bluuebunny/crossref_metadata_2025 huggingface dataset:
The Principles of Validation and the ECVAM Validation Process
Authors: Worth Andrew P. , Balls Michael
Published: April 2019

The validation of a test method is the process by which the relevance and reliability of the method are assessed for a particular purpose. It is an essential stage in the evolution of the method from its development to its acceptance and application for regulatory purposes. The principles according to which alternative tests should be validated have been agreed at an international level, although the actual process by which the validation process is conducted varies between different validation authorities. This paper summarises the principles of alternative test development and validation, and describes how the principles have been applied to the validation of in vitro tests by ECVAM.
ECVAM's Activities in Validating Alternative Tests for Skin Corrosion and Irritation
Authors: Fentem Julia H. , Botham Philip A.
Published: April 2019

ECVAM has funded and managed validation studies on in vitro tests for skin corrosion, resulting in the validities of four in vitro tests being endorsed by the ECVAM Scientific Advisory Committee: the rat skin transcutaneous electrical resistance (TER) assay, two tests based on the use of commercial reconstituted human skin equivalents, EPISKIN™ and EpiDerm™, and another commercially-produced test, CORROSITEX®. In the European Union (EU), a new test method on skin corrosion (B.40), incorporating the rat skin TER and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000, thereby making the use of in vitro alternatives for skin corrosion testing of chemicals mandatory in the EU. At the recommendation of its Skin Irritation Task Force, ECVAM has funded prevalidation studies on five in vitro tests for acute skin irritation: EpiDerm, EPISKIN, PREDISKIN™, the pig-ear test, and the mouse-skin integrity function test (SIFT). However, none of the tests met the criteria (set by the Management Team for the studies) for inclusion in a large-scale formal validation study. Thus, to date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. Following further work on the EPISKIN, EpiDerm and SIFT test protocols and/or prediction models after the completion of the prevalidation studies, it appears that the modified tests could meet the performance criteria defined for progression to a validation study. This will now be assessed independently by the ECVAM Skin Irritation Task Force, with the objective of taking a decision before the end of 2002 on whether to conduct a formal validation study.
The Current Status and Future Applicability of Quantitative Structure–activity Relationships (QSARs) in Predicting Toxicity
Authors: Cronin Mark T.D.
Published: April 2019

The current status of quantitative structure–activity relationships (QSARs) in predicting toxicity is assessed. Widespread use of these methods to predict toxicity from chemical structure is possible, both by industry to develop new compounds, and also by regulatory agencies. The current use of QSARs is restricted by the lack of suitable toxicity data available for modelling, the unsuitability of simplistic modelling approaches for the prediction of certain endpoints, and the poor definition and utilisation of the applicability domain of models. Suggestions to resolve these issues are made.
The Necessity of Biokinetic Information in the Interpretation of In Vitro Toxicity Data
Authors: Blaauboer Bas J.
Published: April 2019

Data derived from in vitro toxicity studies are not directly applicable in an assessment of the toxicity of compounds in intact organisms. The major limitation is the lack of knowledge of biokinetic behaviour in vivo. Since the toxicity of a compound will be determined by the critical concentration (or other dose metric) of the critical compound (or a metabolite thereof) at the critical site of toxic action, biokinetic behaviour must be taken into account. Possibilities of biokinetic modelling on the basis of in vitro and other non-animal data are discussed, and the application of the results in hazard and risk-assessment schedules is considered.
ECVAM's Activities on Computer Modelling and Integrated Testing
Authors: Worth Andrew P.
Published: April 2019

This paper introduces the basic concepts of quantitative structure–activity relationship (QSAR), expert system and integrated testing strategy, and explains how the analogy between QSARs and prediction models leads naturally to criteria for the validation of QSARs. ECVAM's in-house research programme on QSAR modelling and integrated testing is summarised, along with plans for the validation of QSAR models and expert system rulebases at the European Union level.
Human Hepatic Cell Cultures: In Vitro and In Vivo Drug Metabolism
Authors: Gómez-Lechón María José , Donato Teresa , Ponsoda Xavier , Castell José V.
Published: April 2019

Drug metabolism is the major determinant of drug clearance, and the factor most frequently responsible for inter-individual differences in drug pharmacokinetics. The expression of drug metabolising enzymes shows significant interspecies differences, and variability among human individuals (polymorphic or inducible enzymes) makes the accurate prediction of the metabolism of a new compound in humans difficult. Several key issues need to be addressed at the early stages of drug development to improve drug candidate selection: a) how fast the compound will be metabolised; b) what metabolites will be formed (metabolic profile); c) which enzymes are involved and to what extent; and d) whether drug metabolism will be affected directly (drug-drug interactions) or indirectly (enzyme induction) by the administered compound. Drug metabolism studies are routinely performed in laboratory animals, but they are not sufficiently accurate to predict the metabolic profiles of drugs in humans. Many of these issues can now be addressed by the use of relevant human in vitro models, which speed up the selection of new candidate drugs. Human hepatocytes are the closest in vitro model to the human liver, and they are the only model which can produce a metabolic profile of a drug which is very similar to that found in vivo. However, the use of human hepatocytes is restricted, because limited access to suitable tissue samples prevents their use in high throughput screening systems. The pharmaceutical industry has made great efforts to develop fast and reliable in vitro models to overcome these drawbacks. Comparative studies on liver microsomes and cells from animal species, including humans, are very useful for demonstrating species differences in the metabolic profile of given drug candidates, and are of great value in the judicious and justifiable selection of animal species for later pharmacokinetic and toxicological studies. Cytochrome P450 (CYP)-engineered cells (or microsomes from CYP-engineered cells, for example, Supersomes™) have made the identification of the CYPs involved in the metabolism of a drug candidate more straightforward and much easier. However, the screening of compounds acting as potential CYP inducers can only be conducted in cellular systems fully capable of transcribing and translating CYP genes.
Bridging the Gap between In Vitro and In Vivo Toxicology Testing
Authors: Ehrich Marion
Published: April 2019

