production, the activities of membrane ATPases, and protein synthesis. Studies involving the incorporation of 35S-methionine revealed significant protein-synthesising ability in resting PMN cells and in cells treated with lipopolysaccharide (LPS), as well as with opsonised zymosan (OZ). Protein synthesis, as judged by fluorography of the cytosolic fraction, showed more than 12 bands, whilst the cytoskeletal fraction showed 2–3 bands. PMN activation with concanavalin A (ConA), digitonin and sodium nitroprusside (SNP) resulted in increased O2– and H2O2 production. However, in the presence of anti-inflammatory agents such as indomethacin and cortisol, the production of O2– and H2O2 by these cells was found to decline. Studies pertaining to membrane ATPases revealed that verapamil hydrochloride (VpHCl) significantly increased total ATPase and Na+K+ATPase activity. ConA treatment yielded only a moderate level of activity. Similarly, digitonin up to 24μM also caused a significant increase in ATPase activity. Our observations indicate that these membrane-acting agents influenced oxygen-dependent and oxygen-independent microbicidal mechanisms in buffalo PMN cells.
Phototoxicity and Acute Toxicity Studies Conducted by the FRAME Alternatives Laboratory: A Brief Review
Authors: Clothier Richard H.
Published: April 2019

FRAME and the University of Nottingham have been in association for the past 25 years. During this time, the research in the FRAME Alternatives Laboratory (FAL) at the University of Nottingham, which is partly funded by FRAME and also, more recently, by ECVAM, has involved participation in a number of international validation studies. Validation has become a pre-requisite for the regulatory acceptance of in vitro alternative test procedures, and a number of key lessons have been learned from these studies. The directors of validation studies need to ensure that standard operating procedures (SOPs) are fully complied with, and that the equipment used is certified to be of an acceptable standard. Database managers need to be able to check the original data, and to ensure adherence to procedures agreed before the study began. When the validation study is part of an integrated EU Framework Project, such as ACute-Tox, the Workpackage Leader must have the ability to understand and evaluate the data to be presented for inclusion in the study analysis, and to check that it complies with acceptance criteria. The potential to relate observed cellular biochemical changes to morphological endpoints also increases the level of understanding of the relevance and/or limitations of an assay. For example, exposure to a surfactant can induce the temporary loss of adhesion junctions between adjacent epithelial cells, resulting in the loss of barrier integrity and other effects on cell culture activity, which can potentially be restored over time. Unexpected results from the NRU phototoxicity assay with human keratinocytes instead of 3T3 cells, stimulated research into the ability of the in vitro assay, not only to identify phototoxins, but also to identify their possible mechanisms of action and mechanisms underlying the protective capacity within human primary keratinocytes in vitro. The protective effects of UV-filters can also be used to ascertain their effects on the photoactivation of drugs.
An Evaluation of a Novel Chick Cardiomyocyte Micromass Culture Assay with Two Teratogens/Embryotoxins Associated with Heart Defects
Authors: Hurst Helena S. , Clothier Richard H. , Pratten Margaret
Published: April 2019

This study was aimed at determining whether the chick cardiomyocyte micromass (MM) system could be employed to predict the teratogenicity/embryotoxicity of exogenous chemicals. Two documented teratogens/embryotoxins, sodium valproate (the sodium salt of valproic acid; VPA) and all -trans retinoic acid (tRA), were used in the initial phase of the study. White Leghorn 5-day-old embryo hearts were dissociated to produce a cardiomyocyte suspension in Dulbecco's Modified Eagle's Medium. Cultures were incubated at 37°C in 5% CO2 in air, and observations were made every 24 hours over 5 days, for the detection of beating. Culture viability was assessed by using the resazurin reduction assay for determining culture activity and the kenacid blue assay for determining cell number. It was found that tRA significantly reduced cell activity and beating, whilst not affecting total cell number. VPA up to 500μM induced no cytotoxicity in the MM cardiomyocyte cultures, whilst all the VPA concentrations tested reduced beating. The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter functionality, which may result in a teratogenic outcome, whilst not causing cytotoxicity (direct embryotoxicity). This could form part of a screen for developmental toxicity related to cardiac function, whilst limb cultures and brain cultures based on the same system could be relevant to teratogenic effects on those tissues.
Strategies for the Reduction of Live Animal Use in Microsurgical Training and Education
Authors: Schöffl Harald , Froschauer Stefan M. , Dunst Karin M. , Hager Dietmar , Kwasny Oskar , Huemer Georg M.
Published: April 2019

