bluuebunny/crossref_metadata_2025 huggingface dataset:
International Cooperation: An Essential Requirement for Replacing Animal Toxicity Tests
Authors: Spielmann Horst
Published: April 2019

The Three Rs concept, which was developed by Russell & Burch in 1959, was implemented into the legal framework in the European Union (EU) for the protection of vertebrate animals used for experimental and other scientific purposes, when Directive 86/609/EEC was adopted in 1986. One focus of activity under this Directive is the use of animals and alternative methods in regulatory testing. To reduce or replace animal testing for regulatory purposes, non-animal tests must be independently validated to prove that they can provide information that is relevant and reliable for hazard prediction in relation to specific types of toxicity in vivo. At the end of the 1980s, no scientific concept existed for the formal validation of in vitro toxicity tests, so a small group of European and American scientists met to develop a set of principles for experimental validation, which were first adopted by ECVAM in Europe in 1995, and, after harmonisation with experts from the USA and Japan, accepted internationally by the OECD in 1996. ECVAM has directly funded a number of validation studies, and a major breakthrough in the year 2000 was the acceptance for regulatory purposes in the EU of scientifically validated in vitro toxicity tests for phototoxic potential and for skin corrosivity. These, and other examples which are discussed, confirm that the internationally harmonised ECVAM/ICCVAM/OECD validation concept is a practical and effective way of making possible the replacement of regulatory testing in animals.
In Vitro Setting of Dose–effect Relationships of 32 Metal Compounds in the Balb/3T3 Cell Line, as a Basis for Predicting their Carcinogenic Potential
Authors: Mazzotti Francesca , Sabbioni Enrico , Ponti Jessica , Ghiani Michela , Fortaner Salvador , Rossi Gian Luigi
Published: April 2019

The results are reported of the second stage in a programme for a systematic in vitro study on the carcinogenic potential of metal compounds with Balb/3T3 clone A31-1-1 mouse fibroblasts. Nineteen metal compounds that exhibited a strong cytotoxic effect during a previous screening run with a 100μM fixed dose were tested with a 72-hour exposure over a wide range of concentrations (from 0.1μM to 1000μM), to produce dose–effect curves to permit extrapolation of the 50% inhibition concentration (IC50) values for each metal compound. This allows the establishment of a suitable range of doses for individual metal species, for use in the subsequent Balb/3T3 assay based on a two-stage concurrent cytotoxicity and morphological transformation protocol. Another 13 metal compounds were also tested, to determine whether the Balb/3T3 cell transformation assay really is a valuable in vitro model in relation to the problem of metal speciation. Of the metal compounds assayed, 26 showed a dose related cytotoxic response with calculated IC50 values ranging from 0.25μM (CH3HgCl) to 140μM [(C5H5)2TiCl2], whereas six metal compounds, namely (NH4)6Mo7O24·4H2O, CH3AsO(OH)2, (CH3)2AsNaO2·3H2O, KBr, CrCl3·6H2O and (NH4)2[TiO(C2O4)2]·H2O, displayed no observable cytotoxicity or low cytotoxicity at all the doses tested. The determination of IC50 values permits a ranking of the cytotoxicity responses of metal compounds with the highest cytotoxicities. Dose–effect curves and IC50 values of different chemical forms of individual metal compounds of As, Br, Cr, Hg, Ir, Pt, Te, Ti and V (cationic/anionic inorganic or organometallic species) showed clearly how the chemical nature of the metal strongly influences the toxic response. This confirms that the Balb/3T3 cell line is a valuable in vitro model with respect to the problem of metal speciation. This is a fundamental aspect to be considered when incorporating the results from in vitro cell transformation assays of the carcinogenic potential of metal compounds into regulatory testing schemes. In this context, the choice of test metal species for the development and validation of such assays cannot disregard the possibility that humans will be exposed to specific chemical forms of individual metal compounds (different oxidation states, and inorganic or organometallic natures) that can profoundly affect their toxicity.
The Implications of Microarray Technology for Animal Use in Scientific Research
Authors: Jenkins Elizabeth S. , Broadhead Caren , Combes Robert D.
Published: April 2019

Microarray technology has the potential to affect the number of laboratory animals used, the severity of animal experiments, and the development of non-animal alternatives in several areas of scientific research. Microarrays can contain hundreds or thousands of microscopic spots of DNA, immobilised on a solid support, and their use enables global patterns of gene expression to be determined in a single experiment. This technology is being used to improve our understanding of the operation of biological systems during health and disease, and their responses to chemical insults. Although it is impossible to predict with certainty any future trends regarding animal use, microarray technology might not initially reduce animal use, as is often claimed to be the case. The accelerated pace of research as a result of the use of microarrays could increase overall animal use in basic and applied biological research, by increasing the numbers of interesting genes identified for further analysis, and the number of potential targets for drug development. Each new lead will require further evaluation in studies that could involve animals. In toxicity testing, microarray studies could lead to increases in animal studies, if further confirmatory and other studies are performed. However, before such technology can be used more extensively, several technical problems need to be overcome, and the relevance of the data to biological processes needs to be assessed. Were microarray technology to be used in the manner envisaged by its protagonists, there need to be efforts to increase the likelihood that its application will create new opportunities for reducing, refining and replacing animal use. This comment is a critical assessment of the possible implications of the application of microarray technology on animal experimentation in various research areas, and makes some recommendations for maximising the application of the Three Rs.
The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM): A Review of the ICCVAM Test Method Evaluation Process and Current International Collaborations with the European Centre for the Validation of Alternative Methods (ECVAM)
Authors: Stokes William S. , Schechtman Leonard M. , Hill Richard N.
Published: April 2019

