Reduce Animal Use Research Articles

Simultaneous Analysis of Multiple Monoclonal Antibody Biotherapeutics by LC-MS/MS Method in Rat Plasma Following Cassette-Dosing

We have recently developed a general liquid chromatography-tandem mass spectrometric (LC-MS/MS) method using a stable isotope-labeled (SIL) monoclonal antibody (mAb) as an internal standard (IS) for single-analyte quantification of mAb (Li et al. Anal Chem 84(3):1267-1273, 2012). The method offers an advantage over ligand binding assay in reducing the time and resources needed for bioanalytical support in preclinical stages of drug development. In this paper, we report another marked increase in assay efficiency for multi-analyte bioanalysis using unique surrogate peptides for each analyte and the strategic choice of the SIL-IS peptide. The method was qualified for the simultaneous determinations of four mAbs in rat plasma and applied to samples from discrete- and cassette-dosed rats. The pharmacokinetic parameters of the four mAbs of cassette dosing were comparable to those of discrete dosing and of enzyme-linked immunosorbent assay results. Although there may be limitations and special considerations for cassette-dosing of biologics, these results demonstrate the robust performance of the multi-analyte LC-MS/MS method allowing cassette-dosing that would ultimately reduce animal use and improve efficiency.

Establishment of a Molecular Embryonic Stem Cell Developmental Toxicity Assay

The mouse embryonic stem cell test (EST) is a 10-day screen for teratogenic potential developed to reduce animal use for embryotoxicity testing of chemicals (Spielmann, 2005; Spielmann et al., 1997). In this study, we used the cytotoxicity IC(50) values and transcriptional expression changes as primary endpoints in a shorter 4-day version of the EST, the molecular embryonic stem cell assay. Mouse D3 embryonic stem cells were used for cytotoxicity assessment (monolayers) or grown as embryoid bodies in low attachment plates for transcriptional profiling. Sixty-five compounds with known in vivo teratogenicity (33 teratogens and 32 nonteratogens) were evaluated to develop a model for classifying compounds with teratogenic potential. The expression of 12 developmentally regulated gene targets (nanog, fgf5, gsc, cd34, axin2, apln, chst7, lhx1, fgf8, sox17, foxa2, and cxcr4) was measured following exposure of embryoid bodies to a single compound concentration (0.1 × the cytotoxicity IC(20)) for 4 days. In the decision-tree model, compounds with IC(50) values < 22 µM were categorized as teratogens, whereas compounds in the two groups with IC(50) values between 22-200 µM and > 200 µM were categorized as teratogens if ≥ 8 and 12 genes, respectively, were deregulated by at least 10%. Forty-seven of 65 compounds of the training set were correctly identified (72% total concordance). In a test set of 12 additional compounds (5 teratogens, 7 nonteratogens), 10 were correctly classified by this approach (83% concordance). The false positive rate in the training and test sets was 24 and 0%, respectively, indicating that this assay has potential to identify teratogens.

Reducing suffering in animal models and procedures involving seizures, convulsions and epilepsy

This report is based on discussions and submissions from an expert working group consisting of veterinarians, animal care staff and scientists with expert knowledge relevant to the field and aims to facilitate the implementation of the Three Rs (replacement, reduction and refinement) in the use of animal models or procedures involving seizures, convulsions and epilepsy. Each of these conditions will be considered, the specific welfare issues discussed, and practical measures to reduce animal use and suffering suggested. The emphasis is on refinement since this has the greatest potential for immediate implementation, and some general issues for refinement are summarised to help achieve this, with more detail provided on a range of specific refinements.

Don’t Judge A Neuron Only by Its Cover: Neuronal Function in In Vitro Developmental Neurotoxicity Testing

Classical cases of developmental neurotoxicity (DNT) in humans and advances in risk assessment methods did not prevent the emergence of new chemicals with (suspected) DNT potential. Exposure to these chemicals may be related to the increased worldwide incidence of learning and neurodevelopmental disorders in children. DNT is often investigated in a traditional manner (in vivo using large numbers of experimental animals), whereas development of in vitro methods for DNT reduces animal use and increases insight into cellular and molecular mechanisms of DNT. Several essential neurodevelopmental processes, including proliferation, migration, differentiation, formation of axons and dendrites, synaptogenesis, and apoptosis, are already being evaluated in vitro using biochemical and morphological endpoints. Yet, investigation of chemical-induced effects on the development of functional neuronal networks, including network formation, inter- and intracellular signaling and neuronal network function, is underrepresented in DNT testing. This view therefore focuses on in vitro models and innovative experimental approaches for functional DNT testing, ranging from optical and electrophysiological measurements of intra- and intercellular signaling in neural stem/progenitor cells to measurements of network activity in neuronal networks using multielectrode arrays. The development of functional DNT assays will strongly support the decision-making process for measures to prevent potential chemical-induced adverse effects on neurodevelopment and cognition in humans. We therefore argue that for risk assessment, biochemical and morphological approaches should be complemented with investigations of neuronal (network) functionality.