In vitro systems for neurotoxicological studies can be useful for the investigation of events associated with pertubations of cellular and molecular targets that are similar to those in the intact animal. The toxicities of organophosphorus compounds, which inhibit esterases, and 1,2,3,6-tetrahydropyridine (MPTP), which depletes dopamine, can be studied in human neuroblastoma cells.
Refining In Vitro Neurotoxicity Testing — The Development of Blood–Brain Barrier Models
Authors: Tähti Hanna , Nevala Heidi , Toimela Tarja
Published: April 2019

The purpose of this paper is to review the current state of development of advanced in vitro blood–brain barrier (BBB) models. The BBB is a special capillary bed that separates the blood from the central nervous system (CNS) parenchyma. Astrocytes maintain the integrity of the BBB, and, without astrocytic contacts, isolated brain capillary endothelial cells in culture lose their barrier characteristics. Therefore, when developing in vitro BBB models, it is important to add astrocytic factors into the culture system. Recently, novel filter techniques and co-culture methods have made it possible to develop models which resemble the in vivo functions of the BBB in an effective way. With a BBB model, kinetic factors can be added into the in vitro batteries used for evaluating the neurotoxic potential of chemicals. The in vitro BBB model also represents a useful tool for the in vitro prediction of the BBB permeability of drugs, and offers the possibility to scan a large number of drugs for their potential to enter the CNS. Cultured monolayers of brain endothelial cell lines or selected epithelial cell lines, combined with astrocyte and neuron cultures, form a novel three-dimensional technique for the screening of neurotoxic compounds.
Buying or Making, What's it to Be? The Choice between Acquiring or Generating Genetically Modified Animals or Embryos in the Light of the three Rs
Authors: Cohen Nina , de Cock Buning Tjard
Published: April 2019

This paper discusses whether acquiring genetically modified animals or embryos from an existing strain, as opposed to generating a new founder, is to be preferred, with respect to animal use and animal distress. To address this question, the results of an inventory performed in 17 Dutch research institutes are presented. It is argued that the acquisition of embryos is to be preferred to the acquisition or generation of animals. Furthermore, it is argued that patenting may lead to the generation of already existing strains.
Sensory Nerves, Neurogenic Inflammation and Pain: Missing Components of Alternative Irritation Strategies? A Review and a Potential Strategy
Authors: Garle Michael J. , Fry Jeffrey R.
Published: April 2019

The eyes and skin are highly innervated by sensory nerves; stimulation of these nerves by irritants may give rise to neurogenic inflammation, leading to sensory irritation and pain. Few in vitro models of neurogenic inflammation have been described in conjunction with alternative skin and eye irritation methods, despite the fact that the sensory innervation of these organs is well-documented. To date, alternative approaches to the Draize skin and eye irritation tests have proved largely successful at classifying severe irritants, but are generally poor at discriminating between agents with mild to moderate irritant potential. We propose that the development of in vitro models for the prediction of sensory stimulation will assist in the re-classification of the irritant potential of agents that are under-predicted by current in vitro strategies. This review describes the range of xenobiotics known to cause inflammation and pain through the stimulation of sensory nerves, as well as the endogenous mediators and receptor types that are involved. In particular, it focuses on the vanilloid receptor, its activators and its regulation, as these receptors function as integrators of responses to numerous noxious stimuli. Cell culture models and ex vivo preparations that have the potential to serve as predictors of sensory irritation are also described. In addition, as readily available sensory neuron cell line models are few in number, stem cell lines (with the capacity to differentiate into sensory neurons) are explored. Finally, a preliminary strategy to enable assessment of whether incorporation of a sensory component will enhance the predictive power of current in vitro eye and skin testing strategies is proposed.
Growing Old Disgracefully: The Cosmetic Use of Botulinum Toxin
Authors: Bottrill Krys
Published: April 2019

