bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrated Decision-tree Testing Strategies for Environmental Toxicity with Respect to the Requirements of the EU REACH Legislation
Authors: Grindon Christina , Combes Robert , Cronin Mark T.D. , Roberts David W. , Garrod John
Published: April 2019

Liverpool John Moores University and FRAME recently conducted a research project sponsored by Defra on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This paper focuses on the prospects for using alternative methods (both in vitro and in silico) for environmental (aquatic) toxicity testing. The manuscript reviews tests based on fish cells and cell lines, fish embryos, lower organisms, and the many expert systems and QSARs for aquatic toxicity testing. Ways in which reduction and refinement measures can be used are also discussed, including the Upper Threshold Concentration — Step Down (UTC) approach, which has recently been retrospectively validated by ECVAM and subsequently endorsed by the ECVAM Scientific Advisory Committee (ESAC). It is hoped that the application of this approach could reduce the number of fish used in acute toxicity studies by around 65–70%. Decision-tree style integrated testing strategies are also proposed for acute aquatic toxicity and chronic toxicity (including bioaccumulation), followed by a number of recommendations for the future facilitation of aquatic toxicity testing with respect to environmental risk assessment.
Predicting Drug Metabolism–dependent Toxicity for Humans with a Genetically Engineered Cell Battery
Authors: Doehmer Johannes
Published: April 2019

This paper covers the presentation of an invited lecture — the FRAME Annual Lecture — given in London on 8 November 2006. Investigating the metabolism of chemicals in general, and of drugs and pollutants in particular, is of key importance to understanding pharmacological and toxicological effects. Over more than 15 years, the genes encoding the enzymes involved, have been individually cloned and expressed after gene transfer into V79 Chinese hamster cells, yielding a collection of cell lines — the so-called V79 Cell Battery. With this technology, it has become possible to study the relevant enzymes individually, thus avoiding complex in vivo situations. By cloning genes from different species, including humans, species–species comparison became possible, yielding results of immediate predictive value for humans. Since V79 cells had already been approved by the OECD for toxicity studies since the 1980s, the metabolically competent V79 cell lines are of even greater value, as metabolism and toxicity testing are linked in the very same cells in a highly defined fashion. The results obtained so far with the genetically engineered V79 cell lines justify their acceptance as alternatives to animal experimentation in drug development and in the toxicity testing of chemicals, serving the goals of the Three Rs and, in particular, the most important R: Replacement.
The Integrated Acute Systemic Toxicity Project (ACuteTox) for the Optimisation and Validation of Alternative In Vitro Tests
Authors: Clemedson Cecilia , Kolman Ada , Forsby Anna
Published: April 2019

The ACuteTox project is designed to replace animal testing for acute systemic toxicity, as is widely used today for regulatory purposes, by using in vitro and in silico alternatives. In spite of the fact that earlier studies on acute systemic toxicity demonstrated a good correlation between in vitro basal cytotoxicity data (the 50% inhibitory concentration [IC50]) in human cell lines and rodent LD50 values, and an even better correlation between IC50 values and human lethal blood concentrations, very few non-animal tests have been accepted for general use. Therefore, the aim of the ACuteTox project is to adapt new testing strategies, for example, the implementation of new endpoints and new cell systems for toxicity screening, organ-specific models, metabolism-dependent toxicity, tissue absorption, distribution and excretion, and computer-based prediction models. A new database, AcuBase, containing descriptions and results of in vitro tests of the 97 reference chemicals, as well as the results of animal experimentation, and human acute toxicity data, will be generated within the framework of ACuteTox. Scientists from 13 European countries are working together and making efforts to find the most appropriate testing strategies for the prediction of human acute systemic toxicity, and also to select a robust in vitro test battery for cytotoxicity testing of chemicals.
The Use of Bioassays for the Risk Assessment of Toxic Leachates: An Experimental Study
Authors: Irha Natalya , Blinova Irina
Published: April 2019

Solid wastes from the oil-shale industry produce leachates containing toxic compounds such as heavy metals and persistent polycyclic aromatic hydrocarbons (PAH). The hazard to the environment represented by waste leachates depends not only on their chemical composition, but also on the mobility and bioavailability of toxic contaminants in soils. We evaluated the applicability of bioassays for toxicity assessment of the bioavailable fraction of heavy metals and PAH in soils, in experiments with samples of four different soil types (Rendzina, Brown pseudopodzolic, Typical brown, Sodpodzolic), the pH of which ranged from 6.2 to 7.2. The toxicity of the bioavailable fraction of the soil contaminants was assessed with the dehydrogenase enzyme activity assay, and with a Toxkit microbiotest with the crustacean, Thamnocephalus platyurus, after treatment of the soil samples with an artificial solution containing chromium (III), lead (II), copper (II), cadmium (II) and pyrene. The test results confirm those of earlier experiments, which characterised the sorption potential of investigated soils for the same compounds. Both tests turned out to be sufficiently sensitive, and hence can be recommended as effective and useful tools for the assessment of the bioavailable fraction of soil contaminants.
Comparative Quantitative Structure–Activity–Activity Relationships for Toxicity to Tetrahymena pyriformis and Pimephales promelas
Authors: Kahn Iiris , Maran Uko , Benfenati Emilio , Netzeva Tatiana I. , Schultz T. Wayne , Cronin Mark T.D.
Published: April 2019