In vivo flow cytometric Pig‐a and micronucleus assays: Highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated‐dose designs

Combining multiple genetic toxicology endpoints into a single in vivo study, and/or integrating one or more genotoxicity assays into general toxicology studies, is attractive because it reduces animal use and enables comprehensive comparative analysis using toxicity, metabolism, and pharmacokinetic information from the same animal. This laboratory has developed flow cytometric scoring techniques for monitoring two blood-based genotoxicity endpoints-micronucleated reticulocyte frequency and gene mutation at the Pig-a locus-thereby making combination and integration studies practical. The ability to effectively monitor these endpoints in short-term and repeated dosing schedules was investigated with the carcinogen/noncarcinogen pair benzo(a)pyrene (BP) and pyrene (Pyr). Male Sprague-Dawley rats were treated via oral gavage for 3 or 28 consecutive days with several dose levels of Pyr, including maximum tolerated doses. BP exposure was administered by the same route but at one dose level, 250 or 125 mg/kg/day for 3-day and 28-day studies, respectively. Serial blood samples were collected up to Day 45, and were analyzed for Pig-a mutation with a dual labeling method (SYTO 13 in combination with anti-CD59-PE) that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. A mutant cell enrichment step based on immunomagnetic column separation was used to increase the statistical power of the assay. BP induced robust mutant reticulocyte responses by Day 15, and elevated frequencies persisted until study termination. Mutant erythrocyte responses lagged mutant reticulocyte responses, with peak incidences observed on Day 30 of the 3-day study (43-fold increase) and on Day 42 of the 28-day study (171-fold increase). No mutagenic effects were apparent for Pyr. Blood samples collected on Day 4, and Day 29 for the 28-day study, were evaluated for micronucleated reticulocyte frequency. Significant increases in micronucleus frequencies were observed with BP, whereas Pyr had no effect. These results demonstrate that Pig-a and micronucleus endpoints discriminate between these structurally related carcinogenic and noncarcinogenic agents. Furthermore, the high sensitivity demonstrated with the enrichment protocol indicates that the Pig-a endpoint is suitable for both repeated-dose and acute studies, allowing integration of mutagenic and clastogenic endpoints into on-going toxicology studies, and use as a short-term assay that provides efficient screening and mechanistic information in vivo.

Quo vadis integrated testing strategies? Experiences and observations from the work floor

Integrated testing strategies (ITSs) have received much attention as promising tools for more resource-efficient hazard and risk assessment of chemicals and for reducing animal use in toxicological testing. The usage of ITSs crucially depends on their acceptance and application by various stakeholder groups, for example chemical industry, scientific organisations and regulatory authorities. However, little is known about stakeholders’ views on the use and application of ITSs. In this study, we present results from semi-structured interviews with different stakeholder groups. Interviewees were asked to express their personal views and opinions about what an ITS is or should be, about ITS advantages and limitations, about ITS implementation and acceptance and about needs for further research. Using qualitative data analysis, we identified a set of core themes that stakeholders considered most relevant with respect to these six topics. Our results illustrate that stakeholder perspectives differed considerably for each of the topics addressed. We found particularly diverging views across stakeholder groups with respect to ITS limitations and acceptance. This underlines that improving stakeholder integration and intensifying the dialogue about useful and successful ITS applications should receive more attention for strengthening ITSs as effective decision-support tools.

Relative parameter sensitivity in prenatal toxicity studies with substances classified as developmental toxicants

Developmental toxicity testing according to the globally standardized OECD 414 protocol is an important basis for decisions on classification and labeling of developmental toxicants in the European Union (EU). This test requires relatively large animal numbers, given that parental and offspring generations are involved. In vitro assay designs and systems biology paradigms are being developed to reduce animal use and to improve prediction of human hazard. Such approaches could benefit from the long-term experience with animal protocols and more specifically from information on the relevance of effects observed in these tests for developmental toxicity. Therefore, we have analyzed relative parameter sensitivity in 22 publicly available developmental toxicity studies, representing about one third of all classified developmental toxicants under European legislation. Maternal and fetal weight effects and fetal survival were most often affected parameters at the developmental Lowest Observed Adverse Effect Level (dLOAEL), followed by skeletal malformations. Specific end points such as cleft palate were observed in fewer studies at dLOAEL, but if observed may have been crucial in classification and labeling decisions. These results are similar to earlier studies using different selections of chemicals, indicating that in general classified developmental toxicants have a similar pattern of effects at the dLOAEL as chemicals in general. These findings are discussed within the perspective of the development of innovative alternative approaches to developmental hazard assessment.