The explosive growth in the use of botulinum toxin for cosmetic purposes has undoubtedly had an impact on the number of animals used in the potency testing of this product. The test used is a classical LD50, a severe procedure during which animals experience increasing paralysis until the occurrence of death. The enthusiastic adoption by the general public of the use of botulinum toxin as an anti-wrinkle treatment has, at least in Europe, paradoxically taken place against a background of moves to stop animal testing of cosmetics and cosmetic ingredients. There appears to be a dearth of information aimed at the public concerning botulinum toxin testing. Botulinum toxin does have important medical applications; however, the question arises whether a blanket licence for the testing can be justified, when a large proportion of the product is being used cosmetically. A further question is why death continues to be the endpoint of the potency test, when a more-humane endpoint has been proposed. In addition, a number of alternative methods have been developed, which could have the potential to replace the lethal potency test altogether. These methods are discussed in this paper, and the importance of establishing a strategy for their validation is emphasised, a need that has become even more urgent in the light of the recently published draft monograph on botulinum toxin by the European Pharmacopoeia Commission.
Platinum Toxicity and Gene Expression in Xenopus Embryos: Analysis by FETAX and Differential Display
Authors: Monetti Claudio , Bernardini Giovanni , Vigetti Davide , Prati Mariangela , Fortaner Salvador , Sabbioni Enrico , Gornati Rosalba
Published: April 2019

Since the level of platinum in the environment is destined to increase, because of its use in vehicle catalytic converters, the toxicity of platinum needs further investigation. In this study, the frog embryo teratogenesis assay– Xenopus (FETAX) was used to compare the embryotoxicity and teratogenicity of two common platinum species, (NH4)2PtCl4 and (NH4)2PtCl6. The uptake rates of the two platinum species were studied, and also their effects on the expression of genes encoding metallothionein and heat-shock protein 70, which are known to be induced by several stress factors. In addition, the differential display technique was used to search for genes that were specifically induced by platinum. A gene for the type I collagen α-chain and a novel gene were identified.
The Immunohistochemical Characterisation of an SV40-immortalised Human Corneal Epithelial Cell Line
Authors: Huhtala Anne , Nurmi Sami K. , Tähti Hanna , Salminen Lotta , Alajuuma Päivi , Rantala Immo , Helin Heikki , Uusitalo Hannu
Published: April 2019

Alternatives to the Draize rabbit eye irritation test are currently being investigated. Because of morphological and biochemical differences between the rabbit and the human eye, continuous human cell lines have been proposed for use in ocular toxicology studies. Single cell-type monolayer cultures in culture medium have been used extensively in ocular toxicology. In the present study, an SV40-immortalised human corneal epithelial (HCE) cell line was characterised immunohistochemically, by using 13 different monoclonal antibodies to cytokeratins (CKs), ranging from CK3 to CK20. The results from the monolayer HCE cell cultures were compared with those from the corneal epithelium of human corneal cryostat sections. Previous studies have shown that the morphology of the HCE cell is similar to that of primary cultured human corneal epithelial cells, and that the cells express the cornea-specific CK3. In the study reported here, we show that the cell line also expresses CKs 7, 8, 18 and 19. These CKs are typically expressed by simple epithelial cells, and are not found in the human cornea in vivo. Therefore, the monolayer HCE cell line grown in culture medium does not express the CK pattern that is typical of human corneal epithelium. This should be taken into consideration when using HCE cell cultures in similar single cell-type experiments for ocular toxicology.
A Scientific and Animal Welfare Assessment of the OECD Health Effects Test Guidelines for the Safety Testing of Chemicals under the European Union REACH System
Authors: Combes Robert D. , Gaunt Ian , Balls Michael
Published: April 2019

We have assessed each of the OECD Health Effects Test Guidelines (TGs) that were included in an annex to the Internet consultation issued by the European Commission relating to the Registration, Evaluation and Authorisation of Chemicals (REACH) legislation for the testing of new and existing chemical substances. Each guideline has been analysed with respect to its design and its scientific and animal welfare implications, the extent to which it makes use of modern techniques, and its suitability to be used in the REACH system for the testing of large numbers of chemicals. The scientific basis of the test and its justification are considered, as well as the numbers of animals required, and the potential adverse effects on them. The prospects and possibilities for applying the Three Rs ( reduction, refinement and replacement) to each of the TGs are also discussed. We have proposed an overall testing strategy for how these TGs and other methods could best be deployed for chemicals testing, should it be necessary to fill data gaps. Certain TGs have been omitted from the strategy, when we have considered them to be unnecessary for chemicals testing. A series of recommendations has been made for improving the TGs with regard to both their scientific content and ways in which they could be better designed in relation to optimising the use of the animals concerned, and minimising adverse welfare consequences to them. Our investigations show that there is an urgent need to update the TGs to reflect modern techniques and methods, and to use current approaches for applying refinement strategies to improve the scientific and animal welfare aspects of the procedures used. Improvements can and should be made in all aspects of toxicity testing, from sample preparation, and animal housing, care and feeding, to dose formulation, test material administration, and the histopathological and clinical analysis of tissue samples. Opportunities for streamlining individual assays are very limited, but testing could be made more efficient by: a) only undertaking studies that provide relevant data; b) making greater use of screens and preliminary testing; c) applying some tests simultaneously to the same animals; d) using one sex; and e) eliminating redundant tests. In conclusion, it is clear that, as they stand, the OECD Health Effects TGs are unsuitable for use in the European Union REACH system, for which potentially very large numbers of laboratory animals will be needed for the testing of a very large number of chemicals.
Validation of the Embryonic Stem Cell Test in the International ECVAM Validation Study on Three In Vitro Embryotoxicity Tests
Authors: Genschow Elke , Spielmann Horst , Scholz Gabriele , Pohl Ingeborg , Seiler Andrea , Clemann Nicole , Bremer Susanne , Becker Klaus
Published: April 2019