Over the last decade, national authorities in the USA and Europe have launched initiatives to validate new and improved toxicological test methods. In the USA, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and its supporting National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), were established by the Federal Government to work with test developers and Federal agencies to facilitate the validation, review, and adoption of new scientifically sound test methods, including alternatives that can reduce, refine, and replace animal use. In Europe, the European Centre for the Validation of Alternative Methods (ECVAM) was established to conduct validation studies on alternative test methods. Despite differences in organisational structure and processes, both organisations seek to achieve the adoption and use of alternative test methods. Accordingly, both have adopted similar validation and regulatory acceptance criteria. Collaborations and processes have also evolved to facilitate the international adoption of new test methods recommended by ECVAM and ICCVAM. These collaborations involve the sharing of expertise and data for test-method workshops and independent scientific peer reviews, and the adoption of processes to expedite the consideration of test methods already reviewed by the other organisation. More recently, NICEATM and ECVAM initiated a joint international validation study on in vitro methods for assessing acute systemic toxicity. These collaborations are expected to contribute to accelerated international adoption of harmonised new test methods that will support improved public health and provide for reduced and more-humane use of laboratory animals.
Transepithelial Resistance and Inulin Permeability as Endpoints in In Vitro Nephrotoxicity Testing
Authors: Duff Tracey , Carter Simon , Feldman Gemma , McEwan Gordon , Pfaller Walter , Rhodes Pauline , Ryan Michael , Hawksworth Gabrielle
Published: April 2019

Transepithelial electrical resistance (RT) and the flux of fluorescein isothiocyanate (FITC) across Madin Darby canine kidney (MDCK) strain 1 cells and porcine epithelial kidney (LLC-PK1) monolayers were compared between three laboratories for a range of nephrotoxins. The precision of the REMS AutoSampler was similar to that of the Ussing chamber and the ENDOHM® technique, but superior to using chopstick electrodes, for measurements of resistance. The nephrotoxins used were selective for the proximal tubule, and in all cases, LLC-PK1 cells were more sensitive than MDCK cells. In most cases, change in RT was a more sensitive indicator of damage than alterations in FITC flux. The REMS system provides high intra-plate precision for RT measurements and is a higher throughput system, which is applicable to screening for nephrotoxicity in vitro.
ECVAM, ECETOC and the EU Chemicals Policy
Authors: Botham Philip A.
Published: April 2019

ECVAM's initiatives in validation have received significant support from the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), especially through the provision of reference chemical data banks, which contain peer-reviewed, high-quality in vivo data on commercially available chemical substances. Chemicals have been selected from these ECETOC data banks for validation studies on alternative methods for skin corrosion and irritation and for eye irritation and, in addition, an ECETOC task force peer-reviewed the selection and classification, on the basis of in vivo data, of chemicals used in the validation of three alternative methods for developmental toxicity. More recently, ECVAM and ECETOC have been pursuing parallel initiatives on the proposed new EU chemicals policy, with the common goals of ensuring that industry and European Commission resources are used to investigate only those chemicals that pose a significant risk to human health and the environment, and that the Policy requires that any testing that is necessary follows the Three Rs principles of reduction, refinement and replacement.
ECVAM'S Activities in the EU Candidate Countries
Authors: Sladowski Dariusz , Halder Marlies
Published: April 2019

ECVAM has been given a special grant for collaborative projects on alternative/advanced testing methods involving eleven Candidate Countries for membership of the European Union (Bulgaria, Cyprus, the Czech Republic, Estonia, Hungary, Latvia, Lithuania, Poland, Romania, Slovakia and Slovenia). The project involves the promotion of the Three Rs (reduction, refinement, replacement) concept of Russell & Burch, in cooperation with appropriate individuals and national and international organisations in the Candidate Countries themselves, and elsewhere. The scope of the programme's activities covers: conferences in some of the Candidate Countries, workshops, training courses, training visits, and technology development/transfer initiatives. A database of contacts in the Candidate Countries and in relevant institutions in other countries, is being compiled.
The EC White Paper on a Strategy for a Future Chemicals Policy
Authors: Vogelgesang Jürgen
Published: April 2019

