bluuebunny/crossref_metadata_2025 huggingface dataset:
Parathyroid Computed Tomography Angiography: Early Experience with a Novel Imaging Technique in Primary Hyperparathyroidism
Authors: Schwartz Isaac E. , Capra Gregory G. , Mullin David P. , Johnson Terence E. , Boswell Gilbert E.
Published: April 2019

Objectives

To describe parathyroid computed tomography angiography (PCTA), determine its accuracy, and, as a secondary objective, calculate its mean radiation dosimetry.

Study Design

Retrospective chart review of patients who underwent parathyroidectomy for primary hyperparathyroidism from 2007 to 2015.

Setting

Single‐center tertiary care academic military hospital.

Subjects and Methods

PCTA is a 2‐phase computed tomography imaging technique that uses individualized timing of contrast infusion and novel patient positioning to accurately identify parathyroid adenomas. Consecutive patients who underwent parathyroidectomy for primary hyperparathyroidism from 2007 to 2015 were reviewed; 55% of patients were women. The mean age was 50.9 years (range, 26‐68 years). Sensitivity and specificity were calculated as well as mean radiation dosimetry and timing of contrast.

Results

A total of 108 procedures were performed during the study period. Twenty‐one patients undergoing 22 PCTAs after prior sestamibi scans were nonlocalizing or equivocal. In this group, there were 15 true‐positive, 3 false‐positive, 4 true‐negative, and 0 false‐negative PCTAs. This represents a sensitivity of 100% (95% CI, 74.7%‐100%) and a specificity of 57% (95% CI, 20%‐88%). The mean calculated radiation dose was 5.15 mSv. In the most recent studies, a mean dose of 4.1 mSv was calculated. The ideal time of image acquisition contrast administration varied from 20 to 30 seconds after contrast infusion.

Conclusions

PCTA is a new technique in anatomic imaging for hyperparathyroidism. In a single‐center, single‐radiologist retrospective study, it demonstrates excellent accuracy for patients with parathyroid adenomas that are otherwise difficult to localize preoperatively. Preliminary experience suggests that its use may be indicated as a primary imaging modality in the future.
Immigration and Expulsion of ECOWAS Aliens in Nigeria
Authors: Afolayan A.A.
Published: April 2019

The immigration of ECOWAS citizens into Nigeria gathered momentum after the protocol on the free movement of goods, capital and people was ratified in 1980. This period coincided with the era of oil-boom in the country. However, the influx was disrupted by the expulsion orders of January 1983 and April 1985. It is suggested here that even though the protocol is a laudable objective, the spatial mobility needs to be well monitored and the sovereignty of the state clearly delineated.
Minority Practices and Principles of Toleration
Authors: Parekh Bhikhu
Published: April 2019

In a multicultural society, different groups entertain different conceptions of the good life. The wider society cannot be expected indiscriminately to tolerate all kinds of minority practices, nor can it justifiably ban all practices that diverge from its own. This raises the question as to how it should decide which practices to tolerate and which ones to ban. The appeal to such principles as the society's fundamental values, human rights, and no-harm runs into difficulties. This paper suggests that the principles of toleration cannot be laid down in advance, and are best elicited by means of an open-minded intercommunal dialogue aimed at evolving a reasonable consensus.
Effects on Detoxification Enzymes in Different Life Stages of Honey Bees (Apis mellifera L., Hymenoptera: Apidae) Treated with a Synthetic Pyrethroid (Flumethrin)
Authors: Nielsen Søren Achim , Brødsgaard Camilla Juul , Hansen Henrik
Published: April 2019

The activities of two detoxification enzymes, glutathione S-transferase (GST) with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, and glutathione peroxidase with tert-butyl hydroperoxide as substrate (GSH-Px[TBH]), were measured in the larvae, pupae and adults of honey bees ( Apis mellifera ligustica Spinola) originating from two colonies, one untreated acting as a control group and the other treated with flumethrin. The treatment with flumethrin led to increased GST activity in the larval instars, pupae and nurse bees compared with the control group. In particular, the late fifth larval instars (LS) and the pupal instars showed increased GST activities. GSH-Px[TBH] activities were highest in the early larval instars (L1–L2). Treatment with flumethrin resulted in the induction of GSH-Px[TBH] activity in the L4–L5 instars and LS. In the forager bees, the enzyme activity was lower in the group treated with flumethrin than in the control group. This could have been due to unknown extrinsic environmental factors. In general, the average protein contents were lower in the flumethrin-treated group than in the control group. In the LS and pupal stage, and in the adult worker bees, the differences were significantly lower. This inhibition of growth could be due to metabolic costs resulting from exposure to toxicants.
Retinal Müller Cell Culture
Authors: Toimela Tarja , Mäenpää Hanna , Tähti Hanna
Published: April 2019