A detailed report is presented on the performance of the embryonic stem cell test (EST) in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the EST can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.
Validation of the Rat Limb Bud Micromass Test in the International ECVAM Validation Study on Three In Vitro Embryotoxicity Tests
Authors: Spielmann Horst , Genschow Elke , Brown Nigel A. , Piersma Aldert H. , Verhoef Aart , Spanjersberg Marielle Q.I. , Huuskonen Hannele , Paillard Francoise , Seiler Andrea
Published: April 2019

A detailed report is presented on the performance of the rat limb bud micromass (MM) test in a European Centre for the Evaluation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects on animals and/or humans, were tested in four laboratories. The outcome showed that the MM test is an experimentally validated test, which holds promise for use for identifying strongly embryotoxic chemicals, but which needs to be improved before it can be recommended for use for regulatory purposes.
The Use of Live Apes in Research in the Twenty-first Century
Authors: Hagelin Joakim
Published: April 2019

A literature-based survey was conducted on the use of live apes in research between 2000 and 2003. The 599 studies identified and considered were grouped according to area of research, taxonomy and geographic location of the work. The results suggested that behaviour/cognition, conservation and various applications related to virology (most notably, hepatitis and HIV) were the most frequent areas of research. Of the studies, 73% were classified as non-invasive, whereas 27% were classified as invasive. Among the invasive studies, 39% were scored as of mild severity, and 61% were scored as of moderate/substantial severity. Pan species were involved in 65% of the studies, Gorilla species in 15%, Pongo species in 12%, and Hylobates species in 8%. Most of the invasive research was conducted in the USA (60%). The majority of the non-invasive research was conducted in the USA (31%), Japan (13%), or in the animals’ natural habitats in Africa (35%) and Asia (8%).
Psi-Screen, an In Vitro Toxicity Test System: Applications in the Bioassay of Perfumes and Fragrance Chemicals
Authors: Griffiths David E.
Published: April 2019

The effects of 65 perfume formulations (perfume oils, perfumes, eau de parfum, eau de toilette) on mitochondrial membrane potential (ΔΨm) and mitochondrial respiration have been investigated using a mitochondria-based assay for ΔΨm, termed Psi-Screen. All the perfume formulations tested are highly active in the Psi-Screen assay, and the major site of inhibition in all cases is NADH-ubiquinone reductase (Complex I). This is confirmed in studies on the inhibition of NADH oxidase and NADH-ubiquinone reductase. Some formulations also inhibit succinate oxidation at either Complex II or Complex III. Evidence for the inhibition of mitochondrial ATPase is presented, as well as for the induction of reactive oxygen species production by perfume inhibition of Complex I. Thus, perfume formulations are multiple inhibitor mixtures which inhibit multiple bioenergetic functions at high dilutions (103 to 7 × 104). The implications of these findings are discussed with respect to cell toxicity via necrosis and/or apoptosis. Twenty candidate fragrance chemicals were investigated and all inhibited Complex I (5 at < 35μM). Mass screening strategies and high-throughput screening assays are discussed.
Municipal Sludge-induced Phytotoxicity
Authors: Srivastava Richa , Kumar Dinesh , Gupta Shrawan K.
Published: April 2019

Municipal sludge (MS), containing various types of environmental pollutants, can exert phytotoxic effects in plants. Seed germination, chlorophyll content, plant weight, root and shoot growth, and metal levels, were measured as endpoints to assess the phytotoxic effects of leachates of MS from Lucknow city in three plants, Triticum aestivum, Brassica campestris and Phaseolus aureus. Among the five metals analysed, lead was found to be present at the highest concentration, and chromium at the lowest. Aqueous MS leachates had lower amounts of metals than pure MS. Seed germination was inhibited significantly in all three plants. The greatest inhibition of root and shoot growth was observed in B. campestris and T. aestivum. Total chlorophyll was depleted in all three plants, but the maximum depletion was observed in T. aestivum exposed to 10% leachate. Symptoms such as necrosis, leaf discoloration and root curling were also observed after exposure to 10% leachate, and plant weight was reduced significantly. It is evident that MS from Lucknow city exerted phytotoxic effects on all three test plant species.
An Inter-laboratory Collaborative Study by the Non-Genotoxic Carcinogen Study Group in Japan, on a Cell Transformation Assay for Tumour Promoters Using Bhas 42 cells
Authors: Ohmori Kiyomi , Umeda Makoto , Tanaka Noriho , Takagi Hiroki , Yoshimura Isao , Sasaki Kiyoshi , Asada Shin , Sakai Ayako , Araki Harumi , Asakura Masumi , Baba Hiroshi , Fushiwaki Yuichi , Hamada Shuichi , Kitou Nobuko , Nakamura Tetsu , Nakamura Yoshiyuki , Oishi Hidetoshi , Sasaki Satoshi , Shimada Sawako , Tsuchiya Toshiyuki , Uno Yoshifumi , Washizuka Masataka , Yajima Satoshi , Yamamoto Yasuhito , Yamamura Eiji , Yatsushiro Tomoko
Published: April 2019