The European Commission (EC) White Paper on a Strategy for a Future Chemicals Policy calls for the collection of adequate information about chemicals, in order to ensure their appropriate risk management. The White Paper proposes a stepwise and flexible approach to all chemicals produced in amounts above 1 tonne/year/manufacturer, including testing, if information cannot be provided by other means. The required information should be collected by the end of 2012. The EC services are currently preparing the drafts for the future chemicals legislation.
Future Activities: ECVAM and the Quality Control of Biologicals
Authors: Cussler Klaus , Halder Marlies , Hendriksen Coenraad
Published: April 2019

ECVAM's activities in the field of biologicals have contributed in many ways to the successful incorporation of Three Rs methods, as summarised elsewhere in these proceedings. The progress achieved is impressive, but large numbers of animals are still needed in order to meet the requirements stipulated by various regulations. ECVAM's activities in this area should therefore be continued and extended. Besides the well-established organisation of ECVAM workshops and contributions to conferences, further prevalidation and validation studies should be funded. In addition, studies on refinement, and training courses on validated and well-established Three Rs methods, could be initiated. There is a need for more communication and information exchange, especially between regulators and industry concerning the Three Rs. ECVAM could provide a suitable forum for such activities. An ECVAM Biologicals Task Force should be established in order to define a list of priorities.
ECVAM-ICCVAM: Prospects for Future Collaboration
Authors: Schechtman Leonard M. , Stokes William S.
Published: April 2019

The level and complexity of testing for hazard and risk assessment of marketed products and environmental agents has increased substantially over time, resulting in the use of greater numbers of both animals and humans for testing. Today, industry and regulatory bodies worldwide face increasing pressures to demonstrate responsible utilisation of laboratory animals, to limit their use, and to employ alternative non-animal tests. Institutions have also been established to identify, encourage the development of, conduct research on, and validate new, improved, and surrogate test methods that will reduce and replace animal use. Two such organisations are ECVAM and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). As the evolutionary changes occurring in the field of toxicology result in an unprecedented increase in the introduction of alternative methodologies, these will strain the capacities of such alternative-methods institutions. That realisation is causing a shift in thinking and is creating an impetus to seek approaches by which to collaborate and develop more-efficient operational procedures for the validation and regulatory acceptance of alternative methods. Similarities in objectives, functions, scientific standards, and commitment to the principles of validation and animal welfare support the value of a cooperative arrangement between ECVAM and ICCVAM, to minimise duplication of effort, maximise productivity, and influence the international adoption of alternative tests. Opportunities for ECVAM–ICCVAM collaboration are discussed, which illustrate the feasibility and potential benefits of such a partnership.
The Future of ECVAM: A Personal Perspective
Authors: Balls Michael
Published: April 2019

Progress made in the practical application of the validation process is summarised, and some of the remaining problems are considered. Highlights of the first ten years of ECVAM are reviewed, and ECVAM's activities as a route of communication on the Three Rs are discussed. Finally, some suggestions are made for maintaining ECVAM's momentum in the future, especially in relation to the challenge and opportunity for alternative methods afforded by the new EU Chemicals Policy.
The Prediction of Human Acute Systemic Toxicity by the EDIT/MEIC In Vitro Test Battery: The Importance of Protein Binding and of Partitioning into Lipids
Authors: Clemedson Cecilia , Dierickx Paul J. , Sjöström Michael
Published: April 2019

The aim of the two studies presented in this paper was to further improve the predictability of the original Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) in vitro test battery for acute systemic toxicity. In the first study, whether a protein-free cytotoxicity assay could improve the prediction of human acute systemic toxicity was investigated. The cytotoxicity of 39 MEIC reference chemicals was measured by the neutral red uptake inhibition test after 30 minutes in phosphate-buffered saline (PBS), with hepatoma-derived Fa32 cells. The results were compared with the corresponding values obtained in complete culture medium, including 10% fetal calf serum. Mercuric chloride and hexachlorophene were much more cytotoxic in PBS, as was the case, to a lesser extent, for seven other chemicals. Potassium cyanide and eight other chemicals were less cytotoxic in PBS than in complete culture medium, probably because of poor physiological conditions. The correlation between the cytotoxicity measured in PBS and human acute toxicity was rather low, but became of the same order as for other assays, when mercuric chloride and hexachlorophene were withdrawn from the comparison. In the second study, modelling of human lethal blood concentrations by using the results of the three cell line tests of the original MEIC test battery were complemented by logP (octanol–water partition coefficient) values. The introduction of logP into the modelling did not improve the correlations, but some improvement of both R2 and Q2 was obtained by expanding the logP values with logP2 values. The highest R2 (0.84) and Q2 (0.80) values were obtained for a model in which both experimental and calculated (ambiguous) logP values were used. When only experimental logP values were used, the corresponding values were 0.80 and 0.78. These two studies showed that including protein binding and the partition of chemicals in the MEIC in vitro test battery is important, in order to improve the predictability of the results obtained.
SAR Modelling of Complex Phenomena: Probing Methodological Limitations
Authors: Rosenkranz Herbert S.
Published: April 2019