A mini-review is presented of the current techniques for maintaining Müller cells in a culture. Within the retina, Müller cells are the predominant glial cells. These highly specialised cells extend over the entire neural retina. One of the most important of the various physiological functions of Müller cells is to regulate the balance of ions and neurotransmitters in the retina. Disturbance of these regulatory functions may lead to toxic effects on receptor and other neural cells in the neuroretina, and may be a common mechanism of clinical retinal neuropathy. The main excitatory neurotransmitter in the retina is glutamate. Müller cells regulate the amount of glutamate in the synaptic regions of the neural network in the retina. Accumulation of extra glutamate seems to be an important mechanism for initiating pathological changes leading to retinal damage. Many previous in vitro studies on the role of Müller cells in retinal toxicology have been based on the use of morphological and histochemical methods. In cell toxicology studies, it is important to develop culture techniques able to provide more cells for biochemical determinations.
Structural Basis of the Toxicity of Chemicals in Cultured Human HeLa Cells
Authors: Zhu Xinyu , Rosenkranz Herbert S.
Published: April 2019

A coherent structure-activity relationship (SAR) model was derived from data based on the toxicity of chemicals in cultured human HeLa cells. The ability to construct such a model indicates that toxicity is a function of a finite number of mechanisms. A comparison of the HeLa SAR model with models that describe other toxicological phenomena demonstrated significant mechanistic similarities with systemic toxicity in rodents and ocular irritation in rabbits. Overall, the present analyses support the hypothesis that the inclusion of a battery of cell toxicity assays together with toxicokinetic models will result in a more predictive model of systemic toxicity in humans.
A Computer-Based Structure-Activity Relationship Method for Predicting the Toxic Effects of Organic Chemicals from One-dimensional Representations of their Molecular Structures
Authors: Zinke Stephan , Gerner Ingrid
Published: April 2019

A computer-based method is presented for the analysis and interpretation of structural formulae characterising chemical molecules. This method was developed to enable a computer to identify substructures of chemical molecules that are relevant in the context of specific toxicological questions. The new computer-based structure-examination method was used to develop the “structure” parts of several electronic structure-activity relationship models (SAR models) for analysing and interpreting the structural formula of a chemical from its one-dimensional representation, by applying recursive principles and identifying partial isomorphic graphs. The structure-examination method is designed as part of an open-endpoint procedure to be used in expert systems, and could be applied in the construction of SAR models for almost all toxicological endpoints. The system was satisfactorily tested by identifying substructures relevant to severely damaging effects on skin and eyes.
Development of a Decision Support System for the Introduction of Alternative Methods into Local Irritancy/Corrosivity Testing Strategies. Creation of Fundamental Rules for a Decision Support System
Authors: Gerner Ingrid , Zinke Stephan , Graetschel Gabriele , Schlede Eva
Published: April 2019

The notification procedure of the European Union (EU) for new chemicals requires the application of protocols on physicochemical and toxicological tests for the evaluation of physicochemical properties and probable toxic effects of each notified substance. A computerised database was developed from data sets and toxicological test protocols relating to substance properties responsible for skin and eye irritation/corrosion. To develop specific structure-activity relationship (SAR) models and to find rules for a decision support system (DSS) to predict local irritation/corrosion, physical property data, chemical structure data and toxicological data for approximately 1300 chemicals, each having a purity of 95% or more, were evaluated. The evaluation demonstrated that the lipid solubility and aqueous solubility of a chemical are relevant to, or — in some cases — responsible for, the observed local effects of a substance on the skins and eyes of rabbits. The octanol/water partition coefficient and the measured value of the surface tension of a saturated aqueous solution of the substance give additional information that permits the definition of detailed SAR algorithms that use measured solubility values. Data on melting points and vapour pressure can be used to assess the intensity and duration of local contact with a chemical. Considerations relating to the reactivity of a pure chemical can be based on molecular weight and the nature of the heteroatoms present. With respect to local lesions produced following contact with the skin and eyes of rabbits, the data evaluation revealed that no general “local irritation/corrosion potential” of a chemical can be defined. A variety of mechanisms are responsible for the formation of local lesions on the skin or in the eyes: serious lesions are produced by mechanisms different from those that cause moderate irritation in these organs. In order to develop a DSS that uses the information extracted from the database, chemical main groups were categorised on the basis of their empirical formulae, and rules were defined of the type IF (physicochemical property) A, THEN not (toxic) effect B, based on correlations between specific local effects and measured physicochemical values. Other rules of the type IF substructure A, THEN effect B were developed based on correlations between specific local effects and the submitted structural formulae. Reactive chemical substructures relevant to the formation of local lesions and rules for the prediction of the absence of any skin irritation potential were identified. Proposals are made relating to the development of alternatives to eye irritation testing with rabbits.
Application of Modelling Techniques to the Planning of In Vitro Arsenic Kinetic Studies
Authors: Kenyon Elaina M. , Fea Michael , Styblo Mirek , Evans Marina V.
Published: April 2019