Education and training in microsurgical techniques have historically relied on the use of live animal models. Due to an increase in the numbers of microsurgical operations in recent times, the number of trainees in this highly-specialised surgical field has continued to grow. However, strict legislation, greater public awareness, and an increasing sensitivity toward the ethical aspects of scientific research and medical education, emphatically demand a significant reduction in the numbers of animals used in surgical and academic education. Hence, a growing number of articles are reporting on the use of alternatives to live animals in microsurgical education and training. In this review, we report on the current trends in the development and use of microsurgical training models, and on their potential to reduce the number of live animals used for this purpose. We also share our experiences in this field, resulting from our performance of numerous microsurgical courses each year, over more than ten years. The porcine heart, in microvascular surgery training, and the fresh chicken leg, in microneurosurgical and microvascular surgery training, are excellent models for the teaching of basic techniques to the microsurgical novice. Depending on the selected level of expertise of the trainee, these alternative models are capable of reducing the numbers of live animals used by 80–100%. For an even more enhanced, “closer-to-real-life” scenario, these non-animated vessels can be perfused by a pulsatile pump. Thus, it is currently possible to provide excellent and in-depth training in microsurgical techniques, even when the number of live animals used is reduced to a minimum. With these new and innovative techniques, trainees are able to learn and prepare themselves for the clinical situation, with the sacrifice of considerably fewer laboratory animals than would have occurred previously.
A Reassessment of the In Vitro RBC Haemolysis Assay with Defibrinated Sheep Blood for the Determination of the Ocular Irritation Potential of Cosmetic Products: Comparison with the In Vivo Draize Rabbit Test
Authors: Alves Eloísa Nunes , de Farias Presgrave Rosaura , Presgrave Octávio Augusto França , Sabagh Fernanda Peres , de Freitas João Carlos Borges Rolim , Corrado Alexandre Pinto
Published: April 2019

We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (DI); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro ( r = 0.752–0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.
Integrated Decision-tree Testing Strategies for Skin Corrosion and Irritation with Respect to the Requirements of the EU REACH Legislation
Authors: Grindon Christina , Combes Robert , Cronin Mark T.D. , Roberts David W. , Garrod John F.
Published: April 2019

Liverpool John Moores University and FRAME recently conducted a research project, sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This report focuses on how to maximise the use of alternative methods (both in vitro and in silico) for skin corrosion and irritation testing within a tiered testing strategy. It considers the latest developments in in vitro testing, with particular reference to the reconstituted skin models which have now been now been successfully validated and independently endorsed as suitable for both skin corrosivity and irritancy testing within the EU.
The Use of the Integrated Discrete Multiple Organ Co-culture (IdMOC®) System for the Evaluation of Multiple Organ Toxicity
Authors: Li Albert P.
Published: April 2019

The application of the Integrated Discrete Multiple Organ Co-culture (IdMOC®) system in the evaluation of organ-specific toxicity is reviewed. In vitro approaches to predict in vivo toxicity have met with limited success, mainly because of the complexity of in vivo toxic responses. In vivo properties that are not well-represented in vitro include organ-specific responses, multiple organ metabolism, and multiple organ interactions. The IdMOC system has been developed to address these deficiencies. The system uses a ‘wells-within-a-well’ concept for the co-culturing of cells or tissue slices from different organs as physically separated (discrete) entities in the small inner wells. These inner wells are nevertheless interconnected (integrated) by overlying culture medium in the large outer containing well. The IdMOC system thereby models the in vivo situation, in which multiple organs are physically separated but interconnected by the systemic circulation, permitting multiple organ interactions. The IdMOC system, with either cells or tissue slices from multiple organs, can be used to evaluate cell type-specific or organ-specific toxicity.
Immunotoxicology: Opportunities for Non-animal Test Development
Authors: Corsini Emanuela , Roggen Erwin L.
Published: April 2019

