bluuebunny/crossref_metadata_2025 huggingface dataset:
On the World Health Organization’s Neglect of the Role of Medical Doctors in Its Conception and Practice of Primary Health Care
Authors: Litsios Socrates
Published: April 2019

Primary health care (PHC) emerged in the early 1970s as WHO’s response to the failure of its basic health services approach. The Soviet Union succeeded in getting WHO’s governing bodies to agree to hold an international conference on PHC, a conference that was held in Alma-Ata, the capital of the Soviet Republic of Kazakhstan, in September 1978. In 1975, Dr. Halfdan Mahler, WHO’s charismatic director-general, introduced the goal of “health for all” (HFA) by the year 2000. Alma-Ata declared PHC as the key for achieving HFA. Although WHO had promoted the involvement of medical schools in community health, Mahler’s antimedical establishment rhetoric contributed to WHO ignoring the potential role that medical doctors could play in PHC and HFA.
Health-related life events in patients with somatic symptom disorders: A case control study
Authors: Reddy Balaswamy , Chaturvedi Santosh Kumar , Desai Geetha , Manjunatha Narayana , Guruprasad Srinivas
Published: April 2019

Background:

The positive association between adverse life events and somatoform disorders is a consistent observation. But no systematic studies have evaluated the relationship between health-related life events (HLEs) in patients with somatic symptom disorder (SSD)/somatoform disorders.

Aim:

To examine the nature and relationship of HLE in patients with SSD and to assess the correlates of HLE.

Methods:

A total of 50 adult patients with SSD and 50 matched healthy controls from a Neuropsychiatric hospital were recruited. Sociodemographic interview, scale for assessment of somatic symptoms (SASS), Patient Health Questionnaire-15 (PHQ-15) and a semi-structured interview schedule to gather information on HLE were used.

Results:

Patients in the SSD group had a significantly higher number of total HLE in general (4.72 ± 2.63, 3.36 ± 1.92; p = .004), major HLE (1.48 ± 1.45, 0.68 ± 0.94; p = .006), during their lifetime in comparison to controls. Infections, trauma, non-communicable diseases and reproductive system–related diseases were the more common types of HLE in SSD group.

Conclusion:

Our findings may implicate a role of major HLE of patients and HLE of their family members in the pathophysiology of SSD.
Extensive study on electrocution at the Bureau of Legal Medicine of Milan (1993–2017): Determination of the current mark with scanning electron microscope/energy-dispersive X-ray analysis on paraffin-embedded samples
Authors: Boracchi Michele , Domenico Luigi Crudele Graziano , Gentile Guendalina , Maciocco Francesca , Maghin Francesca , Marchesi Matteo , Muccino Enrico , Zoja Riccardo
Published: April 2019

Literature on electrocution in Italy is limited. The authors have focused their attention on the use of scanning electron microscope/energy-dispersive X-ray analysis in order to evaluate the current mark even on paraffin-embedded samples. A total of 24,104 autopsies were performed at the Section of Legal Medicine of Milan (1993–2017); all cases of death caused by electrocution (low and high voltage) were selected. We assessed a regular histological examination, a toxicological examination and detected metallisation with energy-dispersive X-ray spectroscopy analysis on paraffin-embedded tissues of typical current marks. We collected 27 high-voltage fatalities and five cases of low-voltage suicide electrocution; the technique revealed itself as sensitive. In all cases, microscopic examination highlighted the typical signs of electric current and thermal damage but the histochemical specific stainings for copper and iron gave negative results. Scanning electron microscope/energy-dispersive X-ray analysis, assessed on these same samples, even when paraffin-embedded, however, enabled us to detect these elements and differentiate between the various metal residues involved in the electrocution process. Based on their experience, authors advise the use of scanning electron microscope/energy-dispersive X-ray analysis on electrocution deaths to provide information for forensic diagnosis.
Flame behavior and mechanical properties of polyester fabrics coated with intumescent coatings via layer-by-layer assembly
Authors: Wattanatanom Warunee , Charuchinda Sireerat , Potiyaraj Pranut
Published: April 2019

Intumescent coatings consisting of branched-polyethyleneimine (BPEI) and ammonium polyphosphate (APP) solution were successfully applied on polyester fabrics by the layer-by-layer assembly technique to enhance the flame retardant and anti-dripping properties of polyester fabrics. The surface morphology of fabrics revealed that the coatings thoroughly covered the fabrics. The assessment of the vertical burning test of thin material (UL-94 rating VTM 0) indicated that the flame retardancy of coated polyester fabrics was improved significantly as compared with the uncoated fabric. The self-extinguishing ability and anti-dripping behaviors of coated polyester fabrics were also enhanced markedly. The improvement of flame retardancy depends on the number of coating layers and the amount of APP. However, after washing, the flame retardancy decreases to some extent. The morphology of char exhibits the typical structure of intumescent char on coated fabrics. The assessment of mechanical properties revealed that the intumescent coating via layer-by-layer assembly did not deteriorate the strength of the fabric. The increasing number of coated layers leads to the slight increase of the breaking force of the fabric; however, the increasing number of layers and the increasing amount of prepared APP lead to increased rigidity of the fabrics.
A study of fiber configuration in sliver fiber arrangement and its influence on sliver limit irregularity
Authors: Yang Jianping , Shen Yuanying , Zhang Hongqiang
Published: April 2019