The increased acceptance of the use of structure–activity relationship (SAR) approaches to toxicity modelling has necessitated an evaluation of the limitations of the methodology. In this study, the limit of the capacity of the MULTICASE SAR program to model complex biological and toxicological phenomena was assessed. It was estimated that, provided the data set consists of at least 300 chemicals, divided equally between active and inactive compounds, the program is capable of handling phenomena that are even more “complex” than those modelled up to now (for example, allergic contact dermatitis, Salmonella mutagenicity, biodegradability, inhibition of tubulin polymerisation). However, within the data sets currently used to generate SAR models, there are limits to the complexity that can be handled. This may be the situation with regard to the modelling of systemic toxicity (for example, the LD50).
Third EU Report on the Statistics on Animals Used for Experimental and Other Scientific Purposes: Trends, Problems, Conclusions
Authors: Sauer Ursula G. , Spielmann Horst , Rusche Brigitte
Published: April 2019

A brief overview is given of the data published in the third report on the number of animals used for scientific purposes in the European Union (EU), in 1999. In comparison to the second EU statistical report, the meaningfulness of the data has been greatly improved, since 14 EU Member States have now used the “EU tables”. Even though the total number of animals officially reported in the EU for 1999 (9.81 million animals) is lower than the number reported for 1996 in the second report (11.65 million animals), an increase in animal numbers was observed in several Member States. Repeatedly, the use of transgenic animals was considered to be the cause for this. Most national authorities provided some comments on the data they submitted. However, the quality of these accompanying notes varies considerably, with the comments provided by The Netherlands, Finland, Sweden, Austria, Denmark and the United Kingdom containing the most-detailed remarks on their data.
The Detection of Differentiation-inducing Chemicals by using Green Fluorescent Protein Expression in Genetically Engineered Teratocarcinoma Cells
Authors: Adler Sarah , Paparella Martin , Pellizzer Cristian , Hartung Thomas , Bremer Susanne
Published: April 2019

The murine embryonal teratocarcinoma cell line, P19, was genetically manipulated in order to provide preliminary information on compounds that induce differentiation. Without chemical induction, P19 cells remain in an undifferentiated state, but can be induced to differentiate into specific cell types. For example, dimethyl sulphoxide (DMSO) induces cardiac and skeletal muscle differentiation, whereas retinoic acid stimulates neuronal differentiation. P19 cells were transfected with a construct containing a segment of the murineTert (mTert) promoter sequence combined with the green fluorescent protein (GFP) gene, which acts as a reporter gene. mTert expression, the reverse transcriptase component of murine telomerase, is closely linked to telomerase activity and is down-regulated during differentiation. Three retinoids and DMSO induced the differentiation of P19 cells, which was determined by a reduction in mTert_GFP expression, detected by flow cytometry and confocal microscopy as independent methods of detection. A test substance, ethanol, and a control substance, saccharin, did not cause a decrease in mTert_GFP expression. In addition, it could be demonstrated that the mTert_GFP test detects developmentally relevant effects at non-cytotoxic concentrations. The ID50 values derived for the reduction of mTert_GFP expression were lower than the IC50 values detected with the MTT test, by a factor of 21.4 for all-trans retinoic acid, 12.7 for 9-cis retinoic acid, 29.6 for 13-cis retinoic acid, and 8.7 for DMSO. In comparison to the IC50 value for the P19 cell line, a similar IC50 value was obtained with 3T3 cells for ethanol, but there was a 2-fold increase for DMSO. The retinoids were not cytotoxic to 3T3 cells at the concentrations tested. This newly developed test is capable of detecting differentiation-inducing compounds at non-cytotoxic concentrations within 4 days. It offers a method for detecting chemicals with specific toxicological mechanisms, such as the retinoids, which could provide additional information in embryotoxicity testing as different promoters could be employed. Here, we report the use of this novel test system for the successful analysis of DMSO and three retinoids with different in vivo teratogenic potentials.
Increased Heat Shock Protein 70 Expression following Toxicant-mediated Cytotoxicity: A Ubiquitous Marker of Toxicant Exposure?
Authors: Farzaneh Parivash , Allameh Abdolamir , Pratt Steven , Moore Nicholas , Travis Lucy , Gottschalg Elke , Kind Clive , Fry Jeffrey
Published: April 2019