At present, several animal-based assays are used to assess immunotoxic effects such as immunosuppression and sensitisation. The use of whole animals, however, presents several secondary issues, including expense, ethical concerns and relevance to human risk assessment. There is a growing belief that non-animal approaches can eliminate these issues without impairing human safety, provided that biological markers are available to identify the immunotoxic potentials of new chemicals to which humans may be exposed. Driven by the 7th Amendment to the EU Cosmetics Directive, the new EU policy on chemicals (the REACH system), proposals to update the European legislation on the protection of animals used in research, and emerging visions and strategies for predicting toxicity, such in vitro methods are likely to play a major role in the near future. The realisation that the immune system can be the target of many chemicals, resulting in a range of adverse effects on the host's health, has raised serious concerns from the public and within the regulatory agencies. Hypersensitivity and immunosuppression are considered the primary focus for developing in vitro methods in immunotoxicology. However, in vitro assays to detect immunostimulation and autoimmunity are also needed. This review of the state-of-the-art in the field of in vitro immunotoxicity, reveals a lack of cell-based immunotoxicity assays for predicting the toxicity of xenobiotics toward the immune system in a simple, fast, economical and reliable way.
The Extended Welfare Assessment Grid: A Matrix for the Assessment of Welfare and Cumulative Suffering in Experimental Animals
Authors: Honess Paul , Wolfensohn Sarah
Published: April 2019

Combining a range of assessment parameters into one usable entity has been identified as an important goal in providing a practical, objective and robust assessment of welfare, particularly in laboratory animals. This paper refines and extends one such previously published method. The proposed Extended Welfare Assessment Grid provides for the incorporation of changes in the state of an animal over time, allowing for predictive, retrospective, scheduled, or event monitoring. It enables the numeric, as well as visual, representation of the animal's welfare, placing this in the context of the careful and realistic justification for experimental use of the animal. This assessment method represents a valuable tool for those tasked with ensuring ethical oversight, as well as for those planning the use, or monitoring, of animals in research. It is particularly applicable to animals used in long-term studies, especially non-human primates. It is believed that this system will draw attention to the temporal component of suffering that is often overlooked in the planning of research schedules and allow an assessment of cumulative suffering imposed by the events that occur.
Refining Animal Experiments: The First Brazilian Regulation on Animal Experimentation
Authors: Tréz Thales de A. E
Published: April 2019

The very first law on animal experimentation has been approved recently in Brazil, and now is part of a set of the legal instruments that profile the Brazilian government's attitude toward the use of animals in experiments. Law 11794/08 establishes a new legal instrument that will guide new methods of conduct for ethics committees, researchers and representatives of animal protection societies. This comment aims to analyse critically the implications that this law brings to Brazilian reality. The link between it and the Russell & Burch's Three Rs concept is defined, and certain problems are identified. The conclusion is that the body of the law emphasises the refinement of animal experiments, but gives little importance to the principles of reduction and replacement.
FRAME and the Three Rs: Yesterday, Today and Tomorrow
Authors: Balls Michael , Morton David
Published: April 2019

At one of the events to mark FRAME's 40th anniversary, two long-standing trustees of the charity independently answered 20 questions related to FRAME's role in the drafting and passage of the Animals (Scientific Procedures) Act 1986, and on progress made since its introduction in 1987, in terms of the reduction, refinement and replacement of animal procedures in fundamental research and testing.
Evaluation of Anti-inflammatory and Atrophogenic Effects of Glucocorticoids on Reconstructed Human Skin
Authors: Weindl Günther , Castello Francesca , Schäfer-Korting Monika
Published: April 2019