An approach for predicting acute aquatic toxicity, in the form of a quantitative structure–activity–activity relationship (QSAAR), is described. This study assessed relative toxic effects to a fish, Pimephales promelas, and a ciliate, Tetrahymena pyriformis, and attempted to form relationships between them. A good agreement between toxic potencies ( R2 = 0.754) was found for a chemically diverse dataset of 364 compounds, when using toxicity to the ciliate as a surrogate to that for fish. This relationship was extended by adding three theoretical structural descriptors of the molecules. The inclusion of these descriptors improved the relationship further ( R2 = 0.824). The structural features that were found to improve the extrapolation between the toxicity to the two different species were related to the electron distribution of the carbon skeleton of the toxicant, its hydrogen-bonding ability, and its relative nitrogen content. Such a QSAAR approach provides a potential tool for predicting the toxicities of chemicals for environmental risk assessment and thus for reducing animal tests.
Environmental and Synthetic Sulphydryl Group Inhibitors: Effects on Bioluminescence and Respiration in Vibrio fischeri
Authors: Kalcienė Virginija , Četkauskaitė Anolda
Published: April 2019

Elemental sulphur (as S0 and S8) is abundant in anaerobic sediments and soil, and is highly toxic in the Vibrio fischeri bioluminescence test. This mode of S0 action remains uncertain. The objective of this research was the analysis of the toxic effects of S0 on bioluminescence and respiration in V. fischeri, in joint action with N-ethylmaleimide (NEM) or 2,4-dithio-DL-threitol (DTT), which are –SH group inhibiting and maintaining synthetic agents, respectively. Non-toxic DTT immediately protected cell bioluminescence against S0 inhibition at low (5.5ppb) and high (55ppb) concentrations of S0, whilst restoration of the inhibitory effect of S0 took up to 30 minutes. NEM (62.5ppb) diminished cell bioluminescence by up to 50% after 5 minutes, but after 60 minutes, the inhibition reached 100%. DTT restored the bioluminescence function inhibited in vivo and in vitro by S0 and NEM. Enhancement of cell respiration by up to 20% and 33% was observed at 2.2ppm of S0 and 36.8ppm of 2,4-dinitrophenol (2,4-DNP; an uncoupler of oxidative phosphorylation), respectively; whilst NEM (3.1ppm) caused a reduction of up to 40%. This comparative analysis confirmed that S0 has multiple modes of action — it acts as both an –SH group inhibitor and an uncoupler of oxidative phosphorylation in V. fischeri cells.
A Comparative Study of Machine Learning Algorithms Applied to Predictive Toxicology Data Mining
Authors: Neagu Daniel C. , Guo Gongde , Trundle Paul R. , Cronin Mark T.D.
Published: April 2019

This paper reports results of a comparative study of widely used machine learning algorithms applied to predictive toxicology data mining. The machine learning algorithms involved were chosen in terms of their representability and diversity, and were extensively evaluated with seven toxicity data sets which were taken from real-world applications. Some results based on visual analysis of the correlations of different descriptors to the class values of chemical compounds, and on the relationships of the range of chosen descriptors to the performance of machine learning algorithms, are emphasised from our experiments. Some interesting findings relating to the data and the quality of the models are presented — for example, that no specific algorithm appears best for all seven toxicity data sets, and that up to five descriptors are sufficient for creating classification models for each toxicity data set with good accuracy. We suggest that, for a specific data set, model accuracy is affected by the feature selection method and model development technique. Models built with too many or too few descriptors are undesirable, and finding the optimal feature subset appears at least as important as selecting appropriate algorithms with which to build a final model.
The Developmental Expression of Fluorescent Proteins in Organotypic Hippocampal Slice Cultures from Transgenic Mice and its Use in the Determination of Excitotoxic Neurodegeneration
Authors: Noraberg Jens , Jensen Carsten V. , Bonde Christian , Montero Maria , Nielsen Jakob V. , Jensen Niels A. , Zimmer Jens
Published: April 2019