Random fiber arrangement in slivers is the direct and intuitive factor resulting in sliver limit irregularity. In this study, by simulating the fiber arrangement in slivers, the effect of fiber configuration on sliver limit irregularity is investigated. The tracer fiber technique was employed to ascertain fiber straightness and the incidence of different fiber configurations in carded slivers. It is observed from the results that the majority of fibers are hooked in the carded sliver. It is fiber straightness rather than hook type that affects the sliver limit irregularity more; with the increase of fiber straightness, the sliver limit irregularity decreased. In addition, the simulation proposed in this study can not only portray fiber arrangement in slivers more practically, but also present sliver limit irregularity more accurately compared with the method mentioned in previous studies.
Systematic Social Observation in the Study of Civil Society Organizations
Authors: Baggetta Matthew , Bredenkamp David M.
Published: April 2019

Scholars of civil society organizations (CSOs) have long been concerned with face-to-face interactions among participants at meetings, events, and activities—what we collectively call “convenings.” Small-N ethnographies have uncovered substantial differences in the dynamics of convenings within and across CSOs. Large-N quantitative techniques, however, capture little data on convening dynamics; instead, they rely on questionable proxy measures. How much do dynamics vary across CSO convenings? New research tools are needed to answer this question. We adapt systematic social observation (SSO) to the study of CSO convenings as one such tool. SSO uses trained, independent observers and carefully constructed protocols and forms to collect detailed, standardized, quantitative data from many settings. Here, we illustrate what SSO can capture beyond conventional quantitative approaches using data from a pilot study of collegiate CSO convenings. We argue that SSO data can improve on prior quantitative findings about CSOs and extend research in new directions.
Evaluation of the Effect of Various Radiotherapy Modalities on Swallowing Function in Patients With Nasopharyngeal Cancer
Authors: Cetin Zeynep Erdogan , Eyigor Sibel , Ozturk Kerem , Akagunduz Ozlem , Turhal Goksel , Kirazli Tayfun , Esassolak Mustafa
Published: April 2019

The objective of this study was to compare the effect of curative 3-dimensional (3D) conformal radio-chemotherapy and intensity-modulated radiotherapy (IMRT) modalities on swallowing function in patients with nasopharyngeal cancer. Ten patients receiving 3D conformal radiotherapy and 10 patients receiving curative radiotherapy with IMRT, who were admitted for malignancy control for nasopharyngeal cancer, were included in the study. Swallowing functions were determined by flexible fiberoptic endoscopic evaluation. Premature spillage, retention pooling, penetration, aspiration, and reflex cough were evaluated. No statistically significant difference was found between patients receiving 3D conformal radiotherapy and IMRT regarding the scores of premature spillage, retention pooling, penetration, and aspiration with 3, 5, and 10 mL water and 5 mL yoghurt and fish crackers ( P > .05). Velopharyngeal insufficiency or delayed onset of swallowing reflex was not found in any of the patients ( P > .05). No significant difference was found between the groups in terms of the symptoms regarding subjective evaluation of swallowing ( P > .05). Swallowing function did not differ among patients receiving IMRT and 3D conformal radiotherapy. Further studies with a larger sample size are warranted in order to verify the results.
Mapping the efficiency of international scientific collaboration between cities worldwide
Authors: Csomós György , Lengyel Balázs
Published: April 2019

International scientific collaboration, a fundamental phenomenon of science, has been studied from several perspectives for decades. In the spatial aspect of science, cities have generally been considered by their publication output or by their citation impact. Only a minority of scientometric studies focus on exploring collaboration patterns of cities. In this visualisation, we go beyond the well-known approaches and map international scientific collaboration patterns of the most prominent science hubs considering both the quantity and the impact of papers produced in the collaboration. The analysis involves 245 cities and the collaboration matrix contains a total number of 7718 international collaboration links. Results show that US–Europe co-publication links are more efficient in terms of producing highly cited papers than those international links that Asian cities have built in scientific collaboration.
Boobie Miles: Failure and Friday Night Lights
Authors: Sheppard Samantha N.
Published: April 2019