The Bhas promotion assay is a cell culture transformation assay designed as a sensitive and economical method for detecting the tumour-promoting activities of chemicals. In order to validate the transferability and applicability of this assay, an inter-laboratory collaborative study was conducted with the participation of 14 laboratories. After confirmation that these laboratories could obtain positive results with two tumour promoters, 12- O-tetradecanoylphorbol-13-acetate (TPA) and lithocholic acid (LCA), 12 coded chemicals were assayed. Each chemical was tested in four laboratories. For eight chemicals, all four laboratories obtained consistent results, and for two of the other four chemicals, only one of the four laboratories showed inconsistent results. Thus, the rate of consistency was high. During the study, several issues were raised, each of which were analysed step-by-step, leading to revision of the protocol of the original assay. Among these issues were the importance of careful maintenance of mother cultures and the adoption of test concentrations for toxic chemicals. In addition, it is suggested that three different types of chemicals show positive promoting activity in the assay. Those designated as T-type induced extreme growth enhancement, and included TPA, mezerein, PDD and insulin. LCA and okadaic acid belonged to the L-type category, in which transformed foci were induced at concentrations showing growth-inhibition. In contrast, M-type chemicals, progesterone, catechol and sodium saccharin, induced foci at concentrations with little or slight growth inhibition. The fact that different types of chemicals similarly induce transformed foci in the Bhas promotion assay may provide clues for elucidating mechanisms of tumour promotion.
Integrated Testing Strategies for Use in the EU REACH System
Authors: Grindon Christina , Combes Robert , Cronin Mark T.D. , Roberts David W. , Garrod John F.
Published: April 2019

Integrated testing strategies have been proposed to facilitate the process of chemicals risk assessment to fulfil the requirements of the proposed EU REACH system. Here, we present individual, decision-tree style, strategies for the eleven major toxicity endpoints of the REACH system, including human health effects and ecotoxicity. These strategies make maximum use of non-animal approaches to hazard identification, before resorting to traditional animal test methods. Each scheme: a) comprises a mixture of validated and non-validated assays (distinguished in the schemes); and b) decision points at key stages to allow the cessation of further testing, should it be possible to use the available information to classify and label and/or undertake risk assessment. The rationale and scientific justification for each of the schemes, with respect to the validation status of the tests involved and their individual advantages and limitations, will be discussed in detail in a series of future publications.
Human Tissue for In Vitro Research as an Alternative to Animal Experiments: A Charitable “Honest Broker” Model to Fulfil Ethical and Legal Regulations and to Protect Research Participants
Authors: Thasler Wolfgang E. , Schlott Thilo , Kalkuhl Arno , Plän Thomas , Irrgang Bernhard , Jauch Karl-Walter , Weiss Thomas S.
Published: April 2019

Research with human tissue offers the possibility not only of improving preclinical pharmaceutical research and safety assessment, but also of the substitution of some animal experiments. Surgically removed human tissue is discarded after pathological evaluation. This tissue would be of enormous value for research, especially in the pharmaceutical branch, if it were readily available in an ethically and legally approved manner. But there are public concerns about the use of human tissue, especially for “commercial” purposes, such as in the pharmaceutical industry. The question is whether the ethical boundaries are sufficiently respected in the course of striving for industrial profit. To overcome this problem, a clear procedure for tissue donation, collection, supply and allocation must be established, which is guaranteed to be independent of special interests. The persisting problem seems to be the lack of an authority which asks for informed consent, coordinates tissue as well as blinded data collection, and supplies research facilities with tissue samples in a transparent manner. Therefore, a charitable, state-controlled foundation acting as an “honest broker” was initiated, to cover the ethical and legal aspects, as well as to protect the research participants in their use of human tissue as an alternative to animal experiments.
An Assessment of the Role of Chimpanzees in AIDS Vaccine Research
Authors: Bailey Jarrod
Published: April 2019