Transgenic mice, expressing fluorescent proteins in neurons and glia, provide new opportunities for real-time microscopic monitoring of degenerative and regenerative structural changes. We have previously validated and compared a number of quantifiable markers for neuronal damage and cell death in organotypic brain slice cultures, such as cellular uptake of propidium iodide (PI), loss of microtubule-associated protein 2 (MAP2), Fluoro-Jade (FJ) cell staining, and the release of cytosolic lactate dehydrogenase (LDH). An important supplement to these markers would be data on corresponding morphological changes, as well as the opportunity to monitor reversible changes or long-term effects in the event of minor damage. As a first step, we present: a) the developmental expression in organotypic hippocampal brain slice cultures of transgenic fluorescent proteins, useful for the visualisation of neuronal subpopulations and astroglial cells; and b) examples of excitotoxic, glutamate receptor-induced degeneration of hippocampal CA1 pyramidal cells, with corresponding astroglial reactivity in such cultures. The slice cultures were set up according to standard techniques, by using one-week old pups from four transgenic mouse strains which express fluorescent proteins in their neurons and/or astroglial cells. From the time of explantation, and subsequently for up to nine weeks in culture, the transgenic neuronal fluorescence displayed the expected characteristics of a developmental, in vivo-like increase, including both the number and localisation of cells, as well as the intensity of fluorescence. At that stage and later, the transgenic fluorescence clearly permitted the visualisation of cell bodies, larger and smaller dendritic branches, spines and axons. In separate experiments, with a 24-hour exposure of matured sliced cultures to 100μM of the glutamate agonist, N-methyl-D-aspartate (NMDA), we observed, by time-lapse recording, a gradual, but rapid loss of fluorescent CA1 pyramidal cells, accompanied by astrogliosis of transgene fluorescent astroglial cells. Based on these results, we consider that organotypic brain slice cultures from transgenic mice, with fluorescent neurons and glia, combined with detailed visualisation by time-lapse fluorescence microscopy, have great potential for investigating both major irreversible and minor reversible structural changes in neurons and glia, induced by neurotoxins and other neurodegenerative compounds and conditions.
Long-term, Repeated Dose In Vitro Neurotoxicity of the Glutamate Receptor Antagonist L-AP3, Demonstrated in Rat Hippocampal Slice Cultures by Using Continuous Propidium Iodide Incubation
Authors: Kristensen Bjarne W. , Blaabjerg Morten , Noraberg Jens , Zimmer Jens
Published: April 2019

Most in vitro models are only used to assess the short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3), which is known to be toxic in vivo after subchronic, but not acute, administration. Degenerative effects were monitored by measuring the cellular uptake of propidium iodide (PI; continuously present in the medium) and lactate dehydrogenase (LDH) leakage, and by using a panel of histological stains. Hippocampal slices, derived from 2–3 day old rats and grown for 3 weeks, were subsequently exposed for the next 3 weeks to 0, 10 or 100μM L-AP3, with PI (2μM) in the culture medium. Exposure to 100μM L-AP3 induced severe toxicity after 4–6 days, as shown by massive PI uptake, LDH leakage, and changes in MAP2 and GFAP immunostaining and in Nissl and Timm staining. In contrast, 10μM L-AP3 did not induce detectable neuronal degeneration. Treatment with the NMDA receptor antagonist, MK-801, or the AMPA/KA receptor antagonist, NBQX, together with 100μM L-AP3, reduced neurodegeneration to close to control values. It is concluded that the continuous incubation of hippocampal slice cultures with PI is technically feasible for use in studies on inducible neuronal degeneration over time.
The Selection of Serum-independent PC12 Cells for a More-reliable Manganese Cytotoxicity Test
Authors: Bignetti Enrico , Franchi Daniela , Gorreri Alessandra , Fusari Antonella , Del Bono Giovanni , Ubaldi Antonio , Aiello Gaetano L.
Published: April 2019

A major issue concerning the protocols of heavy metal cytotoxicity tests with PC12 cells was the hypothesis that serum in the culture medium might sequester the metal, thus altering the results obtained. However, serum withdrawal impairs the viability of PC12 cells themselves, thus impeding cytotoxicity testing in the absence of serum. In this study, we repeatedly selected undifferentiated, totally non-adherent PC12 cells in Petri dishes. Surprisingly, we discovered that these cells could survive and proliferate in serum-free medium. Moreover, features such as NGF-responsiveness, resazurin reduction potential, doubling rate, protein content, and basal caspase-3 enzyme activity, were equivalent to those exhibited by standard PC12 cultures. Further experiments aimed at fully characterising these serum-independent PC12 cells are in progress. These cells enabled cytotoxicity experiments to be conducted with manganese, both in serum-supplemented and in serum-deprived medium. The results demonstrated that serum removal decreased the LC50 of manganese from 250μM to 32μM, without affecting the internalisation of the metal. The data exclude an early competitive effect of serum on metal internalisation; rather, they suggest a late protective mechanism mediated by serum against the cytotoxic effect of the already-internalised metal.
Working with other Partners — the Retail Sector
Authors: Fridd Petrina
Published: April 2019