This essay examines the sporting failures and racial iconicity of James “Boobie” Miles, whose athletic performance of defeat on the gridiron is chronicled in H. G. Bissinger’s bestselling nonfiction book Friday Night Lights: A Town, A Team, A Dream (1990), Peter Berg’s film adaptation Friday Night Lights (2004), and Big K.R.I.T.’s songs and music videos “Hometown Hero” from his album K.R.I.T. Wuz Here (2010) and “Boobie Miles” from his album 4eva N a Day (2012). Examining how the film displaces defeat, locating its effects and affects in the injured running back, I unpack the ways (Black) popular culture reclaims Boobie’s embodied failures on screen as a site of rhetorical agency and oppositional theorizing.
Developments in the Collection of Statistical Information on the Number of Animals used in Experiments and other Scientific Purposes in the European Union
Authors: Sauer Ursula G. , Kolar Roman
Published: April 2019

In 1999, the European Commission presented its second report on the numbers of laboratory animals used in the European Union (EU). The plausibility of the data and the usefulness of the format of the registration tables remain questionable, for reasons previously discussed in connection with the Commission's first statistical report. In addition, it is impossible to derive sound information on trends in animal use in the EU and its Member States from the second statistical report. The European Commission and the Member States have agreed on new tables to be used for future statistics on the use of experimental animals in the EU. These new tables have been significantly extended and improved. Several categories of little relevance have been revised, and ambiguous expressions have been clarified. However, several problems either persist or have been newly created. Moreover, some important data (i.e. categories for pain and distress, as well as for several specific purposes of use; the origin of some animal species; types of institutions; and the use of genetically engineered animals) are still not required. Nevertheless, these are highly relevant to animal welfare and must be regarded as indispensable for a well-aimed application of the statistics to set priorities concerning the Three Rs.
Development of a Decision Support System for the Introduction of Alternative Methods into Local Irritancy/Corrosivity Testing Strategies. Development of a Relational Database
Authors: Gerner Ingrid , Graetschel Gabriele , Kahl Jürgen , Schlede Eva
Published: April 2019

For new chemical substances that are notified within the European Union, data sets have to be submitted to the National Competent Authorities. The data submitted have to demonstrate the physicochemical and toxic properties of the new chemical, such as solubility, partition coefficients and spectra, as well as acute toxic properties and the potential to cause local irritant or corrosive effects. In order to minimise testing for notification purposes (for example, animal testing), it is necessary to develop stepwise assessment procedures, including structure-activity considerations, alternative methods (for example, in vitro tests), and computerised structure-activity relationship (SAR) models. An electronic database was developed which contains physicochemical and toxicological data on approximately 1300 chemical substances. It is used for regulatory structure-property relationship (SPR) and SAR considerations, and for the development of rules for a decision support system (DSS) for the introduction of alternative methods into local irritancy/corrosivity testing strategies. The information stored in the database is derived from proprietary data, so it is not possible to publish the data directly. Therefore, the database is evaluated by regulators, and the information derived from the data is used for the development of scientific information about SARs. This information can be published, for example, by means of tables correlating measured physicochemical values and specific toxic effects caused by the measured chemical. This information is introduced to the public by means of a DSS that predicts local irritant/corrosive potential of a chemical by listing so-called exception rules of the kind IF (physicochemical property) A THEN not (toxic) Effect B and so-called structural rules of the kind IF Substructure A THEN Effect B. These DSS rules “translate” proprietary data into scientific knowledge that can be published.
The Use of Bootstrap Resampling to Assess the Uncertainty of Cooper Statistics
Authors: Worth Andrew P. , Cronin Mark T.D.
Published: April 2019

The predictive abilities of two-group classification models (CMs) are often expressed in terms of their Cooper statistics. These statistics are often reported without any indication of their uncertainty, making it impossible to judge whether the predicted classifications are significantly better than the predictions made by a different CM, or whether the predictive performance of the CM exceeds predefined performance criteria in a statistically significant way. Bootstrap resampling routines are reported that provide a means of expressing the uncertainty associated with Cooper statistics. The usefulness of the bootstrapping routines is illustrated by constructing 95% confidence intervals for the Cooper statistics of four alternative skin-corrosivity tests (the rat skin transcutaneous electrical resistance assay, EPISKIN™, Skin2™ and CORROSITEX™), and four two-step sequences in which each in vitro test is used in combination with a physicochemical test for skin corrosion based on pH measurements.
Detection of the Embryotoxic Potential of Cyclophosphamide by Using a Combined System of Metabolic Competent Cells and Embryonic Stem Cells
Authors: Bremer Susanne , Pellizzer Cristian , Coecke Sandra , Paparella Martin , Catalani Paolo
Published: April 2019