Prior to Simian Immunodeficiency Virus (SIV)-infected macaques becoming the ‘model of choice’ in the 1990s, chimpanzees were widely used in AIDS vaccine research and testing. Faced with the continued failure to develop an effective human vaccine, some scientists are calling for a return to their widespread use. To assess the past and potential future contribution of chimpanzees to AIDS vaccine development, databases and published literature were systematically searched to compare the results of AIDS vaccine trials in chimpanzees with those of human clinical trials, and to determine whether the chimpanzee trials were predictive of the human response. Protective and/or therapeutic responses have been elicited in chimpanzees, via: passive antibody transfer; CD4 analogues; attenuated virus; many types and combinations of recombinant HIV proteins; DNA vaccines; recombinant adenovirus and canarypox vaccines; and many multi-component vaccines using more than one of these approaches. Immunogenicity has also been shown in chimpanzees for vaccinia-based and peptide vaccines. Protection and/or significant therapeutic effects have not been demonstrated by any vaccine to date in humans. Vaccine responses in chimpanzees and humans are highly discordant. Claims of the importance of chimpanzees in AIDS vaccine development are without foundation, and a return to the use of chimpanzees in AIDS research/vaccine development is scientifically unjustifiable.
Reconstructed Epidermis and Full-Thickness Skin for Absorption Testing: Influence of the Vehicles used on Steroid Permeation
Authors: Schäfer-Korting Monika , Mahmoud Ashraf , Borgia Simone Lombardi , Brüggener Barbara , Kleuser Burkhard , Schreiber Sylvia , Mehnert Wolfgang
Published: April 2019

A protocol for percutaneous absorption studies has been validated, based on the use of reconstructed human epidermis (RHE) and aqueous solutions of test substances. However, it is often the case that it is more-complex formulations of drugs or chemicals which will make contact with the skin surface. To investigate whether RHE and the reconstructed full-thickness skin model (FT-model) can be used to predict uptake from formulations, we compared the permeation of hydrocortisone and testosterone when applied in emulsion form and as a solution containing the penetration enhancer, ethanol. Human and pig skin and a non-cornified alveolar model served as references. The results were compared with steroid release from the formulations. The permeation rates of the steroids were ranked as: alveolar model >> RHE > FT-model, pig skin > human skin. In accordance with the rapid hydrocortisone release from the formulations, the permeation rates of this steroid exceeded those of testosterone. Only minor differences were observed when comparing the testosterone formulations, in terms of release and permeation. However, the ranking of the permeation of the hydrocortisone formulations was: solution > w/o emulsion > o/w emulsion, which permitted the elucidation of penetration enhancing effects, which is not possible with drug release studies. Differences in penetration were most obvious with native skin and reconstructed tissues, which exhibited a well-developed penetration barrier. In conclusion, RHE and skin preparations may be useful in the development of topical dermatics, and in the framework of hazard analysis of toxic compounds and their various formulations.
Home Office Licence Abstracts — An Assessment
Authors: Phillips Barry , Jennings Maggy
Published: April 2019

Since late in 2004, brief abstracts of projects licensed under the Animals (Scientific Procedures) Act 1986 have been published on the Home Office website. These are produced by the Project Licence Holders, and their publication is seen by the Home Office as contributing to greater openness and to greater public understanding of the use of animals in science, and how it is regulated. Here, we assess the value of the database for this purpose, based on an examination of the 1400 abstracts published up to January 2008. The abstracts are generally strong on justification, but often very poor at describing the likely effects on the animals used. In many cases, they lack basic information on the procedures applied, and the numbers, and even the species, of animals involved. A significant number of projects lack abstracts altogether. In order to ensure that the database gives a complete and balanced picture of animal use in research, we consider that it should be mandatory to submit an abstract, which should include at least the species and numbers of animals used, the adverse effects they are likely to experience, and the severity limits assigned to the procedures applied to them. The value of the database would also be improved greatly, if it were more readily searchable, at least by species, level of severity, and broad area of research.
Application of a Systems Biology Approach to Skin Allergy Risk Assessment
Authors: Maxwell Gavin , MacKay Cameron
Published: April 2019

We have developed an in silico model of the induction of skin sensitisation, in order to characterise and quantify the contribution of each pathway to the overall biological process. This analysis has been used to guide our research on skin sensitisation and in vitro test development programmes, and provides a theoretical rationale for the interpretation and integration of non-animal predictive data for risk assessment (RA) purposes. The in vivo mouse Local Lymph Node Assay (LLNA) is now in widespread use for the evaluation of skin sensitisation potential and potency. Recent changes in European Union (EU) legislation (i.e. the 7th Amendment to the EU Cosmetics Directive) have made the development of non-animal approaches to provide the data for skin sensitisation RA a key business need. Several in vitro predictive assays have already been developed for the prediction of skin sensitisation. However, these are based on the determination of a small number of pathways within the overall biological process, and our understanding of the relative contribution of these individual pathways to skin sensitisation induction is limited. To address this knowledge gap, a “systems biology” approach has been used to construct a computer-based mathematical model of the induction of skin sensitisation, in collaboration with Entelos, Inc. The biological mechanisms underlying the induction phase of skin sensitisation are represented by nonlinear ordinary differential equations and defined by using information from over 500 published papers. By using the model, we have identified knowledge gaps for future investigative research, and key factors that have a major influence on the induction of skin sensitisation (e.g. TNF-α production in the epidermis). The relative contribution of each of these key pathways has been assessed by determining their contributions to the overall process (e.g. sensitiser-specific T-cell proliferation in the draining lymph node). This information provides a biologically-relevant rationale for the interpretation and potential integration of diverse types of non-animal predictive data. Consequently, the Skin Sensitisation Physiolab® (SSP) platform represents one approach to integration that is likely to prove an invaluable tool for hazard evaluation in a new framework for consumer safety RA.
Technical Modification of the Balb/c 3T3 Cell Transformation Assay: The use of Serum-reduced Medium to Optimise the Practicability of the Protocol
Authors: Hayashi Kumiko , Sasaki Kiyoshi , Asada Shin , Tsuchiya Toshiyuki , Hayashi Makoto , Yoshimura Isao , Tanaka Noriho , Umeda Makoto
Published: April 2019