Animal testing to demonstrate the safety of cosmetic and toiletries has been used for many years. However, such testing, now viewed by many consumers as an emotive issue, has been banned within the UK, and is severely restricted in the European Union. Consumer knowledge and understanding have been challenged by the use of focus group studies, which showed that consumers were often confused and potentially misled by claims on labels and in leaflets. Despite the fact that some pressure groups had taken positive action, even holding store demonstrations, in attempts to stop such testing or claims, there was still a legal requirement to demonstrate the safety of products, and this involved the use of animal testing. Nevertheless, there was great pressure to move away from animal testing, clarify marketing and pack claims, and provide general transparency to the consumer. Therefore, the decision was taken to actively seek out and work with organisations which were working toward the validation of alternative testing. A number of potential organisations were approached and, after careful consideration of their respective aims and aspirations, it was decided that, for the retailer in question, FRAME was the most appropriate organisation with which to work.
A Review of In Vitro Modelling Approaches to the Identification and Modulation of Squamous Metaplasia in the Human Tracheobronchial Epithelium
Authors: Gray Alison C. , McLeod Julie D. , Clothier Richard H.
Published: April 2019

Squamous metaplasia in the tracheobronchial epithelium (TBE) involves the replacement of the normal pseudostratified mucociliary epithelium with a stratified squamous epithelium. Squamous metaplasia is considered to be an adaptive response that protects the lumen from the effects of inhaled airborne pollutants, but which might also feature as a pre-neoplastic lesion preceding squamous cell carcinoma. With the exception of transglutaminase I, involucrin, and cytokeratins 5, 6 and 13, few markers that contribute to the squamous phenotype have been identified in human TBE that can be used in diagnosis or to monitor its development in laboratory investigations, and current models are inadequate to provide statistically meaningful data. Therefore, new predictive markers have been identified, and new techniques established, in epithelial in vitro models capable of expressing squamous characteristics, which will be used to identify hazardous exposures and elucidate the mechanisms by which they induce their effects. A protocol for the quantitative detection of transglutaminase activity has been standardised in keratinocytes, based on the enzymatic incorporation of fluorescein–cadaverine (FC) into bis(γ-glutamyl) polyamine cross-links. The specificity of this compound as a transglutaminase substrate was demonstrated by using a range of competitive transglutaminase inhibitors, and by modulation of the squamous pathway. FC incorporation was localised to the cell membrane of terminally differentiating cells, and was not visible in basal, proliferating cells. High calcium-containing medium, nicotine and cigarette smoke condensates (CSC) induced an increase in FC incorporation, providing evidence of their role in enhancing the squamous pathway. Analysis by flow cytometry was used to provide a quantitative assessment of a range of optimised squamous differentiation markers, identified in normal human bronchial epithelia and in a bronchial cell line. Transglutaminase I was induced in a time-dependent manner, in post-confluent cells induced to differentiate down the squamous pathway, whereas involucrin was ubiquitously expressed and the levels of cytokeratins 5, 6 and 18 were reduced. The response of these and other differentiation markers to squamous-inducing conditions is being explored.
Development of an Innervated Model of Human Skin
Authors: Khammo Nancy , Ogilvie Jane , Clothier Richard H.
Published: April 2019

Neuronal cell responses and interactions with the epithelial and fibroblastic cells of the skin are a key factor in the production in vivo of the irritation/inflammatory response. Currently, few in vitro models are available that contain dermal, epidermal and the relevant neuronal components. The primary objective of this study was to produce and maintain a 3-D in vitro model of human skin containing these elements. The relevant neuronal component was supplied by adding sensory neurons derived from the dorsal root ganglion (DRG). Since adult neuronal cells do not grow significantly in vivo or in vitro, and since it is very difficult to obtain such cells from humans, it was necessary to employ embryonic rat DRG cells. The ultimate purpose of this model is to improve prediction of the in vivo skin irritancy potential of chemicals and formulations, without the need to use animal models. In addition, this approach has also been applied to the in vitro human eye and bronchial 3-D models being developed in the FRAME Alternatives Laboratory.
An Evaluation of the Effects of Photoactivation of Bithionol, Amiodarone and Chlorpromazine on Human Keratinocytes In Vitro
Authors: Reid Linzi , Khammo Nancy , Clothier Richard H.
Published: April 2019

Human skin is a continual target for chemical toxicity, due to its constant exposure to xenobiotics. The skin possesses a number of protective antioxidant systems, including glutathione and enzymic pathways, which are capable of neutralising reactive oxygen species (ROS). In combination with certain chemicals, the presence of ROS might augment the levels of toxicity, due to photoactivation of the chemical or, alternatively, due to an oxidatively-stressed state in the skin which exisited prior to exposure to the chemical. Bithionol is a phototoxic anti-parasitic compound. The mechanism of its toxicity and the possible methods of protection from its damaging effects have been explored. The capacity of keratinocytes to protect themselves from bithionol and other phototoxic chemicals has been investigated. In addition, the potential of endogenous antioxidants, such as vitamin C and E, to afford protection to the cells, has been evaluated. The intracellular glutathione stores of HaCaT keratinocytes were reduced following treatment with biothionol. Following photoactivation, both bithionol and chlorpromazine had similar effects, which suggests that glutathione is important in the detoxification pathway of these chemicals. This was confirmed by means of the visual identification of fluorescently-labelled glutathione. Endogenous antioxidants were unable to protect the HaCaT keratinocytes from bithionol toxicity or chlorpromazine phototoxicity. Amiodarone was shown to have no effect on cellular glutathione levels, which suggests that an alternative mechanism of detoxification was occurring in this case. This was supported by evidence of the protection of HaCaT cells from amiodarone phototoxicity via endogenous antioxidants. Thus, it appears that amiodarone toxicity is dependent on the levels of non-gluathione antioxidants present, whilst bithionol and chlorpromazine detoxification relies on the glutathione antioxidant system. This type of approach could indicate the likely mechanisms of phototoxicity of chemicals in vitro, with relevance to potential effects in vivo.
An Evaluation of the Predictive Ability of the QSAR Software Packages, DEREK, HAZARDEXPERT and TOPKAT, to Describe Chemically-induced Skin Irritation
Authors: Mombelli Enrico
Published: April 2019