In order to develop a method for detecting metabolism-mediated embryotoxicity, differentiating embryonic stem (ES) cells were exposed to the well-known proteratogen, cyclophosphamide (CPA). CPA was tested in a scientifically validated embryonic stem-cell test (EST), and in the newly developed reporter-gene assay for developmental cardiotoxicity. Both assays gave false-negative results. Because no metabolic competence (cytochrome P450 [CYP] activity) was found in the ES cells under the selected culture conditions, a simple biotransformation system was combined with the reporter-gene assay. As the metabolic pathway of CPA is well characterised, the genetically engineered mammalian cell line V79, transfected with CYP2B1 cDNA, was selected as a biotransformation system. CYP2B1 is responsible for transforming CPA into teratogenically active metabolites. The supernatants of genetically engineered V79 cells were analysed in the reporter-gene assay for developmental cardiotoxicity. In preliminary experiments, the combined system was able to detect the embryotoxic potential of the proteratogen, CPA.
Follow-up to the ECVAM Prevalidation Study on In Vitro Tests for Acute Skin Irritation
Authors: Zuang Valérie , Balls Michael , Botham Philip A. , Coquette Alain , Corsini Emanuela , Curren Rodger D. , Elliott Graham R. , Fentem Julia H. , Heylings Jon R. , Liebsch Manfred , Medina Jesús , Roguet Roland , van de Sandt Johannes J.M. , Wiemann Christianne , Worth Andrew P.
Published: April 2019

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm™ method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN™ and PREDISKIN™, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999–2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig-ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.
Cytotoxicity of Amino Alcohols to Rat Hepatoma-derived Fa32 Cells
Authors: Scheers Ellen M. , Forsby Anna , Dierickx Paul J.
Published: April 2019

Amino alcohols are used as emulsifying agents in dry-cleaning soaps, wax removers, cosmetics, paints and insecticides. The cytotoxicities of 12 amino alcohols, which differed in chain length, position of the amino and alcohol groups, and the presence of an additional phenyl group, were determined by the neutral red uptake inhibition assay with normally cultured, glutathione-depleted or antioxidant-enriched Fa32 rat hepatoma-derived cells. Glutathione depletion and antioxidant enrichment were achieved by including 50μM L-buthionine- S,R-sulphoximine (BSO) or 100μM α-tocopherol acetate (vitamin E) in the culture medium for 24 hours before and during the assay. The cytotoxicity of the amino alcohols observed after treatment for 24 hours was expressed as the concentration of compound needed to induce a 50% reduction in neutral red uptake (NI50). The observed NI50 values ranged from 3mM to 30mM. The individual stereoisomers and a racemic mixture of 1-amino-2-propanol exhibited similar cytotoxicities (with normally cultured Fa32 cells, and vitamin E- and BSO-treated cultures). Similar NI50 values for D-(+)-2-amino-1-propanol, 3-amino-1-propanol and the L-, D- or DL- forms of 1-amino-2-propanol, indicated that the position of the amino group had little influence on the cytotoxicities of the amino alcohols. In contrast, the position of the hydroxyl group appeared to play an important role for the toxicity of the compound, as indicated by the significantly different NI50 values for 4-amino-1-butanol and 4-amino-2-butanol. An additional phenyl group greatly increased the cytotoxicity of 2-amino-1,3-propanediol. For most of the compounds, cytotoxicity increased when GSH was depleted, and decreased when the cells were enriched with vitamin E. This indicated that most of the tested chemicals interact with GSH, either directly or indirectly, by processes which generate oxygen free-radicals. Decreased toxicity was found for most of the chemicals administered to vitamin E-enriched cells, indicating that reactive oxygen species could be involved in the toxicity of the amino alcohols.
Development of an In Vitro Test Battery for the Estimation of Acute Human Systemic Toxicity: An Outline of the EDIT Project
Authors: Clemedson Cecilia , Nordin-Andersson Marika , Bjerregaard Henning F. , Clausen Jørgen , Forsby Anna , Gustafsson Helena , Hansson Ulrika , Isomaa Boris , Jørgensen Carsten , Kolman Ada , Kotova Natalia , Krause Gunter , Kristen Udo , Kurppa Kalle , Romert Lennart , Scheers Ellen
Published: April 2019

The aim of the Evaluation-guided Development of New In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R2 = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity — to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the “missing” data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6–9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood–brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and potassium cyanide) in the MEIC in vivo–in vitro evaluation have a neurotoxic potential, it was proposed that the development within the EDIT target organ programme should initially be focused on the nervous system.
Morphology of Cell Injury: An Approach to the EDIT Programme by the Use of Tobacco Pollen Tubes
Authors: Kristen Udo , Bischoff Natalie , Lisboa Saskia , Schirmer Enno , Witt Sören , Quader Hartmut
Published: April 2019

Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubu-lin immunofluorescence and rhodamin–phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations.
Chemical Tests as Alternatives to Animal Tests in Research on Late Symptoms in Diabetes
Authors: Nielsen Mette , Clausen Jørgen
Published: April 2019