The up-regulation of heat shock protein (HSP) expression has been proposed as a general biomarker of cellular protection against various environmental stresses and chemicals. The present study investigated the possibility of using HSP70 up-regulation as a biomarker of toxicant exposure in vitro. Cells of a rat hepatoma cell line (FGC4) were exposed to concentrations of 1,3-dichloroacetone, duroquinone, diquat dibromide, menadione, hydrogen peroxide, cadmium chloride (CdCl2) and sodium (meta)arsenite (NaAsO2) that elicited 20–50% cytotoxicity over a 24-hour period, and HSP70 levels were measured by ELISA. Up-regulation of HSP70 expression was demonstrated following treatment with menadione, CdCl2 and NaAsO2, but not with the other chemicals tested. A shorter exposure time (6 hours) and/or the use of non-toxic concentrations reduced the level of HSP70 up-regulation with menadione, CdCl2 and NaAsO2, but did not uncover any up-regulation with the other chemicals. Although the toxicity of the majority of the chemicals tested is believed to involve an oxidative stress component, the results of this study clearly demonstrate that up-regulation of HSP70 expression cannot be used as a general biomarker of toxicant exposure in vitro.
The EpiDerm Test Protocol for the Upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests — An Assessment of the Performance of the Optimised Test
Authors: Kandárová Helena , Liebsch Manfred , Gerner Ingrid , Schmidt Elisabeth , Genschow Elke , Traue Dieter , Spielmann Horst
Published: April 2019

During the past decade, several validation studies have been conducted on in vitro methods for discriminating between skin irritating and non-irritating chemicals. The reconstructed human skin models, EpiDerm and EPISKIN, provided the most promising results. Based on experience of the similar performance of the two skin models, it was suggested that a common test protocol and prediction model should be developed for the prediction of skin irritation potential with the two models. When the EPISKIN protocol was applied with the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was low. In 2003, the EPISKIN protocol was further refined by extending the post-incubation period following exposure to test chemicals. This extension and additional technical improvements to the EpiDerm protocol were evaluated with 19 chemicals from the prevalidation study. With the new test design, high sensitivity (80%) and specificity (78%) were obtained. The statistical probability for correct classifications was high, so the test was considered to be ready for formal validation. However, since test optimisation had been conducted with the same test chemicals as were used in the ECVAM prevalidation study, it was decided that the optimisation of the protocol had to be verified with a new set of chemicals. Thus, in the current study, 26 additional chemicals (10 rabbit irritants and 16 non-irritants), which had previously been selected and tested by L'ORΈAL with EPISKIN, were evaluated in three independent experiments with EpiDerm. With this unbalanced testing set, a specificity of 94%, and a sensitivity of 60% were obtained, while the positive and negative predictivity and accuracy remained almost unchanged (around 80%) in comparison to the in vivo rabbit data. Overall, 45 chemicals (20 irritants and 25 non-irritants) were tested according to the final protocol. The resulting high positive (82%) and negative predictive values (79%) confirmed the reliability (accuracy of 80%) of the improved test protocol of the EpiDerm model.
The Use of Non-Human Primates in Biological and Medical Research: Evidence Submitted by FRAME to the Academy of Medical Sciences/Medical Research Council/Royal Society/Wellcome Trust Working Group
Authors: Bhogal Nirmala , Hudson Michelle , Balls Michael , Combes Robert D.
Published: April 2019

The Academy of Medical Sciences, the Medical Research Council, the Royal Society and the Wellcome Trust are undertaking a study into the use of non-human primates in biological and medical research. An independent working group of scientific experts, led by Sir David Weatherall, aims to produce a report summarising the findings of this study, early in 2006. The trends in primate research, and the nature and effects of recent and proposed changes in the global use of non-human primates in research, will be investigated. The associated ethical, welfare and regulatory issues, and the role and impact of the Three Rs principles of refinement, reduction and replacement will also be reviewed. As part of this study, a call for evidence was made. The evidence submitted by FRAME emphasised that the use of non-human primates for fundamental research or for regulatory testing still fails to take into account the fact that, although non-human primates are anatomically and physiologically similar to humans, they are not necessarily relevant models for studies on human disease or human physiology. FRAME continues to believe that we have a duty to ensure that these animals are not used without overwhelming evidence that they are the only suitable and relevant models for use in work of undeniable significance.
Attitudes in China toward the use of Animals in Laboratory Research
Authors: Davey Gareth , Wu Zhihui
Published: April 2019

Public support is a strong impetus for the adoption of alternatives to laboratory animals. It is therefore important to find out what a society thinks about ethical animal use. In the case of China, a useful line of enquiry was to survey Chinese people's views, as their country is renowned for the deplorable conditions under which animals are kept. This report concerns an investigation into the attitudes of Chinese university students toward the use of animals in laboratory research. The survey revealed a moderate concern amongst students; for example, they agreed that the use of animals for testing cosmetics and household products is unnecessary and should be stopped, and disagreed that humans have the right to use animals as they see fit. This finding is very encouraging. Further research is needed, in order to understand Chinese views about the justification of using animals in research.
β-Defensin Antibiotic Peptides in the Innate Immunity of the Buffalo: In Vivo and In Vitro Studies
Authors: Das Hemen , Swamy Naganath , Sahoo Gyanaranjan , Ahmed Shahaj Uddin , More Tukaram
Published: April 2019