According to the REACH chemicals legislation, formally adopted by the EU in 2006, Quantitative Structure–Activity Relationships (QSARs) can be used as alternatives to animal testing, which itself poses specific ethical and economical concerns. A critical assessment of the performance of the QSAR models is therefore the first step toward the reliable use of such computational techniques. This article reports the performance of the skin irritation module of three commercially-available software packages: DEREK, HAZARDEXPERT and TOPKAT. Their performances were tested on the basis of data published in the literature, for 116 chemicals. The results of this study show that only TOPKAT was able to predict the irritative potential for the majority of chemicals, whereas DEREK and HAZARDEXPERT could correctly identify only a few irritant substances.
Repeated Exposure to Benzalkonium Chloride in the Ex Vivo Eye Irritation Test (EVEIT): Observation of Isolated Corneal Damage and Healing
Authors: Frentz Markus , Goss Miriam , Reim Martin , Schrage Norbert F.
Published: April 2019

The prediction of side-effects is a key issue in the REACH initiative on chemicals, in the production of cosmetics and in the preclinical testing of drugs. A new ex vivo test for repeated substance application is presented, that is able to identify corrosive and irritant effects on the eye by using crucial endpoints, such as cellular and morphological damage, and healing characteristics. The test is intended to replace the Draize eye test and to improve the preclinical testing of drugs and chemicals that are likely to come into direct contact with the cornea. The Ex Vivo Eye Irritation Test (EVEIT) is a self-healing system, involving living corneas obtained from abattoir rabbit eyes. The corneas are cultured in a similar way to the method used during the transplantation of corneal grafts. The corneas are exposed to multiple small, mechanical abrasions, and then test substances are repeatedly dropped onto the centres of the corneas. The test substances applied in this study were citrate-buffered hyaluronate eye drops and an artificial tear replacement, with increasing concentrations of up to 0.1% benzalkonium chloride. A dose-dependent inhibition of recovery and impairment of the lactate production mechanism in the cornea was observed with benzalkonium chloride treatment.
An Integrated Decision-tree Testing Strategy for Repeat Dose Toxicity with Respect to the Requirements of the EU REACH Legislation
Authors: Grindon Christina , Combes Robert , Cronin Mark T.D. , Roberts David W. , Garrod John F.
Published: April 2019

This paper presents some results of a joint research project conducted by FRAME and Liverpool John Moores University, and sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity end-points associated with REACH. This paper focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for repeat dose (sub-acute, sub-chronic and chronic) toxicity testing. It reviews the limited number of in silico and in vitro tests available for this endpoint, and outlines new technologies which could be used in the future, e.g. the use of biomarkers and the ‘omics’ technologies. An integrated testing strategy is proposed, which makes use of as much non-animal data as possible, before any essential in vivo studies are performed. Although none of the non-animal tests are currently undergoing validation, their results could help to reduce the number of animals required for testing for repeat dose toxicity.
The Physicochemical Parameters of Marker Compounds and Vehicles for Use in In Vitro Percutaneous Absorption Studies
Authors: Kaca Monika , Bock Udo , Jalal Mohamed Tawfik , Harms Meike , Hoffmann Christine , Müller-Goymann Christel , Netzlaff Frank , Schäfer Ulrich , Lehr Claus-Michael , Haltner-Ukomadu Eleonore
Published: April 2019

In order to prepare for a validation study to compare percutaneous absorption through reconstructed human epidermis with ex vivo skin absorption through human and animal skin, nine test compounds, covering a wide range of physicochemical properties were selected, namely: benzoic acid; caffeine; clotrimazole; digoxin; flufenamic acid; ivermectin; mannitol; nicotine; and testosterone. The donor and receptor media for the test substances, the addition of a solubiliser for the lipophilic compounds, as well as the stability and solubility of the test substances in the vehicles, were systematically analysed. Hydrophilic molecules, being freely soluble in water, were applied in buffered saline solutions. In order to overcome solubility restrictions for lipophilic compounds, the non-ionic surfactant, Igepal® CA-630, was added to the donor vehicle, and, in the case of clotrimazole and ivermectin, also to the receptor fluid. The model molecules showed a suitable solubility and stability in the selected donor and receptor media throughout the whole duration of the test.
The Automated, Accurate and Reproducible Determination of Steady-state Permeation Parameters from Percutaneous Permeation Data
Authors: Niedorf Frank , Schmidt Elisabeth , Kietzmann Manfred
Published: April 2019