Topical glucocorticoids (GCs) are extensively used in the treatment of inflammatory skin diseases. However, their long-term use is often accompanied by severe and eventually irreversible adverse effects, with atrophy being the most important limitation. Currently, most non-clinical studies involve animal testing, so the results are not always representative of the situation in humans. The aim of this project was to establish an in vitro test protocol for the evaluation of the anti-inflammatory and atrophic potential of topically applied GCs in reconstructed human skin. Initial studies with fibroblasts and keratinocytes confirmed the anti-inflammatory and atrophogenic effects of GCs, as evidenced by decreased cytokine production and collagen mRNA expression. In non-pretreated reconstructed human skin (EpiDermFT™), the topical application of GCs for seven days strongly reduced the secretion of interleukin (IL)-6. GC-induced skin atrophy, known to appear only after prolonged treatment, was not detected by the analysis of epidermal thickness and collagen mRNA expression. However, reproducible epidermal inflammation was established for the first time in reconstructed human skin. Topical treatment with tumour necrosis factor (TNF) increased IL-6 release and strongly reduced epidermal thickness accompanied by severe parakeratosis. GC treatment of reconstructed human skin reduced IL-6 levels and completely resolved parakeratosis, leading to the normalisation of epidermal thickness. These induced inflammatory conditions mimic more closely the clinical situations in which GCs are used, and therefore appear to be more suitable for future investigations for the establishment of a human-based in vitro test protocol for evaluating wanted and unwanted GC effects.
Hallmarks of Atopic Skin Mimicked In Vitro by Means of a Skin Disease Model Based on FLG Knock-down
Authors: Küchler Sarah , Henkes Dominika , Eckl Katja-Martina , Ackermann Katharina , Plendl Johanna , Korting Hans-Christian , Hennies Hans-Christian , Schäfer-Korting Monika
Published: April 2019

Loss-of-function mutations in the filaggrin gene (FLG) are a strong predisposing factor for atopic dermatitis, although their relevance to the disease pathomechanism needs further elucidation. The generation of an in vitro model of atopic skin would not only permit further evaluation of the underlying pathogenetic mechanisms and the testing of new treatment options, but would also allow toxicological studies to be performed in a simple, rapid and inexpensive manner. In this study, we have knocked down FLG expression in human keratinocytes and created three-dimensional skin models, which we used to investigate the impact of FLG on epidermal maturation and on skin absorption and its response to irritation. Histopathological evaluation of the skin models showed impaired epidermal differentiation in the FLG knockdown model. In addition, skin irritation induced by an application of sodium dodecyl sulphate resulted in significantly higher lactate dehydrogenase leakage, and interleukin (IL)-6 and IL-8 levels, than in the control model. To assess the effect of filaggrin deficiency on skin absorption of topically applied agents, we quantified the percutaneous absorption of lipophilic and hydrophilic model drugs, finding clinical relevance only for lipophilic drugs. This study clearly demonstrates that important clinical characteristics of atopic skin can be mimicked by using in vitro skin models. The FLG knock-down construct is the first step toward an in vitro model that allows clinical and toxicological studies of atopic-like skin.
‘Human-on-a-chip’ Developments: A Translational Cutting-edge Alternative to Systemic Safety Assessment and Efficiency Evaluation of Substances in Laboratory Animals and Man?
Authors: Marx Uwe , Walles Heike , Hoffmann Silke , Lindner Gerd , Horland Reyk , Sonntag Frank , Klotzbach Udo , Sakharov Dmitry , Tonevitsky Alexander , Lauster Roland
Published: April 2019

Various factors, including the phylogenetic distance between laboratory animals and humans, the discrepancy between current in vitro systems and the human body, and the restrictions of in silico modelling, have generated the need for new solutions to the ever-increasing worldwide dilemma of substance testing. This review provides a historical sketch on the accentuation of this dilemma, and highlights fundamental limitations to the countermeasures taken so far. It describes the potential of recently-introduced microsystems to emulate human organs in ‘organ-on-a-chip’ devices. Finally, it focuses on an in-depth analysis of the first devices that aimed to mimic human systemic organ interactions in ‘human-on-a-chip’ systems. Their potential to replace acute systemic toxicity testing in animals, and their inability to provide alternatives to repeated dose long-term testing, are discussed. Inspired by the latest discoveries in human biology, tissue engineering and microsystems technology, this review proposes a paradigm shift to overcome the apparent challenges. A roadmap is outlined to create a new homeostatic level of biology in ‘human-on-a-chip’ systems in order to, in the long run, replace systemic repeated dose safety evaluation and disease modelling in animals.
A Risk Assessment Approach to Severity Classification in Animal Research
Authors: Morton David B.
Published: April 2019

Assessment of pain, suffering, distress and lasting harm in animal research could be carried out more successfully by adopting the practical risk assessment approach currently in use in farming
Medical Waste Tissues — Breathing Life back into Respiratory Research
Authors: BéruBé Kelly A.
Published: April 2019