The two-stage Balb/c 3T3 model of cell transformation can mimic the two-stage carcinogenicity bioassay, and has been recognised as a screening method for detecting potential tumour initiators and promoters. A technical modification to the original protocol (which involved the use of M10F medium, consisting of MEM plus 10% fetal bovine serum [FBS]) has been previously proposed, in order to increase its efficacy, namely: the introduction of enriched, serum-reduced medium (DF2F medium, comprising DMEM/F12 plus 2% FBS and other supplements). The aim of this study was to further modify the protocol, so as to attain higher practicability for the assay. The protocol was further optimised by: a) reducing the number of plates required, through the use of larger plates; b) reducing the cost of the assay by retaining the reduced serum concentration and by using 2μg/ml insulin, rather than the more-complex insulin–transferrin–ethanolamine–sodium selenite (ITES) supplement (i.e. DF2F2I medium); and c) extending the culture period from 24–25 days to 31–32 days, resulting in clearer foci (the number of medium changes did not increase, as less-frequent medium changes were performed during the extended culture period). Growth curve construction revealed that variations in the saturation densities of the parental Balb/c 3T3 cell line and its three transformed clones were highest when M10F medium was replaced with DF2F2I medium just before cells reached confluence. We applied this newly-optimised protocol to the assessment of: a) the tumour initiating activity of 3-methylcholanthrene (MCA), N-methyl- N’-nitro- N-nitrosoguanidine, mitomycin C, methylmethane sulphonate, CdCl2 and phenacetin, combining a post-treatment of 100ng/ml 12- O-tetradecanoylphorbol-13-acetate at the promotion stage; and b) the tumour promoting activity of insulin, lithocholic acid, CdCl2 and phenobarbital, with pre-treatment of 0.2μg/ml MCA at the initiation stage. In the present study, only phenobarbital was negative when tested by using the modified protocol.
Planning for Reduction
Authors: Das Rose Gaines , Fry Derek , Preziosi Richard , Hudson Michelle
Published: April 2019

Reduction is one of the Three Rs which can be readily achieved in practice. This can be done by careful consideration of the experimental strategy and the implementation of good experimental design. Moreover, strategic planning leads to ‘best’ scientific practice and can have a positive impact on both refinement and replacement. The FRAME Reduction Steering Committee has developed a flow chart for an overall strategy for planning and conducting biomedical research. This, and important planning considerations for each of the steps proposed, are discussed. The strategy involves taking an initial overview and undertaking related background research, then planning a sequence of experiments expected to give satisfactory results with the least animal use and minimal severity, choosing an efficient design for each experiment in the sequence, reviewing the results of one experiment before progressing to the next, and conducting an overall analysis at the end of the programme. This approach should minimise animal use and maximise the quality of the resultant scientific output.
Biochemical Characterisation of an In Vitro Model of Hepatocellular Apoptotic Cell Death
Authors: Vinken Mathieu , Decrock Elke , De Vuyst Elke , Leybaert Luc , Vanhaecke Tamara , Rogiers Vera
Published: April 2019

This study was set up to critically evaluate a commonly-used in vitro model of hepatocellular apoptotic cell death, in which freshly isolated hepatocytes, cultured in a monolayer configuration, are exposed to a combination of Fas ligand and cycloheximide for six hours. A set of well-acknowledged cell death markers was addressed: a) cell morphology was studied by light microscopy; b) apoptotic and necrotic cell populations were quantified by in situ staining with Annexin-V, Hoechst 33342 and propidium iodide (PI); c) apoptotic and necrotic activities were monitored by probing caspase 3-like activity and measuring the extracellular leakage of lactate dehydrogenase (LDH), respectively; and d) the expression of apoptosis regulators was investigated by immunoblotting. The initiation of apoptosis was evidenced by the activation of caspase 8 and caspase 9, and increased Annexin-V reactivity. Progression through the apoptotic process was confirmed by the activation of caspase 3 and Bid, the enhanced expression of Bax, and the occurrence of nuclear fragmentation. Late transition to a necrotic appearance was demonstrated by an increased number of PI-positive cells and augmented extracellular release of LDH. Thus, the in vitro model allows the study of the entire course of Fas-mediated hepatocellular apoptotic cell death, which is not possible in vivo. This experimental system can serve a broad range of in vitro pharmaco-toxicological purposes, thereby directly assisting in the reduction of animal experimentation.
The Progress of Humane Experimental Technique since 1959: A Personal View
Authors: Burch Rex L.
Published: April 2019