Procedures for the in vitro determination of percutaneous permeation with Franz diffusion cells are widely accepted. However, the calculation of relevant endpoints, such as the steady-state flux ( J) and the permeation coefficient ( Papp), still depends on visual data inspection or an approximation of the steady-state flux as the maximum observed absorption rate. As both these approaches must be considered inappropriate, an automated and reproducible algorithm to analyse permeation data is presented. The method detects both lag-times and non-linear data resulting from substance accumulation in the acceptor compartment of static diffusion cells. It was evaluated by using simulated data, and data from experiments with caffeine and testosterone on bovine udder skin and human reconstituted epidermis (SkinEthic®), which represent model barriers with high and low barrier strengths, respectively. It was shown that the algorithm is a suitable method for the identification of steady-state ranges in permeation data. If used on data generated with appropriate experimental approaches, it is a reproducible and time-saving alternative to the visual analysis of diffusion data.
Integrated Testing Strategies for Use with Respect to the Requirements of the EU REACH Legislation
Authors: Grindon Christina , Combes Robert , Cronin Mark T.D. , Roberts David W. , Garrod John F.
Published: April 2019

Integrated testing strategies have been proposed to facilitate the process of chemicals risk assessment to fulfil the requirements of the proposed EU REACH system. Here, we present individual, decision-tree style, strategies for the eleven major toxicity endpoints of the REACH system, including human health effects and ecotoxicity. These strategies make maximum use of non-animal approaches to hazard identification, before resorting to traditional animal test methods. Each scheme: a) comprises a mixture of validated and non-validated assays (distinguished in the schemes); and b) decision points at key stages to allow the cessation of further testing, should it be possible to use the available information to classify and label and/or undertake risk assessment. The rationale and scientific justification for each of the schemes, with respect to the validation status of the tests involved and their individual advantages and limitations, will be discussed in detail in a series of future publications.
Integrated Decision-tree Testing Strategies for Mutagenicity and Carcinogenicity with Respect to the Requirements of the EU REACH Legislation
Authors: Combes Robert , Grindon Christina , Cronin Mark T.D. , Roberts David W. , Garrod John F.
Published: April 2019

Liverpool John Moores University and FRAME recently conducted a research project sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This paper focuses on the prospects for using alternative methods (both in vitro and in silico) for mutagenicity (genotoxicity) and carcinogenicity testing — two toxicity endpoints, which, together with reproductive toxicity, are of pivotal importance for the REACH system. The manuscript critically discusses well-established testing approaches, and in particular, the requirement for short-term in vivo tests for confirming positive mutagenicity, and the need for the rodent bioassay for detecting non-genotoxic carcinogens. Recently-proposed testing strategies focusing on non-animal approaches are also considered, and our own testing scheme is presented and supported with background information. This scheme makes maximum use of pre-existing data, computer ( in silico) and in vitro methods, with weight-of-evidence assessments at each major stage. The need for the improvement of in vitro methods, to reduce the generation of false-positive results, is also discussed. Lastly, ways in which reduction and refinement measures can be used are also considered, and some recommendations are made for future research to facilitate the implementation of the proposed testing scheme.
Twenty-First Century Challenges for In Vitro Neurotoxicity
Authors: Smith Robert A.
Published: April 2019

During the last 40 years, studies incorporating in vitro methodologies have greatly advanced our understanding of human nerve cell biology. Attempts have been made to apply these to investigations of neurotoxicity. Due to the complexity of the nervous system, underpinned by an array of integrated interactions between a host of cell types, it is concluded that, at present, alternative neural models are most successful in determining the underlying mechanisms which can cause perturbation of normal functioning of the nervous system, both in adults and during the embryonic period. The use of tiered batteries of test models has been proposed in screening programmes for neurotoxicity, with the generation of much encouraging data in laboratories across the globe. This review aims to discuss the development of neural alternatives, considers the various model systems available, and highlights specific neuronal endpoints which can be tested, in addition to the cytotoxic evaluation of neuronal viability. Developments in molecular and stem cell biology, which are appropriate to neural tissue, and which offer the prospect of exciting advances for the next decade, are cited.
The SCHER Report on Non-human Primate Research —Biased and Deeply Flawed
Authors: Bailey Jarrod , Taylor Katy
Published: April 2019