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn)

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO–BP) were identified after 24h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥687 genes) as well as a variety of GO–BP, as compared to MEHP, PHE and PENG (≤55 genes with no significant changes in GO–BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO–BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

Inroads to Predict in Vivo Toxicology—An Introduction to the eTOX Project

There is a widespread awareness that the wealth of preclinical toxicity data that the pharmaceutical industry has generated in recent decades is not exploited as efficiently as it could be. Enhanced data availability for compound comparison (“read-across”), or for data mining to build predictive tools, should lead to a more efficient drug development process and contribute to the reduction of animal use (3Rs principle). In order to achieve these goals, a consortium approach, grouping numbers of relevant partners, is required. The eTOX (“electronic toxicity”) consortium represents such a project and is a public-private partnership within the framework of the European Innovative Medicines Initiative (IMI). The project aims at the development of in silico prediction systems for organ and in vivo toxicity. The backbone of the project will be a database consisting of preclinical toxicity data for drug compounds or candidates extracted from previously unpublished, legacy reports from thirteen European and European operation-based pharmaceutical companies. The database will be enhanced by incorporation of publically available, high quality toxicology data. Seven academic institutes and five small-to-medium size enterprises (SMEs) contribute with their expertise in data gathering, database curation, data mining, chemoinformatics and predictive systems development. The outcome of the project will be a predictive system contributing to early potential hazard identification and risk assessment during the drug development process. The concept and strategy of the eTOX project is described here, together with current achievements and future deliverables.

Development of a Streamlined Rat Whole Embryo Culture Assay for Classifying Teratogenic Potential of Pharmaceutical Compounds

This study describes a novel rat whole embryo culture (rWEC) teratogenicity assay that applies a simplified experimental design and statistical prediction model, resulting in reduced animal requirements and increased throughput with low prediction error rate for classifying teratogenic potential of compounds. A total of 70 compounds (38 teratogens and 32 nonteratogens) were evaluated, and the prediction model was generated from a dataset of 59 compounds. The rWEC assay requires only one test concentration (1μM) and three structural endpoints (group average morphological scores of spinal cord, heart, and number of somite pairs), which are used in a recursive partition model for classifying teratogenic liability. The model fitting concordance between the WEC assay and in vivo outcome was 83% with a standard deviation (SD) of 4.9%. The predictivity for future compounds was evaluated by using two statistical methods. Fivefold cross-validation estimated the predictivity of this model at 73% (SD 5.8%). A second estimation of predictivity was obtained from an independent test set of 11 compounds that were not used to build the prediction model and reached 82% (SD 11.6%). The overall estimate for prediction concordance is 74% (SD 5.2%). There is no statistically significant difference (p value > 0.50) in the predictivity between this model and the model supporting European Center for the Validation of Alternative Methods WEC assay with predictivity of 80% (SD 10.6%). Overall, the streamlined WEC assay is estimated to reduce animal use and operational costs by more than 50%. It substantially improves results turnaround with no loss of predictivity.

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India Unveils Roadmap for Cooperation with Africa. Indian Agriculture Minister Calls For Improved Soil Health. A*STAR, GE Global Research To Develop Integrated Advanced Medical Imaging. PRA Opens Office in Singapore. Bristol-Myers Squibb Announces Grants to Tackle Hepatitis B and Hepatitis C in Asia. Burrill &amp; Company Expands into Taiwan. Medistem Initiates Collaboration With Shanghai Jia Fu Medical Apparatus. Advinus, SignalChem Commence Collaboration On New Anti-Cancer Drugs. 3D Petri Dish to Reduce Animal Use in Research. Vermillion Receives Patent on Platelet Biomarkers of Angiogenesis. Biotech R&amp;D Outsourcing Set to Increase and Shift Overseas.

Dried Blood Spot Bioanalysis: An Evaluation of Techniques and Opportunities for Reduction and Refinement in Mouse and Juvenile Rat Toxicokinetic Studies

Toxicokinetic investigations are an essential component of nonclinical toxicology studies and generally rely on bioanalysis of plasma samples, which requires relatively large volumes of blood and, often, additional numbers of animals. Dried blood spot (DBS) analysis can substantially reduce the volume of blood needed and, therefore, presents a particular opportunity for reducing animal use in studies involving small animals; an approach consistent with industry objectives to reduce animal use and refine methods that are minimally invasive and improve animal welfare. Investigations using mice and juvenile rats indicate that implementation of DBS technology can reduce the number of animals used, and data are as good as those derived from whole blood in terms of AUC, drug-concentration-over-time curves, quantitation, accuracy, variability, and precision. These factors, and the improved data quality arising from less reliance on composite data, suggest that DBS analysis should be considered from the early stages of nonclinical development.