A kinetic model describing the hepatic methylation of arsenite ([As[III]) was developed on the basis of limited data from in vitro mechanistic studies. The model structure is as follows: sequential enzymic methylation of arsenite to its monomethylated (MMA) and dimethylated (DMA) products by first-order and Michaelis-Menten kinetics, respectively; uncompetitive inhibition of the formation of DMA by As(III); and first-order reversible binding of As(III), MMA and DMA to cytosolic proteins. Numerical sensitivity analysis was used to evaluate systematically the impact of changes in input parameters on model responses. Sensitivity analysis was used to investigate the possibility of designing experiments for robust testing of the uncompetitive inhibition hypothesis, and for further refining the model. Based on the sensitivity analysis, the MMA concentration is the most important response on which to focus. The parameters Vmax and ki can be reliably estimated by using the same concentration time-course data at intermediate initial arsenite concentrations of 1–5μM at 30 ± 5 minutes. Km must be estimated independently of Vmax, since the two parameters are highly correlated at all times, and the optimal experimental conditions would include lower initial concentrations of arsenite (0.1–0.5μM) and earlier time-points (about 8–18 minutes). The use of initial arsenite concentrations much above 5μM would not yield additional useful information, because the sensitivity coefficients for MMA, protein-bound MMA, DMA and protein-bound DMA tend to become extremely small or exhibit erratic trends. Overall trends in the sensitivity analysis indicated the desirability of performing measurements at times shorter than 60 minutes. This work demonstrates that physiological modelling and sensitivity analysis can be efficient tools for experimental planning and hypothesis testing when applied in the earliest phases of kinetic model development, thus allowing more-efficient and more-directed experimentation, and minimising the use of laboratory animals.
Collagen Type I Gel Cultures of Adult Rat Hepatocytes as a Screening Induction Model for Cytochrome P450-dependent Enzymes
Authors: De Smet Karen , Cavin Christophe , Vercruysse Antoine , Rogiers Vera
Published: April 2019

Albumin secretion, expression of cytochrome P450-dependent mono-oxygenases (CYPs) and their inducibility by well-known inducers were evaluated during 1 week in collagen type I gel sandwich and immobilisation cultures of adult primary rat hepatocytes. Albumin secretion increased during culture and, following an initial decrease, CYP biotransformation activities remained stable for at least 7 days. Better preservation results were observed in the collagen gel sandwich culture than in the immobilisation model. The inducibility of CYPs by β-naphthoflavone (β-NF), 3-methylcholanthrene (3-MC), phenobarbital (PB) and dexamethasone (DEX) was studied in both collagen gel hepatocyte cultures. Exposure of the cells to either 5μM 3-MC or 25μM β-NF, added to the culture medium, resulted in strong increases of CYP1A1/2 activity in both culture models. Treatment with PB (3.2mM) resulted in an increase in the CYP2B activity and a higher hydroxylation of testosterone in the 16α-position (CYP2B1/2 and CYP2C11), the 7α-position (CYP2A1/2), and the 6β-position (CYP3A1). DEX (10μM) markedly increased testosterone 6β- and 7α-hydroxylation. Expression and induction experiments on CYP proteins exposed to these molecules confirmed the results of the CYP activity measurements. The patterns of CYP induction in collagen gel cultures of rat hepatocytes were similar to those observed in vivo. Consequently, collagen gel cultures and, more specifically, collagen gel sandwich cultures seem to be suitable as in vitro models for evaluating xenobiotics as potential inducers of CYP-enzymes.
The Red Blood Cell Phototoxicity Test (Photohaemolysis and Haemoglobin Oxidation): EU/COLIPA Validation Programme on Phototoxicity (Phase II)
Authors: Pape Wolfgang J.W. , Maurer Thomas , Pfannenbecker Uwe , Steiling Winfried
Published: April 2019

In the EU/COLIPA validation programme on “Photoirritation in vitro”, two core tests and a number of mechanistically based tests were carried out to examine their suitability as regulatory tests for phototoxicity testing. In the meantime, one core test, the 3T3 neutral red uptake phototoxicity test (NRU PT) has been validated and has been accepted by ECVAM and the European Commission. The second core test, the red blood cell phototoxicity test (Photo-RBC test), has passed through a prevalidation process during this programme. This test protocol combines two endpoints, photohaemolysis and met-haemoglobin (met-Hb) formation. These endpoints are determined by measuring changes in the optical density of the haemoglobin spectrum at 525nm and 630nm, respectively. In addition, a prediction model was inserted into the Standard Operating Procedure (SOP) with two cut-off values: a photohaemolysis factor (PHF) ≥ 3.0 for photohaemolysis, and a ΔODmax ≥ 0.05 for met-Hb formation. Three laboratories agreed to implement the SOP and to perform the study by testing 30 selected test chemicals (25 phototoxicants and 5 non-phototoxic chemicals). The outcome of the study presents a good overall fit, including acceptable accuracy, sensitivity, and positive predictivity. The specificity and the negative predictivity are comparably low, due to the low number of non-phototoxic substances among the test chemicals. Further analysis of the data showed that the transfer of the SOP from between laboratories could have been more efficient. The results, especially of the lead laboratory, clearly indicate that an experienced laboratory can handle the SOP with high predictivity for phototoxicants and non-phototoxic substances. Finally, it was concluded that the combined Photo-RBC test can be considered as a second in vitro test, which can be used advantageously to obtain some mechanistic information, in particular on photodynamic effects on cellular proteins and biomembranes.
Uniform Expression of Alcohol Dehydrogenase 3 in Epithelia Regenerated with Cultured Normal, Immortalised and Malignant Human Oral Keratinocytes
Authors: Hedberg Jesper J. , Hansson Annette , Nilsson Jan A. , Höög Jan-Olov , Grafström Roland C.
Published: April 2019