The European Commission's Scientific Committee on Health and Environmental Risks (SCHER) recently issued an Opinion on the need for non-human primate (NHP) use in biomedical research, and the possibilities of replacing NHP use with alternatives, as part of the Directive 86/609/EEC revision process. Here, we summarise our recent complaint to the European Ombudsman about SCHER's Opinion and the entire consultation process. It is our opinion that the Working Group almost entirely failed to address its remit, and that the Group was unbalanced and contained insufficient expertise. The Opinion presumed the validity of NHP research with inadequate supporting evidence, and ignored substantial evidence against the need for NHP research and examples of valid alternatives that could replace the use of NHPs. Because the European Commission and others might base their approach to NHP research directly on the inquiry's findings during the revision of Directive 86/609/EEC, the implications of a flawed analysis of the efficacy of NHP research are extremely serious, both for animal welfare and for human health and safety. The conduct of the SCHER inquiry, and its published Opinion, should therefore be of major and widespread concern, and should not be given any political, scientific or legislative credibility.
Physiologically-based Simulation Modelling for the Reduction of Animal Use in the Discovery of Novel Pharmaceuticals
Authors: Thomas Simon
Published: April 2019

The global pharmaceutical industry is estimated to use close to 20 million animals annually, in in vivo studies which apply the results of fundamental biomedical research to the discovery and development of novel pharmaceuticals, or to the application of existing pharmaceuticals to novel therapeutic indications. These applications of in vivo experimentation include: a) the use of animals as disease models against which the efficacy of therapeutics can be tested; b) the study of the toxicity of those therapeutics, before they are administered to humans for the first time; and c) the study of their pharmacokinetics —i.e. their distribution throughout, and elimination from, the body. In vivo pharmacokinetic (PK) studies are estimated to use several hundred thousand animals annually. The success of pharmaceutical research currently relies heavily on the ability to extrapolate from data obtained in such in vivo studies to predict therapeutic behaviour in humans. Physiologically-based modelling has the potential to reduce the number of in vivo animal studies that are performed by the pharmaceutical industry. In particular, the technique of physiologically-based pharmacokinetic (PBPK) modelling is sufficiently developed to serve as a replacement for many in vivo PK studies in animals during drug discovery. Extension of the technique to incorporate the prediction of in vivo therapeutic effects and/or toxicity is less well-developed, but has potential in the longer-term to effect a significant reduction in animal use, and also to lead to improvements in drug discovery via the increased rationalisation of lead optimisation.
Definition of the Applicability Domains of Knowledge-based Predictive Toxicology Expert Systems by Using a Structural Fragment-based Approach
Authors: Ellison Claire M. , Enoch Steven J. , Cronin Mark T.D. , Madden Judith C. , Judson Philip
Published: April 2019

The applicability domain of a (quantitative) structure–activity relationship ([Q]SAR) must be defined, if a model is to be used successfully for toxicity prediction, particularly for regulatory purposes. Previous efforts to set guidelines on the definition of applicability domains have often been biased toward quantitative, rather than qualitative, models. As a result, novel techniques are still required to define the applicability domains of structural alert models and knowledge-based systems. By using Derek for Windows as an example, this study defined the domain for the skin sensitisation structural alert rule-base. This was achieved by fragmenting the molecules within a training set of compounds, then searching the fragments for those created from a test compound. This novel method was able to highlight test chemicals which differed from those in the training set. The information was then used to designate chemicals as being either within or outside the domain of applicability for the structural alert on which that training set was based.
The use of Mechanisms and Modes of Toxic Action in Integrated Testing Strategies: The Report and Recommendations of a Workshop held as part of the European Union OSIRIS Integrated Project
Authors: Vonk J. Arie , Benigni Romualdo , Hewitt Mark , Nendza Monika , Segner Helmut , van de Meent Dik , Cronin Mark T.D.
Published: April 2019

This report on The Potential of Mode of Action (MoA) Information Derived from Non-testing and Screening Methodologies to Support Informed Hazard Assessment, resulted from a workshop organised within OSIRIS (Optimised Strategies for Risk Assessment of Industrial Chemicals through Integration of Non-test and Test Information), a project partly funded by the EU Commission within the Sixth Framework Programme. The workshop was held in Liverpool, UK, on 30 October 2008, with 35 attendees. The goal of the OSIRIS project is to develop integrated testing strategies (ITS) fit for use in the REACH system, that would enable a significant increase in the use of non-testing information for regulatory decision making, and thus minimise the need for animal testing. One way to improve the evaluation of chemicals may be through categorisation by way of mechanisms or modes of toxic action. Defining such groups can enhance read-across possibilities and priority settings for certain toxic modes or chemical structures responsible for these toxic modes. Overall, this may result in a reduction of in vivo testing on organisms, through combining available data on mode of action and a focus on the potentially most-toxic groups. In this report, the possibilities of a mechanistic approach to assist in and guide ITS are explored, and the differences between human health and environmental areas are summarised.
The Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) In Vitro System: Design and Experimental Protocol
Authors: Hein Stephanie , Bur Michael , Kolb Tobias , Muellinger Bernhard , Schaefer Ulrich F. , Lehr Claus-Michael
Published: April 2019