With the advent of biobanks to store human lung cells and tissues from patient donations and from the procurement of medical waste tissues, it is now possible to integrate (both spatially and temporally) cells into anatomically-correct and physiologically-functional tissues. Modern inhalation toxicology relies on human data on exposure and adverse effects, to determine the most appropriate risk assessments and mitigations for beneficial respiratory health. A point in case is the recapitulation of airway tissue, such as the bronchial epithelium, to investigate the impact of air pollution on human respiratory health. The bronchi are the first point of contact for inhaled substances that bypass defences in the upper respiratory tract. Animal models have been used to resolve such inhalation toxicology hazards. However, the access to medical waste tissues has enabled the Lung Particle Research Group to tissue-engineer the Micro-Lung™ and Metabo-Lung™ cell culture models, as alternatives to animals in basic research and in the safety testing of aerosolised consumer goods. The former model favours investigations focused on lung injury and repair mechanisms, and the latter model provides the element of metabolism, through the co-culturing of lung and liver (hepatocyte) cells. These innovations represent examples of the animal-free alternatives advocated by the 21st century toxicology paradigm, whereby human-derived cell/tissue data will lead to more-accurate and more-reliable public health risk assessments and therapeutic mitigations (e.g. exposure to ambient air pollutants and adverse drug reactions) for lung disease.
Update from the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Authors: Kojima Hajime
Published: April 2019

The Japanese Center for the Validation of Alternative Methods (JaCVAM) was established in 2005 to promote the use of alternatives to animal testing in regulatory studies, thereby replacing, reducing, or refining the use of animals, according to the Three Rs principles. JaCVAM assesses the utility, limitations and suitability for use in regulatory studies, of test methods needed to determine the safety of chemicals and other materials. JaCVAM also organises and performs validation studies of new test methods, when necessary. In addition, JaCVAM co-operates and collaborates with similar organisations in related fields, both in Japan and internationally, which also enables JaCVAM to provide input during the establishment of guidelines for new alternative experimental methods. These activities help facilitate application and approval processes for the manufacture and sale of pharmaceuticals, chemicals, pesticides, and other products, as well as for revisions to standards for cosmetic products. In this manner, JaCVAM plays a leadership role in the introduction of new alternative experimental methods for regulatory acceptance in Japan.
Brain-derived Neurotrophic Factor as an Indicator of Chemical Neurotoxicity: An Animal-free CNS Cell Culture Model
Authors: Woehrling Elizabeth K. , Hill Eric J. , Nagel David , Coleman Michael D.
Published: April 2019

Recent changes to the legislation on chemicals and cosmetics testing call for a change in the paradigm regarding the current ‘whole animal’ approach for identifying chemical hazards, including the assessment of potential neurotoxins. Accordingly, since 2004, we have worked on the development of the integrated co-culture of post-mitotic, human-derived neurons and astrocytes (NT2.N/A), for use as an in vitro functional central nervous system (CNS) model. We have used it successfully to investigate indicators of neurotoxicity. For this purpose, we used NT2.N/A cells to examine the effects of acute exposure to a range of test chemicals on the cellular release of brain-derived neurotrophic factor (BDNF). It was demonstrated that the release of this protective neurotrophin into the culture medium (above that of control levels) occurred consistently in response to sub-cytotoxic levels of known neurotoxic, but not non-neurotoxic, chemicals. These increases in BDNF release were quantifiable, statistically significant, and occurred at concentrations below those at which cell death was measureable, which potentially indicates specific neurotoxicity, as opposed to general cytotoxicity. The fact that the BDNF immunoassay is non-invasive, and that NT2.N/A cells retain their functionality for a period of months, may make this system useful for repeated-dose toxicity testing, which is of particular relevance to cosmetics testing without the use of laboratory animals. In addition, the production of NT2.N/A cells without the use of animal products, such as fetal bovine serum, is being explored, to produce a fully-humanised cellular model.
The Application of QSAR Approaches to Nanoparticles
Authors: Ehret Jacques , Vijver Martina , Peijnenburg Willie
Published: April 2019