The human oral epithelium is a target for damage from the inhalation of formaldehyde. However, most experimental studies on this chemical have relied on laboratory animals that are obligatory nose breathers, including rats and mice. Therefore, in vitro model systems that mimic the structure of the human oral epithelium and which retain normal tissue-specific metabolic competence are desirable. Based on the established role of alcohol dehydrogenase 3 (ADH3), also known as glutathione-dependent formaldehyde dehydrogenase, as the primary enzyme catalysing the detoxification of formaldehyde, the aim of this study was to investigate the expression of ADH3 in organotypic epithelia regenerated with normal (NOK), immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes. Organotypic epithelia, usually consisting of 5–10 cell layers, were produced at the air–liquid interface of collagen gels containing human oral fibroblasts, after culture for 10 days in a standardised serum-free medium. Immunochemical staining demonstrated uniform expression of ADH3 in these organotypic epithelia, as well as in the epithelial cells of oral tissue. The specificity of the ADH3 antiserum was ascertained from the complete neutralisation of the immunochemical reaction with purified ADH3 protein. Assessment of the staining intensities indicated that the expression levels were similar among the regenerated epithelia. Furthermore, the regenerated epithelia showed similar ADH3 expression to the epithelium in oral tissue. Therefore, a tissue-like expression pattern for ADH3 can be generated from the culture of various oral keratinocyte lines in an organotypic state. Similar expression levels among the various cell lines indicate the preservation of ADH3 during malignant transformation, and therefore that NOK, SVpgC2a and SqCC/Y1 represent functional models for in vitro studies of formaldehyde metabolism in human oral mucosa.
Evaluation of the Toxicity of Substituted Benzthioanilides by Using In Vitro Tests
Authors: Kowalska-Pylka Halina , Majer-Dziedzic Barbara , Niewiadomy Andrzej , Matysiak Joanna
Published: April 2019

The cytotoxicity of 12 benzthioanilides substituted in the N-aromatic ring, and of two commercial preparations (imaverol and thiuram) for comparison, was studied with clone 81 cat cells, by determining the highest tolerated dose, and by using the neutral red uptake assay and the kenacid blue assay for total protein. The concentrations that induced 20%, 50% and 80% (IC20, IC50 and IC80) inhibition relative to controls were calculated from dose–response curves. For some compounds, rat LD50 values were also determined. All the benzthioanilide preparations showed in vitro toxicities lower than those of the fungicides imaverol and thiuram. It was confirmed that the cytotoxicities of the compounds depend on the type of substituent. The least toxic compound contained a CONHCH2CO2H substituent in the para position of the N-aromatic ring, and the most toxic compounds contained chloro and fluoro, or three chloro substituents in the anilide moiety. All the benzthioanilides tested showed fungistatic activity for dermatophytes; two of the compounds (compound 5 and compound 12) also inhibited the development of yeasts at concentrations lower than those which caused toxicity in vitro. The LD50 values and the cytotoxic concentrations in vitro were linearly related.
A Genetically Engineered Cell-based System for Detecting Metabolism-mediated Toxicity
Authors: Bull Sarah , Langezaal Ingrid , Clothier Richard , Coecke Sandra
Published: April 2019

Xenobiotics undergong bioactivation by CYP450 enzymes form reactive metabolites that may exert direct metabolism-mediated toxicity. An in vitro model was developed to study the direct toxic effects that follow the metabolic activation of chemicals. The model uses monolayer cultures of genetically engineered NIH-3T3 or V79 cells that express individual human or rat CYP450 isoforms, respectively. Following exposure to 1,3-dichloropropanol or cyclophosphamide, basal cytotoxicity endpoints, including neutral red uptake and Alamar Blue™ reduction were used to assess changes in cell number and functional viability resulting from the formation of metabolites. Cell lines that express cytochrome P450 enzymes metabolised the test compounds, leading to increased toxicity compared with that observed in the control cell line. The use of specific inhibitors confirmed that the formation of reactive metabolites was CYP450-isoform dependent. These results indicate that a panel of genetically engineered cell lines expressing various cytochrome P450 enzyme isoforms can be used to reveal measurable metabolising capabilities, and could become a useful tool for the detection and possible determination of CYP450 isoforms in human liver metabolism-mediated toxicity.
Using In Vitro Prediction Models Instead of the Rabbit Eye Irritation Test to Classify and Label New Chemicals: A Post Hoc Data Analysis of the International EC/HO Validation Study
Authors: Moldenhauer Ferdinand
Published: April 2019