Best practices for the use of animals in toxicological research and testing

Animal models serve an important role in assessing preclinical safety and efficacy of new medicines and vaccines; however, such assessments can involve significant pain and distress and large numbers of animals. Best practice approaches seek to enhance animal well-being, minimize or avoid pain and distress, and use fewer animals. Advances in science and technology are providing opportunities for improved mechanism-based models and integrated safety assessments that will support improved animal welfare and reduce animal use.

Bridging the Gap Between Validation and Implementation of Non-Animal Veterinary Vaccine Potency Testing Methods.

Simple SummaryMany vaccines are tested for quality in experiments that require the use of large numbers of animals in procedures that often cause significant pain and distress. Newer technologies have fostered the development of vaccine quality control tests that reduce or eliminate the use of animals, but the availability of these newer methods has not guaranteed their acceptance by regulators or use by manufacturers. We discuss a strategic approach that has been used to assess and ultimately increase the use of non-animal vaccine quality tests in the U.S. and U.K.In recent years, technologically advanced high-throughput techniques have been developed that replace, reduce or refine animal use in vaccine quality control tests. Following validation, these tests are slowly being accepted for use by international regulatory authorities. Because regulatory acceptance itself has not guaranteed that approved humane methods are adopted by manufacturers, various organizations have sought to foster the preferential use of validated non-animal methods by interfacing with industry and regulatory authorities. After noticing this gap between regulation and uptake by industry, we began developing a paradigm that seeks to narrow the gap and quicken implementation of new replacement, refinement or reduction guidance. A systematic analysis of our experience in promoting the transparent implementation of validated non-animal vaccine potency assays has led to the refinement of our paradigmatic process, presented here, by which interested parties can assess the local regulatory acceptance of methods that reduce animal use and integrate them into quality control testing protocols, or ensure the elimination of peripheral barriers to their use, particularly for potency and other tests carried out on production batches.

Immunotoxicant Screening and Prioritization in the Twenty-first Century

Current immunotoxicity testing guidance for drugs, high production-volume chemicals, and pesticides specifies the use of animal models to assess potential biomarkers of immune system effects (e.g., lymphoid organ and bone marrow indices, histopathology) or actual measures of immune function (e.g., responses to challenge with antigens or pathogens). These assays are resource intensive and often require special training or experience to ensure reliable results. Alternative in vitro assays to detect immunosuppression and allergic hypersensitivity have the potential to reduce animal use and testing costs and increase immunotoxicity screening and prioritization efforts. Alternative models to detect immunosuppression tend to address broad modes of action because suppression may be caused by a wide variety of events; current in vitro models access the supply of innate and adaptive immune system cells as well as cellular markers associated with function, including gene expression, protein synthesis, and proliferation. Events leading to the induction of allergic hypersensitivity, particularly contact hypersensitivity, are more restricted, and alternative methods currently exploit chemical properties and activation of defined cell populations to detect and estimate the potency of skin sensitizers.

Transcriptomic Concentration-Response Evaluation of Valproic Acid, Cyproconazole, and Hexaconazole in the Neural Embryonic Stem Cell Test (ESTn)

Alternative developmental toxicity assays are urgently needed to reduce animal use in regulatory developmental toxicology. We previously designed an in vitro murine neural embryonic stem cell test (ESTn) as a model for neurodevelopmental toxicity testing (Theunissen et al., 2010). Toxicogenomic approaches have been suggested for incorporation into the ESTn to further increase predictivity and to provide mechanistic insights. Therefore, in this study, using a transcriptomic approach, we investigated the concentration-dependent effects of three known (neuro) developmental toxicants, two triazoles, cyproconazole (CYP) and hexaconazole (HEX), and the anticonvulsant valproic acid (VPA). Compound effects on gene expression during neural differentiation and corresponding regulated gene ontology (GO) terms were identified after 24 h of exposure in relation to morphological changes on day 11 of culture. Concentration-dependent responses on individual gene expression and on biological processes were determined for each compound, providing information on mechanism and concentration-response characteristics. All compounds caused enrichment of the embryonic development process. CYP and VPA but not HEX significantly enriched the neuron development process. Furthermore, specific responses for triazole compounds and VPA were observed within the GO-term sterol metabolic process. The incorporation of transcriptomics in the ESTn was shown to enable detection of effects, which precede morphological changes and provide a more sensitive measure of concentration-dependent effects as compared with classical morphological assessments. Furthermore, mechanistic insight can be instrumental in the extrapolation of effects in the ESTn to human hazard assessment.