Nanoparticles (NPs) are increasingly used throughout the world for many purposes. The resulting exposure increases the relevance of efforts to assess their effects. The activities of NPs are related to many structural features, including their shape, composition and size. Applying Quantitative Structure–Activity Relationship (QSAR) methods to nanoscale systems becomes challenging, due to the lack of data and insight into the fate and effects of NPs. In this study, the possible use of QSAR methods on NPs is investigated. To this intent, several ways of representing and describing NPs were tested by using different data mining methods. The main conclusion is that QSAR methods are relevant for the study of the activity of NPs, but this should be confirmed by using larger and more diverse sets of data. Moreover, representing the constitution of NPs (in terms of core, coating and surface modification) significantly increases the prediction accuracy of the models. In our case, the most significant features to be represented were found to be the core and surface modification.
Ethical Animal use in Education and Training: From Clinical Rotations to Ethically Sourced Cadavers
Authors: Jukes Nick
Published: April 2019

The InterNICHE Policy defines the concept of ‘ethically sourced’ material to encourage replacement and enhance the acquisition of knowledge, skills and attitudes
Development of an In Silico Profiler for Respiratory Sensitisation
Authors: Enoch Steven J. , Roberts David W. , Madden Judith C. , Cronin Mark T.D.
Published: April 2019

In this article, we outline work that led the QSAR and Molecular Modelling Group at Liverpool John Moores University to be jointly awarded the 2013 Lush Science Prize. Our research focuses around the development of in silico profilers for category formation within the Adverse Outcome Pathway paradigm. The development of a well-defined chemical category allows toxicity to be predicted via read-across. This is the central approach used by the OECD QSAR Toolbox. The specific work for which we were awarded the Lush Prize was for the development of such an in silico profiler for respiratory sensitisation. The profiler was developed by an analysis of the mechanistic chemistry associated with covalent bond formation in the lung. The data analysed were collated from clinical reports of occupational asthma in humans. The impact of the development of in silico profilers on the Three Rs is also discussed.
A Campaign to End Animal Testing: Introducing the PETA International Science Consortium Ltd
Authors: Stoddart Gilly , Brown Jeffrey
Published: April 2019

The successful development and validation of non-animal techniques, or the analysis of existing data to satisfy regulatory requirements, provide no guarantee that this information will be used in place of animal experiments. In order to advocate for the replacement of animal-based testing requirements, the PETA International Science Consortium Ltd (PISC) liaises with industry, regulatory and research agencies to establish and promote clear paths to validation and regulatory use of non-animal techniques. PISC and its members use an approach that identifies, promotes and verifies the implementation of good scientific practices in place of testing on animals. Examples of how PISC and its members have applied this approach to minimise the use of animals for the Registration, Evaluation, Authorisation and Restriction of Chemicals regulation in the EU and testing of cosmetics on animals in India, are described.
The Protozoan, Paramecium primaurelia, as a Non-sentient Model to Test Laser Light Irradiation: The Effects of an 808nm Infrared Laser Diode on Cellular Respiration
Authors: Amaroli Andrea , Ravera Silvia , Parker Steven , Panfoli Isabella , Benedicenti Alberico , Benedicenti Stefano
Published: April 2019

Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. Unfortunately, conflicting literature has led to the labelling of PBM as a complementary or alternative medicine approach. However, past and ongoing clinical and research studies by reputable investigators have re-established the merits of PBM as a genuine medical therapy, and the technique has, in the last decade, seen an exponential increase in the numbers of clinical instruments available, and their applications. This resurgence has led to a clear need for appropriate experimental models to test the burgeoning laser technology being developed for medical applications. In this context, an ethical model that employs the protozoan, Paramecium primaurelia, is proposed. We studied the possibility of using the measure of oxygen consumption to test PBM by irradiation with an infrared or near-infrared laser. The results show that an 808nm infrared laser diode (1W; 64J/cm2) affects cellular respiration in P. primaurelia, inducing, in the irradiated cells, a significantly ( p < 0.05) increased oxygen consumption of about 40%. Our findings indicate that Paramecium can be an excellent tool in biological assays involving infrared and near-infrared PBM, as it combines the advantages of in vivo results with the practicality of in vitro testing. This test represents a fast, inexpensive and straightforward assay, which offers an alternative to both traditional in vivo testing and more-expensive mammalian cellular cultures.