β-Defensin antimicrobial peptides are multifunctional biomolecules, which are a major component of the oxygen-independent microbicidal system of buffalo polymorphonuclear (PMN) cells. They have great potential for use as proteomic biomarkers of host cell responses in the presence of microbial agents. On purifying these peptides by RP-HPLC, four defensin peptides were revealed. The results from testing against Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Candida albicans, Rinderpest Virus (RPV) and Newcastle Disease Virus (NDV), showed that the peptides possessed antimicrobial and antiviral activities. Minimum inhibitory concentration (MIC) values varied according to the peptide amounts and the exposure time. Furthermore, an increase in the levels of these cationic antimicrobial peptides was apparent in milk obtained from natural cases of mastitis, as compared to the levels in normal milk. MALDI-TOF-based amino acid sequencing confirmed the expression of two β-defensins (LAP and BNBD-2) in mastitis milk. A comparison of peptide sequences revealed that buffalo LAP and BNBD-2 share 98.6% and 100% sequence identity, respectively, with those of cattle. In vitro, Bovine Viral Diarrhoea Virus (BVDV) infection was shown to induce the expression of the β-defensin gene, as evidenced by the PCR amplification of cDNA with specific primers. The determination of the enhanced expression of β-defensin peptides in mastitis milk and in PMN cells, can be considered as an advanced approach to the assessment of cellular and molecular responses to cell injury. It is hoped that in vitro studies on phagocytes such as PMN cells and other cell lines, will eventually replace the use of animals in elucidating the roles of these cytokines in response to microbe-derived cell damage. It will also be possible to use defensins as biomarkers to correlate failure in host cell defence systems with peptide heterogeneity.
A Database of IC50 Values and Principal Component Analysis of Results from Six Basal Cytotoxicity Assays, for Use in the Modelling of the In Vivo and In Vitro Data of the EU ACuteTox Project
Authors: Clothier Richard , Dierickx Paul , Lakhanisky Thaly , Fabre Myriam , Betanzos Monica , Curren Rodger , Sjöström Michael , Raabe Hans , Bourne Nicola , Hernandez Vanessa , Mainez Jessica , Owen Monika , Watts Sarah , Anthonissen Roel
Published: April 2019

The main aim of the ACuteTox project (part of the EU 6th Framework programme) is to demonstrate that animal tests for acute systemic toxicity can be replaced by alternative in vitro assays. In this project, data for 97 reference chemicals were collected in the AcuBase database, designed to handle deposited in vitro and in vivo (human and animal) data. To demonstrate the applicability of in vitro basal cytotoxicity tests and in vitro– in vivo modelling, it was deemed necessary to obtain data that were generated via defined standard operating procedures. The molar basal cytotoxicity IC50 values (the 50% inhibitory concentrations for the endpoint measured) for a mouse fibroblast cell line (3T3), a human hepatic cell line (HepG2), a rat hepatic cell line (Fa32), and a human neutrophil cell line (HL-60), were compared, and gave an R2 correlation of 0.83. To identify chemicals that showed differential cytotoxicity to the various cell types involved, principal component analysis (PCA) was undertaken independently, once all the results had been returned. This showed that colchicine, cycloheximide, digoxin, 5-fluorouracil and hexachlorobenzene gave the lowest correlations with the first score vector of the PCA. The results presented are to be used to identify outliers that need to be further studied via the use of tissue-specific in vitro assays.
Assuring Consumer Safety without Animal Testing: A Feasibility Case Study for Skin Sensitisation
Authors: Maxwell Gavin , Aleksic Maja , Aptula Aynur , Carmichael Paul , Fentem Julia , Gilmour Nicola , MacKay Cameron , Pease Camilla , Pendlington Ruth , Reynolds Fiona , Scott Daniel , Warner Guy , Westmoreland Carl
Published: April 2019

Allergic Contact Dermatitis (ACD; chemical-induced skin sensitisation) represents a key consumer safety endpoint for the cosmetics industry. At present, animal tests (predominantly the mouse Local Lymph Node Assay) are used to generate skin sensitisation hazard data for use in consumer safety risk assessments. An animal testing ban on chemicals to be used in cosmetics will come into effect in the European Union (EU) from March 2009. This animal testing ban is also linked to an EU marketing ban on products containing any ingredients that have been subsequently tested in animals, from March 2009 or March 2013, depending on the toxicological endpoint of concern. Consequently, the testing of cosmetic ingredients in animals for their potential to induce skin sensitisation will be subject to an EU marketing ban, from March 2013 onwards. Our conceptual framework and strategy to deliver a non-animal approach to consumer safety risk assessment can be summarised as an evaluation of new technologies (e.g. ‘omics’, informatics), leading to the development of new non-animal ( in silico and in vitro) predictive models for the generation and interpretation of new forms of hazard characterisation data, followed by the development of new risk assessment approaches to integrate these new forms of data and information in the context of human exposure. Following the principles of the conceptual framework, we have been investigating existing and developing new technologies, models and approaches, in order to explore the feasibility of delivering consumer safety risk assessment decisions in the absence of new animal data. We present here our progress in implementing this conceptual framework, with the skin sensitisation endpoint used as a case study.
The Need for the Establishment of a Brazilian Centre for the Validation of Alternative Methods (BraCVAM)
Authors: Presgrave Octavio Augusto França
Published: April 2019