The international validation study on alternative methods to replace the Draize rabbit eye irritation test, funded by the European Commission (EC) and the British Home Office (HO), took place during 1992–1994, and the results were published in 1995. The results of this EC/HO study are analysed by employing discriminant analysis, taking into account the classification of the in vivo data into eye irritation classes A (risk of serious damage to eyes), B (irritating to eyes) and NI (non-irritant). A data set for 59 test items was analysed, together with three subsets: surfactants, water-soluble chemicals, and water-insoluble chemicals. The new statistical methods of feature selection and estimation of the discriminant function's classification error were used. Normal distributed random numbers were added to the mean values of each in vitro endpoint, depending on the observed standard deviations. Thereafter, the reclassification error of the random observations was estimated by applying the fixed function of the mean values. Moreover, the leaving-one-out cross-classification method was applied to this random data set. Subsequently, random data were generated r times (for example, r = 1000) for a feature combination. Eighteen features were investigated in nine in vitro test systems to predict the effects of a chemical in the rabbit eye. 72.5% of the chemicals in the undivided sample were correctly classified when applying the in vitro endpoints lgNRU of the neutral red uptake test and lgBCOPo5 of the bovine opacity and permeability test. The accuracy increased to 80.9% when six in vitro features were used, and the sample was subdivided. The subset of surfactants was correctly classified in more than 90% of cases, which is an excellent performance.
The Prediction of Human Skin Responses by Using the Combined In Vitro Fluorescein Leakage/Alamar Blue (Resazurin) Assay
Authors: Clothier Richard , Starzec Gemma , Pradel Lionel , Baxter Victoria , Jones Melanie , Cox Helen , Noble Linda
Published: April 2019

A range of cosmetics formulations with human patch-test data were supplied in a coded form, for the examination of the use of a combined in vitro permeability barrier assay and cell viability assay to generate, and then test, a prediction model for assessing potential human skin patch-test results. The target cells employed were of the Madin Darby canine kidney cell line, which establish tight junctions and adherens junctions able to restrict the permeability of sodium fluorescein across the barrier of the confluent cell layer. The prediction model for interpretation of the in vitro assay results included initial effects and the recovery profile over 72 hours. A set of the hand-wash, surfactant-based formulations were tested to generate the prediction model, and then six others were evaluated. The model system was then also evaluated with powder laundry detergents and hand moisturisers: their effects were predicted by the in vitro test system. The model was under-predictive for two of the ten hand-wash products. It was over-predictive for the moisturisers, (two out of six) and eight out of ten laundry powders. However, the in vivo human patch test data were variable, and 19 of the 26 predictions were correct or within 0.5 on the 0–4.0 scale used for the in vivo scores, i.e. within the same variable range reported for the repeat-test hand-wash in vivo data.
Evaluation and Prevalidation of an Immunotoxicity Test Based on Human Whole-blood Cytokine Release
Authors: Langezaal Ingrid , Hoffmann Sebastian , Hartung Thomas , Coecke Sandra
Published: April 2019

Immunotoxicology is a relatively new field in toxicology, and is one of emerging importance, because immunotoxicity appears to contribute to the development of cancer, autoimmune disorders, allergies and other diseases. At present, there is a lack of human cell-based immunotoxicity assays for predicting the toxicity of xenobiotics toward the immune system in a simple, fast, economical and reliable way. Existing immunotoxicity tests are mainly performed in animals, although species differences favour human-based testing. Whole-blood cytokine release models have attracted increasing interest, and are broadly used for pharmacological in vitro and ex vivo studies, as well as for pyrogenicity testing. We have adapted those methods for immunotoxicity testing, to permit the potency testing of immunostimulants and immunosuppressants. Following stimulation with a lipopolysaccharide or staphylococcal enterotoxin B, monocytes and lymphocytes release interleukin-1β and interleukin-4, respectively. Thirty-one pharmaceutical compounds, with known effects on the immune system, were used to optimise and standardise the method, by analysing their effects on cytokine release. The in vitro results were expressed as IC50 values for immunosuppression, and SC4 (fourfold increase) values for immunostimulation, and compared with therapeutic serum concentrations of the compounds in patients, and in vivo LD50 values from animal studies. The in vitro results correlated well with the in vivo data, so the test appears to reflect immunomodulation. Results were reproducible (CV = 20 ± 5%), and the method could be transferred to another laboratory (r2 = 0.99). We therefore propose this method for further validation and for use in immunotoxicity testing strategies.
Comparison and Validation of Novel Pyrogen Tests Based on the Human Fever Reaction
Authors: Hartung Thomas ,   Members of the Human(e) Pyrogen Test Study Group
Published: April 2019

The Limulus amoebocyte lysate (LAL) test has replaced about 80% of the use of the rabbit pyrogen test. Ideally, human-based in vitro tests are needed, to replace the remaining use of the rabbit test and the use of the LAL test. The progress of an EU-funded project is described, in which a number of in vitro tests based on the human fever reaction are passing through a prevalidation study on the way to evaluation in a formal validation study.
Ocular Safety: A Silent (In Vitro) Success Story
Authors: Curren Rodger D. , Harbell John W.
Published: April 2019

Ocular irritation testing has been one of the animal test methods most criticised by animal welfare advocates. Additional criticism has arisen from within the scientific community, based on the variability of the animal test results and the questionable relevance of the extremely high dose levels employed. As a result, the Draize eye irritation test has been one of the main targets for in vitro replacement. Despite extensive efforts, however, there is still no in vitro method that is fully validated as a regulatory replacement. In spite of this, many individual companies are using diverse in vitro ocular irritation tests to gain important safety and efficacy information about their products and raw materials, eliminating the need for animal testing in the process. This is done in a safe fashion by applying intelligent testing paradigms. ECVAM has played a major role in this success, through its many programmes that have emphasised the importance of understanding the true toxicological need, and then using in vitro tests to provide that information. Thus, even in the absence of a successfully validated regulatory assay, the desired result of reducing animal testing is being met.
Haematotoxicology: Scientific Basis and Regulatory Aspects
Authors: Gribaldo Laura
Published: April 2019