Glycation reactions, such as those seen in late diabetes, can be mimicked in purely chemical systems. The glycation is time-dependent, and in in vitro systems it may continue for days. Ascorbate seems to enhance the reactions. The reactions are associated with free-radical formation through transformation of an Amadori product to (deoxy-)glycoson, catalysed by heavy metals. Ascorbate enhances the reaction by a factor of 5–10 compared with in vitro systems without ascorbate. In vitro systems containing bovine serum albumin retard the formation of free-radicals, because of the formation of advanced glycation products.
Cryopreservation of Organotypic Brain Spheroid Cultures
Authors: Purcell Wendy M. , Atterwill Christopher K. , Xu Jinsheng
Published: April 2019

The cryopreservation of hen and rat brain spheroids was investigated. Brain spheroid cultures were prepared from 7-day-old hen embryos or 16-day-old rat embryos, by using a rotation-mediated culture system. The spheroids were cryopreserved in medium containing 5–15% dimethyl sulphoxide (DMSO) and stored in liquid nitrogen, by using a two-stage cooling procedure. The results show that the viability, as indicated by the total protein content of hen embryo brain spheroids at 24 hours, and at 3, 7 and 28 days after thawing, ranged from 45.5% to 64.2% of control values. It took 3 days for the post-thaw brain spheroids to stabilise, as indicated by their morphology and selected neural markers of functionality. These functions were maintained over a 28-day observation period. Spheroids cultured for 12–15 days in vitro before cryopreservation survived better than those that were cryopreserved after 5–7 days in vitro. The viability and biochemical functionality of spheroids after long-term (up to 6 months) storage were similar to those following short-term storage. The viability of rat brain spheroids cryopreserved in 15% DMSO, as indicated by total protein content, at 24 hours, and at 3 or 7 days after thawing, ranged from 23.1% to 32.1% of control values. This study shows for the first time that brain spheroids prepared from primary tissue can be successfully cryopreserved.
Report of a Meeting to Discuss a National Centre for the Replacement of Animals in Experiments
Authors: Brock Christine , Langley Gill , Newman Carol
Published: April 2019

Following the publication of their joint proposal for a National Centre for the Replacement of Animals in Experiments in 2002, the Dr Hadwen Trust and the Lord Dowding Fund organised a meeting, held on 18 November 2003 at Portcullis House, Westminster, in London, in order to discuss the concept further. A one-page summary of their proposal is attached as an appendix, and full copies are available from the Lord Dowding Fund and the Dr Hadwen Trust. The meeting aimed to discuss the need to stimulate and promote research to replace animal experiments by means of a National Centre (a coordinating body), and how this should be established and funded. Participants, numbering about 80 in total, included politicians (national and European), government officials, scientists, funding bodies and animal welfare representatives. This report is a summary of the issues raised by speakers and other participants at the meeting.
Ischaemia–Reperfusion Injury in the Isolated Haemoperfused Bovine Uterus: An In Vitro Model of Acute Inflammation
Authors: Braun Michael , Kietzmann Manfred
Published: April 2019

Following on from previous studies on dermal inflammation in the isolated perfused bovine udder, a new in vitro model of the isolated haemoperfused bovine uterus was established for studies on acute inflammatory reactions (for example, eicosanoid synthesis and regulation of cyclooxygenase-1 [COX-1] and COX-2) caused by ischaemia–reperfusion (I–R) injury. The organs and blood used in this study were obtained from a slaughterhouse. Within 2 hours of slaughter, uterine perfusion was re-established, by using a mixture of homologous blood and Tyrode solution (4:1). After equilibration, several deposits of arachidonic acid (5mg and 0.1mg) and arachidonylethanolamide (0.1mg) were injected into the myometrial tissue. Tissue biopsies were taken from treated and untreated areas at 180 and 300 minutes after the onset of haemoperfusion, for measuring prostaglandin E2 (PGE2) levels. In addition, the regulation of COX-1 and COX-2 mRNA was investigated by using the reverse transcriptase-polymerase chain reaction. Eicosanoid levels were determined by using an enzyme immunoassay (ELISA). Because both an increase in PGE2 concentration and up-regulation of COX mRNA were observed, the inhibitory effects of dexamethasone, added to the perfusion medium, were studied. Dexamethasone caused a significant decrease in tissue PGE2 production, but did not induce down-regulation of COX-2 mRNA. In conclusion, the isolated haemoperfused bovine uterus was introduced as an in vitro model of acute inflammation, induced by I–R injury. The suitability of the model for investigating anti-inflammatory substances was demonstrated. Use of the isolated haemoperfused bovine uterus in pharmacological research and drug screening may contribute to reducing the number of animals used for testing.
Drug Metabolism by Cultured Human Hepatocytes: How Far are we from the In Vivo Reality?
Authors: Ponsoda Xavier , Donato M. Teresa , Perez-Cataldo Gabriela , Gómez-Lechón Maria José , Castell José V.
Published: April 2019