Brazil has recently approved legislation for regulating experimental animal use. On the other hand, it is a requirement that the safety of many products on the market is controlled on the basis of animal testing. Some groups at official laboratories, universities and industries are studying alternative methods, but there is no approved mechanism for funding collaborative studies, nor is there an institution responsible for coordinating these studies. These shortcomings obstruct the development of these assays in Brazil. The creation of a Brazilian Centre for the Validation of Alternative Methods (BraCVAM) would facilitate the development and validation of tests in all the institutions working on alternative methods in Brazil, and could also offer support to other Latin American countries.
Developmental Toxicity Testing: Protecting Future Generations?
Authors: Bailey Jarrod
Published: April 2019

A recent editorial is discussed, which implied that animal-based developmental and reproductive toxicology tests will continue to be crucial, that the thalidomide disaster could have been prevented by more animal testing, and that tests on juvenile animals would help to protect children (as developing adults) from the adverse effects of pharmaceuticals. It is argued that animal tests in these scientific areas do not provide reliable data that are predictive for human responses and, even if they did, the tests are too expensive and time-consuming for application to the very large number of substances that need to be tested. It is estimated there are already more than 100,000 man-made chemicals to which humans may be exposed on a regular basis, and it is therefore widely accepted that in vivo developmental toxicology could not possibly be used to assess all new and existing chemical substances, due to the scale of its demand upon time and resources. It is therefore imperative that alternatives such as those outlined above are embraced, further developed, accepted and used — as a matter of urgency.
Animal Studies in Spinal Cord Injury: A Systematic Review of Methylprednisolone
Authors: Akhtar Aysha Z. , Pippin John J. , Sandusky Chad B.
Published: April 2019

The objective of this study was to examine whether animal studies can reliably be used to determine the usefulness of methylprednisolone (MP) and other treatments for acute spinal cord injury (SCI) in humans. This was achieved by performing a systematic review of animal studies on the effects of MP administration on the functional outcome of acute SCI. Data were extracted from the published articles relating to: outcome; MP dosing regimen; species/strain; number of animals; methodological quality; type of injury induction; use of anaesthesia; functional scale used; and duration of follow-up. Subgroup analyses were performed, based on species or strain, injury method, MP dosing regimen, functional outcome measured, and methodological quality. Sixty-two studies were included, which involved a wide variety of animal species and strains. Overall, beneficial effects of MP administration were obtained in 34% of the studies, no effects in 58%, and mixed results in 8%. The results were inconsistent both among and within species, even when attempts were made to detect any patterns in the results through subgroup analyses. The results of this study demonstrate the barriers to the accurate prediction from animal studies of the effectiveness of MP in the treatment of acute SCI in humans. This underscores the need for the development and implementation of validated testing methods.
Welfare Phenotyping of Genetically-Modified Mice
Authors: Rose Margaret
Published: April 2019

Technologies that enable the targeted manipulation of the genome have created new opportunities to study the role and interplay of specific genes in both the regulation and function of physiological and behavioural processes and in the development of pathological conditions. Despite the potential benefits, there are ethical issues in relation to the application of these technologies, some of which relate to the impact on the welfare of the animals involved. Matters of concern include the methods involved in the derivation and production of genetically-modified (GM) animals and resulting phenotypes, where animal welfare is compromised. In the case of the latter, this may be the predicted consequence of the genetic modification, but the occurrence of unforeseen animal welfare complications is a major challenge in the management of GM animals. There has been a rapid escalation in the development of new GM lines, most of them involving mice. Databases of available lines have been developed by national and international consortia, and researchers have developed standard protocols to describe the phenotype of a new line; increasingly, such data are entered into these databases. The inclusion of animal welfare assessments with these data would provide a powerful and sophisticated tool to promote refinement. The scope, level and frequency of monitoring would facilitate the identification of unpredicted effects and the management of humane endpoints, and would identify opportunities to manage the animals so as to ameliorate negative impacts. Furthermore, by highlighting the subtleties of gene–environment interactions, such data have wider implications in achieving the goals of refinement.
The Evolution of the Three Rs
Authors: Farnaud Sebastien
Published: April 2019

Whilst the whole world is celebrating the bicentenary of the birth of Charles Darwin and the 150th anniversary of the publication of his renowned book, The Origin of Species, another anniversary should not be forgotten — the publication of The Principles of Humane Experimental Technique by W.M.S. Russell and R.L. Burch. The concomitance of the anniversaries of the two publications is not a coincidence, since, as reflected by the numerous quotes chosen by Russell from Darwin's masterpiece, numerous analogies can be found between the two works and the new ideas they describe. From a discrete birth, and after decades of struggle, the Three Rs concept can now celebrate its 50th anniversary, the result of its evolution through harsh selection and adaptation. The emergence of new types of techniques, in combination with the descent of modified old ones, testify to the undeniable change in our society toward a more efficient and more ethical science, through the progressive replacement of animal models. Both Darwin and Russell would no doubt have welcomed such progress, not only in terms of science, but also of moral values. One could also expect that, if Russell could have foreseen the incredible technological advances achieved 50 years later, where Replacement becomes a reality, as illustrated by some edifying examples, The Principles of Humane Experimental Technique would have probably been defined as the One R concept.
The MatTek Story — How the Three Rs Principles Led to 3-D Tissue Success!
Authors: Sheasgreen John , Klausner Mitch , Kandárová Helena , Ingalls David
Published: April 2019