Haematopoietic tissues are the targets of numerous xenobiotics. The purpose of in vitro haematotoxicology is the prediction of adverse haematological effects from toxicants on human haematopoietic targets under controlled experimental conditions in the laboratory. Building on its foundations in experimental haematology and the wealth of haematotoxicological data found in experimental oncology, this field of alternatives toxicology has developed rapidly during the past decade. Preclinical and clinical drug development for anti-cancer drugs differs from that for other pharmaceuticals, because of the life-threatening nature of the disease. Treatment with anti-cancer drugs at clinically efficacious doses usually induces serious side-effects. The design of preclinical toxicology studies for anti-cancer drugs is intended to identify a safe clinical starting dose, characterise toxicities that could be encountered in human clinical trials, and determine whether these toxicities are reversible, manageable, and predictable. Although the myeloid colony-forming unit (CFU-GM) progenitor is most frequently evaluated, other defined progenitors and stem cells, as well as cell types found in the bone-marrow stroma, can now be evaluated in vitro. Genetic damage to haematopoietic cells can occur in the absence of any overt haematological signs. The development of tissue-specific screening systems that are able to give information about the toxic effects of chemicals, drugs and environmental hazards on target genes is needed, in order to make preliminary decisions or to set priorities for selection among large groups of chemicals and possible drugs.
An Overall Strategy for the Testing of Chemicals for Human Hazard and Risk Assessment under the EU REACH System
Authors: Combes Robert , Barratt Martin , Balls Michael
Published: April 2019

In its White Paper, Strategy for a Future Chemicals Policy, published in 2001, the European Commission (EC) proposed the REACH (Registration, Evaluation and Authorisation of CHemicals) system to deal with both existing and new chemical substances. This system is based on a top-down approach to toxicity testing, in which the degree of toxicity information required is dictated primarily by production volume (tonnage). If testing is to be based on traditional methods, very large numbers of laboratory animals could be needed in response to the REACH system, causing ethical, scientific and logistical problems that would be incompatible with the time-schedule envisaged for testing. The EC has emphasised the need to minimise animal use, but has failed to produce a comprehensive strategy for doing so. The present document provides an overall scheme for predictive toxicity testing, whereby the non-animal methods identified and discussed in a recent and comprehensive ECVAM document, could be used in a tiered approach to provide a rapid and scientifically justified basis for the risk assessment of chemicals for their toxic effects in humans. The scheme starts with a preliminary risk assessment process (involving available information on hazard and exposure), followed by testing, based on physicochemical properties and (Q)SAR approaches. (Q)SAR analyses are used in conjunction with expert system and biokinetic modelling, and information on metabolism and identification of the principal metabolites in humans. The resulting information is then combined with production levels and patterns of use to assess potential human exposure. The nature and extent of any further testing should be based strictly on the need to fill essential information gaps in order to generate adequate risk assessments, and should rely on non-animal methods, as far as possible. The scheme also includes a feedback loop, so that new information is used to improve the predictivity of computational expert systems. Several recommendations are made, the most important of which is that the European Union (EU) should actively promote the improvement and validation of (Q)SAR models and expert systems, and computer-based methods for biokinetic modelling, since these offer the most realistic and most economical solution to the need to test large numbers of chemicals.
The Registry of Cytotoxicity: Toxicity Testing in Cell Cultures to Predict Acute Toxicity (LD50) and to Reduce Testing in Animals
Authors: Halle Willi , Halder Marlies , Worth Andrew , Genschow Elke
Published: April 2019

This is a translation of a report on the Registry of Cytotoxicity (RC), originally published in German in 1998. The report presented an advanced in vitro method, which can significantly reduce the number of animals needed for the toxicity testing of a broad range of compounds/xenobiotics. With the RC method, it was possible to predict the oral or intravenous acute toxicity (LD50) — which is a regulatory requirement for newly developed pharmaceuticals and industrial and household chemicals — from the cytotoxicity data (mean IC50 = IC50X) obtained with mammalian cells. The RC method can be used before the in vivo test, and it does not pose any additional harm or suffering to laboratory animals. The RC method is of broad practical use: it can be applied, for example, in the pharmaceutical industry or the chemical industry in regulatory testing or in research. It is ready for validation, and could then be incorporated into OECD guidelines, thus reducing the total number of animals needed for regulatory toxicity testing. The RC method is based on the comparison of the IC50X values and the LD50 values by using linear regression analysis. With the RC method, it was possible to predict, within a predefined dose range, the acute oral LD50 for 252 of 347 xenobiotics, and the intravenous LD50 for rats and/or mice for 117 of 150 xenobiotics. Comparative studies showed that these results are highly reproducible.
A Survey of Consumer Attitudes to the Supply and Use of Human Hepatocytes in the United Kingdom
Authors: Lloyd Tom D.R. , Orr Samantha , Dennison Ashley R.
Published: April 2019