This is very much a personal interpretation of the Three Rs and the efforts by scientists connected with the more humane use of animals. It is intended to illustrate that scientists are given a job to do and, if it involves animals, they have little choice but to use them. It is also intended to illustrate that so many of those so involved are the very people who have made gigantic efforts in finding ways of replacing, reducing and/or refining techniques which require animals.
Non-Animal Approaches for Consumer Safety Risk Assessments: Unilever's Scientific Research Programme
Authors: Carmichael Paul , Davies Michael , Dent Matt , Fentem Julia , Fletcher Samantha , Gilmour Nicola , MacKay Cameron , Maxwell Gavin , Merolla Leona , Pease Camilla , Reynolds Fiona , Westmoreland Carl
Published: April 2019

Non-animal based approaches to risk assessment are now routinely used for assuring consumer safety for some endpoints (such as skin irritation) following considerable investment in developing and applying new methods over the past 20 years. Unilever's research programme into non-animal approaches for safety assessment is currently focused on the application of new technologies to risk assessments in the areas of skin allergy, cancer and general toxicity (including inhalation toxicity). In all of these areas, a long-term investment is essential to increase the scientific understanding of the underlying biological and chemical processes that we believe will ultimately form a sound basis for novel risk assessment approaches. Our research programme in these priority areas consists of in-house research as well as Unilever-sponsored academic research, involvement with EU-funded projects (e.g. Sens-it-iv, carcino-GENOMICS), participation in cross-industry collaborative research (e.g. COLIPA, EPAA) and ongoing involvement with other scientific initiatives on non-animal approaches to risk assessment (e.g. UK NC3Rs, US ‘Human Toxicology Project’ consortium).
An In Vitro Perfusion System to Examine the Responses of Endothelial Cells to Simulated Flow and Inflammatory Stimulation
Authors: Cockcroft Natalia Y. , Oke Oluwatobiloba , Cunningham Fiona , Bishop Emma , Fearon Ian M. , Zantl Roman , Gaça Marianna D.
Published: April 2019

Atherosclerosis is a disease process which develops at the arterial branches and curvatures of medium to large arteries. Local haemodynamic flow patterns in these vessels play an essential role in the formation of atherosclerotic lesions. To simulate pro-atherogenic blood flow patterns, we have developed a perfusion system with the ability to simulate in vivo patterns of blood flow in vitro. In this system, human umbilical vein endothelial cells were seeded in y-shaped microslides, in which they were exposed to a variety of flow patterns. Besides being able to reproduce the disturbed flow involved in the development of pro-atherogenic events within the arterial wall, the system also permitted the assessment of the pre-conditioning/priming effect of oscillatory flow on endothelial cells. The system was further capable of integrating multi-endpoint assays relevant to cardiovascular disease. We show that oscillatory flow primed endothelial cells, making them more sensitive to subsequent treatments. The treatment of oscillatory flow-primed cells with TNFα resulted in the detection of enhanced levels of pro-inflammatory and chemoattractant factors such as IL-8 and MCP-1. These measurements were facilitated by the small volumes of medium circulating within the perfusion system. Oscillatory flow also altered the characteristics of mono-cyte adhesion to the endothelial layer. In summary, this system allows the monitoring of multiple endpoints and biomarkers, and provides an alternative to the use of in vivo and ex vivo models of cardiovascular disease.
De-Risking Drug Discovery with ADDME — Avoiding Drug Development Mistakes Early
Authors: Tsaioun Katya , Jacewicz Mary
Published: April 2019

The advent of early Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) screening has increased the elimination rate of weak drug candidates early in the drug-discovery process, and decreased the proportion of compounds failing in clinical trials for ADMET reasons. This paper reviews the history of ADMET screening and why it has become so important in drug discovery and development. Assays that have been developed in response to specific needs, and improvements in technology that result in higher throughput and greater accuracy of prediction of human mechanisms of toxicity, are discussed. The paper concludes with the authors’ forecast of new models that will better predict human efficacy and toxicity.
What Kind of Animal is the “Three Rs”?
Authors: Hobson-West Pru
Published: April 2019

Fifty years after the publication of The Principles of Humane Experimental Technique by Russell and Burch, this paper explores the contemporary role of the Three Rs. This is illustrated by reference to a recent social scientific study, which involved a total of 50 in-depth interviews with scientists who use animals and with other stakeholders in the debate. The data analysis shows how the Three Rs are conceptualised in at least three ways: firstly, as an ethical animal, either as a shorthand for a moral imperative, or as a route to managing an ethical dilemma; secondly, as a scientific animal, internal to the scientific method; and finally, as a political animal, with some stakeholders referring to the Three Rs as a way to promote consensus in a controversial domain. Pushing the metaphor a little further, the paper concludes that the Three Rs concept has become a kind of hybrid animal.
Raising the Voice of Reason
Authors: Jeffery Anne
Published: April 2019

In the current economic climate, even the best known charities are facing reduced income and need to raise their profiles to ensure continued support. FRAME has always been well known among scientific organisations, but is less familiar to the public. The traditional sources of funding are drying up, and FRAME needs to promote itself to new audiences, if it is to succeed for another 40 years.