MatTek Corporation has been working diligently for over 15 years to replace traditional animal-based toxicity and efficacy tests with alternative test methods based on human-cell derived, three-dimensional (3-D) tissue models. First discussed in detail by W.M.S. Russell and R.L. Burch 50 years ago in their book, The Principles of Humane Experimental Technique, and now fully integrated into forward-looking publications such as Toxicity Testing in the 21st Century: A Vision and a Strategy, the concept of replacing animals in test procedures with human cells and/or human cell-derived in vitro 3-D tissues is being embraced by the world's research scientists and toxicologists at an ever-increasing rate. 3-D in vitro models are being utilised not only for humanitarian reasons, but also because human 3-D tissues, in particular, produce more-physiologically relevant scientific data. Early on in MatTek's efforts to develop this alternative test method, senior management sought the assistance of experts within the in vitro testing and animal rights communities, to help define the specific in vitro human 3-D tissue products needed and navigate the regulatory landscape, especially in Europe where the replacement of animal-based testing with non-animal alternative test methods was well underway. MatTek was fortunate to receive that expert assistance on both fronts from Professor Michael Balls, who at that time was the newly-elected first director of ECVAM. In 1997, with the guidance and support of Professor Balls and others in the animal rights community, MatTek began the effort to validate several of its human 3-D tissue-based alternative test methods. Today, two MatTek human cell-derived 3-D tissue-based test methods are validated as full replacements for existing animal-based tests, with more tests in the validation pipeline. In addition, MatTek in vitro tissue models are in use worldwide by chemical, pharmaceutical and consumer product companies, as evidenced by citations in hundreds of patents and scientific articles from these industries. This article concludes with MatTek's thoughts on the direction that human 3-D tissue-based in vitro testing will take in the future.
Could Fresh Human Tissues Play a Key Role in Drug Development?
Authors: Hillier Chris , Bunton David
Published: April 2019

Biopta was founded in 2002, to provide human tissue-based drug development and testing services to the pharmaceutical industry. Although animal tissues are readily available and are relatively inexpensive, they frequently fail to faithfully predict the results seen in the clinic. Human tissues can provide integrated responses to test drugs in a manner more representative than individual cell types or cell lines alone, and more-directly relevant to the species of interest — Homo sapiens. In order to expand the use of human tissues, however, an improved infrastructure for the collection and distribution of fresh, functional tissues is highly desirable. Moreover, where there is the potential to obtain tissue from various locations, it becomes possible to test tissue that is specific to the site of drug activity. This is important, as differences may occur between the same tissue types in different locations in the body. The detection of adverse effects is greatly helped by knowledge of how existing drugs behave in the human body. These drugs can act as reference compounds, so that new compounds can then be compared, by using standard concentration–response type studies, in a huge variety of tissues, and their effects extrapolated from what is known of the reference compounds.
Improving the Design and Analysis of Animal Experiments: A Personal Odyssey
Authors: Festing Michael F.W.
Published: April 2019

Everybody's career depends on many chance factors: the people one meets, the opportunities which are available, or the state of a scientific discipline. Mine is no exception. I started out in agriculture, obtained a PhD in quantitative genetics, and spent most of my career concerned with the use of animals in biomedical research. Soon after I joined the Medical Research Council Laboratory Animals Centre in 1966, as their geneticist in charge of many species and strains of laboratory animals, I was introduced to Russell and Burch's book, The Principles of Humane Experimental Technique. It had a significant effect on my future, which has encompassed two related themes: the need for better experimental design, and the conviction that, in most research, inbred strains of rats and mice should normally be used in preference to genetically undefined outbred stocks. The establishment of the FRAME Reduction Committee has helped me to pursue both of these, although toxicologists continue to ignore basic design principles, by using outbred stocks.
FRAME — Reflections on the Future and Challenges to Success
Authors: Flower Chris
Published: April 2019

FRAME is 40 years of age and has contributed much to reasoned debate on the need to use animals in responsible scientific enquiry. It has done this through the promotion of good science as best practice, as well as promotion of the Three Rs of Reduction, Refinement and Replacement of animal experimentation. However, unless the debate widens to question society at large over its attitudes to personal risk, to accepting responsibility rather than apportioning blame, and to its simplistic adoption of regulation as the panacea, we could see the pursuit of the Three Rs degenerate into a formulaic process, leading to imperfect animal models being replaced by imperfect alternatives which would not serve to improve our science. We must not lose sight of the ultimate goal of conducting good science without the need to use animals.