The development of aerosol medicines typically involves numerous tests on animals, due to the lack of adequate in vitro models. A new in vitro method for testing pharmaceutical aerosol formulations on cell cultures was developed, consisting of an aerosolisation unit fitting a commercial dry powder inhaler (HandiHaler®, Boehringer Ingelheim, Germany), an air-flow control unit (Akita®, Activaero, Germany) and a custom-made sedimentation chamber. This chamber holds three Snapwell® inserts with monolayers of pulmonary epithelial cells. The whole set-up, referred to as the Pharmaceutical Aerosol Deposition Device On Cell Cultures (PADDOCC) system, aims to mimic the complete process of aerosol drug delivery, encompassing aerosol generation, aerosol deposition onto pulmonary epithelial cells and subsequent drug transport across this biological barrier, to facilitate the investigation of new aerosol formulations in the early stages of development. We describe here, the development of the design and the protocol for this device. By testing aerosol formulations of budesonide and salbutamol sulphate, respectively, reproducible deposition of aerosol particles on, and the integrity of, the pulmonary cell monolayer could be demonstrated.
Combined In Vitro Tests as an Alternative to In Vivo Eye Irritation Tests
Authors: Ying Yang , Xingfen Yang , Wengai Zhang , Jinheng Cai , Jinyu Xue , Guangyu Yang , Xiaohua Tan , Xiaoping Xie , Xikun Xiong , Junming Huang , Xiang Guo
Published: April 2019

Accurate methods that test the eye irritation potential of chemicals, which do not involve the use of animals, are needed to meet new regulatory standards. We evaluated the applicability and predictive capacity of five in vitro tests for eye irritation: the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) assay; the Chorioallantoic Membrane-Trypan Blue Staining (CAM-TBS) assay; the Fluorescein Leakage Test (FLT); the 3T3-Neutral Red Uptake (3T3-NRU) cytotoxicity assay; and the red blood cell (RBC) haemolysis assay. A panel of 16 chemicals (some at multiple concentrations) was assessed by using the five tests, and the results were compared with historical in vivo Draize test data. The results showed rank correlation and class concordance between the five alternative methods and the Draize test for the 16 chemicals. These in vitro assays had good predictive capacity, reproducibility and reliability when compared to the Draize test. The best relationship was between the HET-CAM, CAM-TBS and FLT results, and the modified maximum average score(s) (MMAS). A prediction model (PM) was developed, based on the maximum possible correlation between the MMAS and the HET-CAM, CAM-TBS and FLT results. The PM had a good predictive capacity when compared to the results of animal tests, indicating its potential value for the in vitro screening of chemicals for eye irritation effects.
Lessons from Fruit Fly Models for PolyQ Neurodegenerative Disorders
Authors: Mallik M. , Lakhotia S.C.
Published: April 2019

Polyglutamine (polyQ) diseases, resulting from a dynamic expansion of glutamine repeats in a polypeptide, are a class of genetically inherited, late-onset neurodegenerative disorders which, despite wide expression of the mutated gene in brain and other tissues, affect defined subpopulations of neurons in a disease-specific manner. This presentation will review the advantages of modelling these neurodegenerative diseases in Drosophila, and the insights obtained. Studies with the fruit fly models have successfully identified a variety of genetic modifiers, and have helped to further our understanding of some of the molecular events that follow expression of the abnormal polyQ proteins. Expression of the mutant polyQ proteins causes, as a consequence of intra-cellular and inter-cellular networking, mis-regulation in the sensitive neurons at multiple points, such as during transcriptional regulation, post-transcriptional regulation, cell signalling, protein quality control (protein folding and degradation networks), and axonal transport, resulting ultimately in cell death. The diversity of genetic modifiers of polyQ toxicity, identified through extensive genetic screening in the fruit fly and in other models, clearly reflects a complex network effect of the presence of the mutated protein. Such a network effect poses a major challenge for therapeutic applications.
The Use of a Chemistry-based Profiler for Covalent DNA Binding in the Development of Chemical Categories for Read-across for Genotoxicity
Authors: Enoch Steven J. , Cronin Mark T.D. , Ellison Claire M.
Published: April 2019

An important molecular initiating event for genotoxicity is the ability of a compound to bind covalently with DNA. However, not all compounds that can undergo covalent binding mechanisms will result in genotoxicity. One approach to solving this problem, when in silico prediction techniques are being used, is to develop tools that allow chemicals to be grouped into categories based on their ability to bind covalently to DNA. For this analysis to take place, compounds need to be placed within categories where the trend in toxicity can be explained by simple descriptors, such as hydrophobicity. However, this can occur only when the compounds within a category are structurally and mechanistically similar. Chemistry-based profilers have the ability to screen compounds and highlight those with similar structures to a target compound, and are thus likely to act via a similar mechanism of action. Here, examples are reported to highlight how structure-based profilers can be used to form categories and hence fill data gaps. The importance of developing a well-defined and robust category is discussed in terms of both mechanisms of action and structural similarity.