The investigation of metabolism is an important milestone in the course of drug development. Drug metabolism is a determinant of drug pharmacokinetics variability in human beings. Fundamental to this are phenotypic differences, as well as genotypic differences, in the expression of the enzymes involved in drug metabolism. Genotypic variability is easy to identify by means of polymerase chain reaction-based or DNA chip-based methods, whereas phenotypic variability requires direct measurement of enzyme activities in liver, or, indirectly, measurement of the rate of metabolism of a given compound in vivo. There is a great deal of phenotypic variability in human beings, only a minor part being attributable to gene polymorphisms. Thus, enzyme activity measurements in a series of human livers, as well as in vivo studies with human volunteers, show that phenotypic variability is, by far, much greater than genotypic variability. In vitro models are currently used to investigate the hepatic metabolism of new compounds. Cultured human hepatocytes are considered to be the closest model to the human liver. However, the fact that hepatocytes are placed in a microenvironment that differs from that of the cells in the liver raises the question of to what extent drug metabolism variability observed in vitro actually reflects that in the liver in vivo. This issue has been examined by investigating the metabolism of the model compound, aceclofenac (an approved analgesic/anti-inflammatory drug), both in vitro and in vivo. Hepatocytes isolated from programmed liver biopsies were incubated with aceclofenac, and the metabolites formed were investigated by HPLC. The patients were given the drug during the course of clinical recovery, and the metabolites, largely present in urine, were analysed. In vitro and in vivo data from the same individual were compared. There was a good correlation between the in vitro and in vivo relative abundance of oxidised metabolites (4′-OH-aceclofenac + 4′-OH-diclofenac; Spearman's ρ = 0.855), and the hydrolysis of aceclofenac (diclofenac + 4′-OH-aceclofenac + 4′-OH-diclofenac; ρ = 0.691), while the conjugation of the drug in vitro was somewhat lower than in vivo. Globally, the metabolism of aceclofenac in vitro correlated with the amount of metabolites excreted in urine after 16 hours (ρ = 0.95). Overall, although differing among assays, the in vitro/ in vivo metabolism data for each patient were surprisingly similar. Thus, the variability observed in vitro appears to reflect genuine phenotypic variability among the donors.
The Case for Taking Account of Metabolism when Testing for Potential Endocrine Disruptors In Vitro
Authors: Combes Robert D.
Published: April 2019

Legislation in the USA, Europe and Japan will require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Such chemicals are known as endocrine disruptors (EDs), and will require extensive testing as part of the new European Union Registration, Evaluation and Authorisation of Chemicals (REACH) system for the risk assessment of chemicals. Both in vivo and in vitro tests are proposed for this purpose, and there has been much discussion and action concerning the development and validation of such tests. However, to date, little interest has been shown in incorporating metabolism into in vitro tests for EDs, in sharp contrast to other areas of toxicity testing, such as genotoxicity, and, ironically, such in vitro tests are criticised for not modelling in vivo metabolism. This is despite the existence of much information showing that endogenous and exogenous steroids are extensively metabolised by Phase I and Phase II enzymes both in the liver and in hormonally active tissues. Such metabolism can lead to the activation or detoxification of steroids and EDs. The absence of metabolism from these tests could give rise to false-positive data (due to lack of detoxification) or false-negative data (lack of activation). This paper aims to explain why in vitro assays for EDs should incorporate mammalian metabolising systems. The background to ED testing, the test methods available, and the role of mammalian metabolism in the activation and detoxification of both endogenous and exogenous steroids, are described. The available types of metabolising systems are compared, and the potential problems in incorporating metabolising systems into in vitro tests for EDs, and how these might be overcome, are discussed. It is recommended that there should be: a) an assessment of the intrinsic metabolising capacity of cell systems used in tests for EDs; b) an investigation into the relevance of using the prostaglandin H synthase system for metabolising EDs; and c) a feasibility study into the generation of genetically engineered mammalian cell lines expressing specific metabolising enzymes, which could also be used to detect EDs.
Development of a Sensory Neuronal Cell Model for the Estimation of Mild Eye Irritation
Authors: Lilja Johanna , Forsby Anna
Published: April 2019