Human hepatocytes are the model of choice for pharmacotoxicological studies, but their acquisition is often problematic due to ethical and logistical difficulties. The UK Human Tissue Bank is a not-for-profit organisation that acquires and processes human tissue, with a specialist interest in the isolation of human hepatocytes. A recent in-house survey of the processing of liver tissue over 1 year revealed that freshly isolated hepatocytes were underutilised due to mismatched consumer demand, despite the published need for them. We present the results of a telephone survey to investigate the reasons behind this paradox. This survey highlighted some problem areas, including “out of hours” availability of cells and personnel difficulties, but overall, demonstrated the value of such a service, with numerous researchers taking advantage of available good quality human hepatocytes. Although further work is required in optimising long-term storage protocols through cryopreservation, we have demonstrated that tissue handling of this type can be successful and beneficial to the pharmaceutical and biotechnology industries.
Statistics of Interlaboratory In Vitro Toxicological Studies
Authors: Hothorn Ludwig A.
Published: April 2019

Interlaboratory studies are common in toxicology, particularly for the introduction of alternative assays. Numerous papers are available on the statistical analysis of interlaboratory studies, but these deal primarily with the case of a replicated single sample studied in several laboratories. This approach can be used for some assays, but for the majority, the results will be unsatisfactory, i.e. involving great variability between both the dose groups and the laboratories. However, the primary objective of toxicological assays is to achieve similarity between the sizes of effects, rather than to determine absolute values. In the parametric model, the sizes of effects are the studentised differences from the negative control or, for the commonly used dose–response designs, the similarity of the slopes of the dose–response curves. Standard approaches for the estimation of intralaboratory and interlaboratory variability, including Mandel plots, are introduced, and new approaches are presented for demonstrating similarity of effect sizes, with or without assuming a dose–response model. One approach is based on a modification of the parallel-line assay, the other is based on a modification of the interaction contrasts of the analysis of variance. SAS programs are given for all approaches, and real data from an interlaboratory immunotoxicological study are analysed as a demonstration.
Lack of Predictivity of the Rat Lethality (LD50) Test for Ecological and Human Health Effects
Authors: Rosenkranz Herbert S. , Cunningham Albert R.
Published: April 2019

The relationship between acute toxicity in rats (LD50 values) and indicators of potential health hazards in humans was investigated, based on a chemical population-based paradigm (i.e. the “chemical diversity approach”). These structure–activity relationship-based analyses indicate that high toxicity in rats (i.e. a low LD50 value) is not a good predictor of health effects in humans. In fact, it was found that high acute toxicity to minnows, as well as toxicity to cultured cells, showed significantly greater associations with the potential for health effects than rat LD50 values.
An Evaluation of the Metabolic Basis of Aflatoxin B1 Toxicity by Using Buffalo Granulocytes and Agranulocytes In Vitro
Authors: More Tukaram , Reddy Golla Ramalinga , Kumar Sandeep
Published: April 2019

This study was aimed at monitoring cytotoxic changes in buffalo leukocyte subpopulations exposed to aflatoxin B1 (AFB1), since no such information is available for this species. The effects of AFB1 on glutathione (GSH) S-transferase, Ca2+Mg2+ATPase and protein synthesis in leukocyte subpopulations, namely, mononuclear (MN) cells and polymorphonuclear (PMN) cells isolated from the blood of the domestic buffalo ( Bos bubalis), were studied. The cells were separated by using Ficoll-Paque and incubated in the presence of AFB1. GSH S-transferase activity was found to increase in cells exposed to AFB1, but there was no difference in activity between MN and PMN cells. PMN cell ATPase activity increased after AFB1 treatment, whereas no such effect was observed in the MN cells, which showed higher basal levels of ATPase activity. In the presence of AFB1, all the cells showed significant decreases in 14C-leucine incorporation, but the MN cells showed higher 14C-leucine incorporation than the PMN cells. Nevertheless, both cell types were affected by AFB1 and participated in its detoxification. There was also an appreciable decrease in the release of myeloperoxidase by activated PMN cells in the presence of AFB1.
QSAR Applicability Domain Estimation by Projection of the Training Set in Descriptor Space: A Review
Authors: Jaworska Joanna , Nikolova-Jeliazkova Nina , Aldenberg Tom
Published: April 2019

As the use of Quantitative Structure Activity Relationship (QSAR) models for chemical management increases, the reliability of the predictions from such models is a matter of growing concern. The OECD QSAR Validation Principles recommend that a model should be used within its applicability domain (AD). The Setubal Workshop report provided conceptual guidance on defining a (Q)SAR AD, but it is difficult to use directly. The practical application of the AD concept requires an operational definition that permits the design of an automatic (computerised), quantitative procedure to determine a model's AD. An attempt is made to address this need, and methods and criteria for estimating AD through training set interpolation in descriptor space are reviewed. It is proposed that response space should be included in the training set representation. Thus, training set chemicals are points in n-dimensional descriptor space and m-dimensional model response space. Four major approaches for estimating interpolation regions in a multivariate space are reviewed and compared: range, distance, geometrical, and probability density distribution.
Insulin and IGF-1 Mediated Inhibition of Apoptosis in CHO Cells Grown in Suspension in a Protein-free Medium
Authors: Adamson Lars , Walum Erik
Published: April 2019