In an attempt to improve the in vitro test strategy for the estimation of eye irritation, a neuronal cell model has been developed, with cells expressing vanilloid receptor type 1 (VR1) nociceptors. The currently accepted method for measuring eye irritancy is the ethically and scientifically criticised Draize rabbit eye test, despite the fact that alternative in vitro methods are available which have proved to be reliable and reproducible for predicting severe ocular toxicity. However, no alternative tests for measuring neuronal stimulation have yet been developed, and the prediction of eye irritation in the mild range is therefore insufficient. VR1 is a nociceptor localised in C-fibre neurons innervating the cornea and the surrounding tissue, and it responds to potentially damaging stimuli by releasing Ca2+ into the cytoplasm. As a sensory endpoint, [Ca2+]i was measured in VR1 transfected cells, as well as in control cells. Short-term cell cytotoxicity studies (cell membrane rupture and morphological divergence) were used to determine the non-corrosive concentrations of the test chemicals. Preliminary results indicated that hygiene products used daily may induce eye irritation via VR1 nociceptors. The lowest toxic concentration (0.025%) of liquid hand soap, as determined by morphologic divergences of cells, generated an 80% increase in [Ca2+]i over the basal [Ca2+]i in VR1 transfected cells, whereas the non-specific [Ca2+]i increased by 33%. Furthermore, all the endpoints studied indicated that shampoo for children was less active than shampoo for adults. If this method is successfully validated with standardised reference chemicals, the model could complete the test battery of in vitro alternatives, resulting in the saving of thousands of laboratory animals.
Oestradiol Potentiates the Effects of Certain Pyrethroid Compounds in the MCF7 Human Breast Carcinoma Cell Line
Authors: Kakko Irma , Toimela Tarja , Tähti Hanna
Published: April 2019

Pyrethroids are the most widely used insecticides for indoor pest control, so human exposure to them is common. The main target of pyrethroids is the nervous system, but their endocrine disrupting capabilities may also be of toxicological concern. In the present study, the proliferation of the breast cancer cell line, MCF7, was studied after a 7-day exposure to various concentrations of pyrethrin, permethrin and cypermethrin. The effects of oestradiol and the combined effects of oestradiol (0.10nM) and pyrethroids (0.1–100μM) on MCF7 cell proliferation were also evaluated. Proliferation and cell toxicity were studied by measuring the ATP content with a luminescence method, and mitochondrial metabolic enzyme activity with the WST-1 test. In the ATP test, low concentrations (0.1–1μM) of pyrethroids in co-exposure with oestradiol caused a clear statistically significant increase in the proliferation of MCF7 cells. This was evident when compared to the proliferative effect caused by 0.1nM oestradiol alone. High concentrations were cytotoxic, and the greatest cell toxicity was that of cypermethrin, which has a cyano group in its molecular structure.
Microinjection of Living Adherent Cells by Using a Semi-automatic Microinjection System
Authors: Viigipuu Katrin , Kallio Pasi
Published: April 2019

Testing in vitro is an alternative to animal experimentation. The capillary pressure microinjection technique is a supporting technology for efficient in vitro testing. The main benefit of the technique is the possibility of injecting large molecules into a single living cell. The ultimate goal of the research discussed in this paper is to increase the cell survival rate in capillary pressure microinjection. A method to reliably evaluate cell survival rate is therefore needed. A three-phase evaluation process is presented in this paper. The first phase determines the success rate of the injection capillary to penetrate the cell membrane. The second phase studies the success rate of delivering the injection substance inside the cell, while the third phase studies cell survival after the microinjection. In addition to the three-phase evaluation process, this paper describes the initial results of penetration and injection tests performed by using a semi-automatic capillary pressure microinjection system developed by the research group. Three adherent cell lines, namely, retinal pigment epithelial cells, MCF-7 human breast cancer cells and SH-SY5Y neuroblastoma cells, were used in the experiments. The results of the penetration tests show that the average success rate of penetrating the cell membrane using the micromanipulator was 87%. The goal of the injection tests was to demonstrate the successful microinjection of living cells and to study the injection success rate. Fluorescein dextran was injected into MCF-7 cells, and preliminary results showed an injection success rate of 49%. In the survival tests, the neuronal cells were microinjected with KCl. During long-term observation after the microinjection, the microinjected cells first decreased their adhesion to the plate, but later adhered to the bottom of the plate and even grew some dendrites. In the next phase of the study, more tests will be performed in order to obtain a statistically reliable value for the survival rate.
A Modular Approach to the ECVAM Principles on Test Validity
Authors: Hartung Thomas , Bremer Susanne , Casati Silvia , Coecke Sandra , Corvi Raffaella , Fortaner Salvador , Gribaldo Laura , Halder Marlies , Hoffmann Sebastian , Roi Annett Janusch , Prieto Pilar , Sabbioni Enrico , Scott Laurie , Worth Andrew , Zuang Valérie
Published: April 2019

The European Centre for the Validation of Alternative Methods (ECVAM) proposes to make the validation process more flexible, while maintaining its high standards. The various aspects of validation are broken down into independent modules, and the information necessary to complete each module is defined. The data required to assess test validity in an independent peer review, not the process, are thus emphasised. Once the information to satisfy all the modules is complete, the test can enter the peer-review process. In this way, the between-laboratory variability and predictive capacity of a test can be assessed independently. Thinking in terms of validity principles will broaden the applicability of the validation process to a variety of tests and procedures, including the generation of new tests, new technologies (for example, genomics, proteomics), computer-based models (for example, quantitative structure–activity relationship models), and expert systems. This proposal also aims to take into account existing information, defining this as retrospective validation, in contrast to a prospective validation study, which has been the predominant approach to date. This will permit the assessment of test validity by completing the missing information via the relevant validation procedure: prospective validation, retrospective validation, catch-up validation, or a combination of these procedures.
Scientific Procedures on Living Animals in Great Britain in 2003: The Facts, Figures and Consequences
Authors: Hudson Michelle , Bhogal Nirmala
Published: April 2019

The statistics for animal procedures performed in 2003 were recently released by the Home Office. They indicate that, for the second year running, there was a significant increase in the number of laboratory animal procedures undertaken in Great Britain. The species and genera used, the numbers of toxicology and non-toxicology procedures, and the overall trends, are described. The implications of these latest statistics are discussed with reference to key areas of interest and to the impact of existing regulations and pending legislative reforms.
Development and Prevalidation of a List of Structure–Activity Relationship Rules to be Used in Expert Systems for Prediction of the Skin-sensitising Properties of Chemicals
Authors: Gerner Ingrid , Barratt Martin D. , Zinke Stephan , Schlegel Kerstin , Schlede Eva
Published: April 2019

The new European Union (EU) chemicals policy, as described in the White Paper entitled Strategy for a Future Chemicals Policy, has identified a need for computer-based tools suitable for predicting the hazardous properties of chemicals. Two sets of structural alerts (fragments of chemical structure) for the prediction of skin sensitisation hazard classification (“R43, may cause sensitisation by skin contact”) have been drawn up, based on sensitising chemicals from a regulatory database (containing data for the EU notification of new chemicals). These alerts comprise 15 rules for chemical structures deemed to be sensitising by direct action of the chemicals with cells or proteins within the skin, and three rules for substructures that act indirectly, i.e. requiring chemical or biochemical transformation. The predictivity rates of the rules were found to be good (positive predictivity, 88%; false-positive rate, 1%; specificity, 99%; negative predictivity, 74%; false-negative rate, 80%; sensitivity, 20%). Because of the confidential nature of the regulatory database, the rules are supported by examples of sensitising chemicals taken from the “Allergenliste” now held by the Federal Institute for Risk Assessment (BfR) and the DEREK for Windows expert system. The rules were prevalidated against data not used for their development. As a result of the prevalidation study, it is proposed that the two sets of structural alerts should be taken forward for formal validation, with a view to incorporating them into regulatory guidelines.
Toward a More Humane Intracerebral Pathogenicity Index that Does Not Compromise Virus Pathogenicity Ratings
Authors: Trajstman Albert C.
Published: April 2019

Determination of the intracerebral pathogenicity index (ICPI) of a virus requires tracking sick animals until death or until the eighth day of the standard observation period. A more humane procedure would be to painlessly kill sick animals, but such intervention in the standard protocol could compromise the rating of the virus. In this paper, a modified scoring system is proposed, whereby humanely killed animals are given a score that, on average, does not alter the ICPI. The variance of an animal's contribution to the optimum modified ICPI is never greater than the variance of its contribution to the standard ICPI.
Cytotoxic and Apoptotic Effects on Hepatocytes of Secondary Metabolites Obtained from Lichens
Authors: Correché Estela Raquel , Enriz Ricardo Daniel , Piovano Marisa , Garbarino Juan , Gómez-Lechón María José
Published: April 2019

There are a large number of species of Antarctic lichens, and several studies describing the secondary metabolites present in these lichens, as well as the advances in understanding the chemistry of these metabolites, have been reported. In addition, some derivatives displaying interesting antibacterial effects have been described. The cytotoxic and apoptotic effects of 15 secondary metabolites (depsides, depsidones and usnic acid) obtained from Continental (Chilean) and Antarctic lichens were evaluated in primary cultures of rat hepatocytes. Intracellular lactate dehydrogenase release, caspase 3 activation and DNA fragmentation were measured. In this study, we have evaluated a set of markers associated with pivotal steps in the execution phase of apoptosis, in order to detect compounds with apoptotic effects on hepatocytes before significant necrosis takes place. Flow cytometric analysis of DNA fragmentation revealed an increase in apoptotic nuclei with sub-diploid DNA content after the exposure of hepatocytes to sub-cytotoxic concentrations of the compounds. Among these, salazinic acid, stictic acid and psoromic acid displayed significant apoptotic activities. Divaricatic acid showed only moderate apoptotic effects at sub-cytotoxic concentrations.