When Chinese hamster ovary (CHO) cells were grown in suspension and deprived of serum, 40% of them became apoptotic after 72 hours, as determined by flow cytometry analysis of TUNEL-labelled cells. Cell viability, assessed by erythrocin B staining, decreased correspondingly. An increase in the total fraction of cells expressing interleukin converting enzyme (ICE; caspase 1), B-cell lymphoma 2 protein (Bcl-2,) and Bcl-2 associated x protein (Bax) was shown by antibody probing and subsequent flow cytometry. The p53 tumour suppressor gene product level remained low within the cell population. Insulin-like growth factor-1 (IGF-1) inhibited cell death in a concentration-dependent manner, and at 20ng/ml, cell viability was maintained close to 100% and no apoptotic cells were detected. Also, insulin was shown to inhibit cell death — at 1.0μg/ml, cell viability was 95%, whereas 10% of the cells stained for apoptosis. At the highest concentrations of IGF-1 and insulin, the expression of ICE, Bcl-2 and Bax was fully suppressed, whereas the p53 product level increased, despite still being detectable in a minority of cells. Under these conditions, IGF-1 may increase p53 expression to restrain abnormal cell proliferation. It is concluded that special attention should be paid to exposure and culture conditions that induce acquired susceptibility to a toxic insult, during the development and validation of cell-based assays.
Effects of Cadmium on Differentiation and Cell Cycle Progression in Cultured Xenopus Kidney Distal Epithelial (A6) Cells
Authors: Bjerregaard Henning F.
Published: April 2019

Cadmium (Cd) is an important industrial and environmental pollutant, and the kidney is the primary organ to be affected. To elucidate the effects of Cd on cell proliferation, an epithelial cell line (A6) originally derived from the distal part of the Xenopus laevis kidney was cultured in media containing 10% fetal bovine serum. The effects of Cd (added as CdCl2) on cellular growth and differentiation from single cells to confluent epithelia were investigated by visual inspection and by measurement of the degree to which living cells covered a unit area. Over a concentration range from 5 to 50μM, Cd did not affect the settling and adherence of single cells to the bottom of the culture well. The addition of 5μM Cd for 4 days did not affect the ability of the A6 cells to develop confluent epithelia, measured as the area covered by adherent living epithelial cells (99 ± 4% of the control value). However, 10μM Cd did effectively inhibit development of confluent epithelia to 13 ± 5% compared to control. Visual inspection of adherent cells exposed to 50μM Cd for 7 days revealed no increase in cell number or in cell death, which indicated the induction of cell cycle arrest. Flow cytometric analysis showed that treatment of cells with Cd (0.4mM) for 24 hours induced a significant increase in the proportion of G1 phase cells from 58.6 ± 3.9 to 80.6 ± 3.7%, and a corresponding reduction in the proportion of cells in both the S and G2 phases from 24.0 ± 3.6 to 13.4 ± 3.3% and 17.2 ± 1.7 to 5.8 ± 2.1%, respectively. This study showed that Cd stopped cell proliferation in a very narrow concentration range, between 5 and 10μM, and cell cycle analysis indicated that Cd arrested the cells in the G1 phase of the cell cycle.
Evaluation of Mitochondrial Function in Isolated Rat Hepatocytes and Mitochondria during Oxidative Stress
Authors: Červinková Zuzana , Lotková Halka , Křivaková Pavla , Roušar Tomáš , Kučera Otto , Tichý Lukas , Červinka Miroslav , Drahota Zdeněk
Published: April 2019

The majority of toxic agents act either fully or partially via oxidative stress, the liver, specifically the mitochondria in hepatocytes, being the main target. Maintenance of mitochondrial function is essential for the survival and normal performance of hepatocytes, which have a high energy requirement. Therefore, greater understanding of the role of mitochondria in hepatocytes is of fundamental importance. Mitochondrial function can be analysed in several basic models: hepatocytes cultured in vitro; mitochondria in permeabilised hepatocytes; and isolated mitochondria. The aim of our study was to use all of these approaches to evaluate changes in mitochondria exposed in vitro to a potent non-specific peroxidating agent, tert-butylhydroperoxide (tBHP), which is known to induce oxidative stress. A decrease in the mitochondrial membrane potential (MMP) was observed in cultured hepatocytes treated with tBHP, as illustrated by a significant reduction in Rhodamine 123 accumulation and by a decrease in the fluorescence of the JC-1 molecular probe. Respiratory Complex I in the mitochondria of permeabilised hepatocytes showed high sensitivity to tBHP, as documented by high-resolution respirometry. This could be caused by the oxidation of NADH and NADPH by tBHP, followed by the disruption of mitochondrial calcium homeostasis, leading to the collapse of the MMP. A substantial decrease in the MMP, as determined by tetraphenylphosphonium ion-selective electrode measurements, also confirmed the dramatic impact of tBHP-induced oxidative stress on mitochondria. Swelling was observed in isolated mitochondria exposed to tBHP, which could be prevented by cyclosporin A, which is evidence for the role of mitochondrial permeability transition. Our results demonstrate that all of the above-mentioned models can be used for toxicity assessment, and the data obtained are complementary.