Toward an integrated human clearance prediction strategy that minimizes animal use

Prediction of human clearance plays a critical role in early drug discovery and development. However, there is no consensus on the most appropriate method to gather such data. A variety of in vivo and in vitro methods exist and a comparison of in vitro data from a standard set of compounds has been called for. This paper compares the predictive capacity of human liver microsomes and single‐species scaling (SSS) from rat and non‐human primate for a standard set of compounds representing marketed drugs. The results enable a framework to be proposed wherein compounds are selected using in vitro methods alone or in vitro methods combined with SSS in the rat. Further investigation of this framework has the potential to increase the efficiency of drug discovery and reduce animal use. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4518–4535, 2011

On the impact of second generation mating and offspring in multi-generation reproductive toxicity studies on classification and labelling of substances in Europe

The possible impact on classification and labelling decisions of effects observed in second generation parental (P1) and offspring (F2) parameters in multi-generation studies was investigated. This was done for 50 substances classified as reproductive toxicants in Europe, for which a multi-generation study was available. The P1 and F2 effects were compared to parental (P0) and first generation offspring (F1) effects with regard to type of effect as well as incidence, magnitude and severity (IMS), at any dose level. For every study with unique P1/F2 effects, or differences in IMS, the influence of the P1/F2 findings on the classification decision was investigated. Unique P1/F2 generation findings did not play a crucial role in the classification decision of any of the 50 classified substances, except for fenarimol. This substance however provided abundant alerts on the basis of its endocrine activity and developmental neurotoxicity and would therefore also be expected to be identified as a developmental neurotoxicant in an Extended One Generation Reproductive Toxicity Study (EOGRTS). These findings, in addition to the increased number of parameters analysed, increased statistical power and reduced animal use, provide strong further support for replacement of the classical two-generation reproductive toxicity study by the EOGRTS in regulatory reproductive toxicity assessment.

Opportunities to minimise animal use in pharmaceutical regulatory general toxicology: A cross-company review

Toxicity studies in animals are carried out to identify the intrinsic hazard of a substance to support risk assessment for humans. In order to identify opportunities to minimise animal use in regulatory toxicology studies, a review of current study designs was carried out. Pharmaceutical companies and contract research organisations in the UK shared data and experience of standard toxicology studies (ranging from one to nine months duration) in rodents and non-rodents; and carcinogenicity studies in the rat and mouse. The data show that variation in study designs was primarily due to (i) the number of animals used in the main study groups, (ii) the use of animals in toxicokinetic (TK) satellite groups, and (iii) the use of animals in off-treatment recovery groups. The information has been used to propose a series of experimental designs where small adjustments could reduce animal use in practice, while maintaining the scientific objectives.

Time-Response Evaluation by Transcriptomics of Methylmercury Effects on Neural Differentiation of Murine Embryonic Stem Cells

Current globally harmonized Organisation for Economic Co-operation and Development (OECD) animal test guidelines for developmental toxicity require high numbers of experimental animals. To reduce animal use in this field, alternative developmental toxicity assays are highly desirable. We previously developed a dynamic in vitro model for screening effects of possible neurodevelopmental toxicants, using neural cell differentiation of pluripotent murine embryonic stem cells. To further mechanistically characterize the mouse neural embryonic stem cell test (ESTn) and to improve detection of possible neurodevelopmental toxicants, gene expression patterns were studied describing neural cell differentiation over time, as well as the impact on gene expression of exposure to the well-known neurotoxicant methylmercury (MeHg). A transcriptomics study was performed to examine whole-genome expression changes during the first 7 days of the cell differentiation protocol. Specific gene clusters were identified and enrichment analysis of Gene Ontology (GO) terms and gene sets derived from literature was performed using DAVID and T-profiler. Over time, a decrease of blastocyst and trophectoderm GO terms was observed, which included well-characterized pluripotency genes. Furthermore, an increase in the range of neural development-related GO terms, such as neuron differentiation and the wnt pathway, was observed. Analysis of gene expression using principle component analysis showed a time-dependent track in untreated cells, describing the process of neural differentiation. Furthermore, MeHg was shown to induce deviation from the predefined differentiation track. The compound inhibited general development GO terms and induced neural GO terms over time. This system appears promising for studying compound effects on neural differentiation in a mechanistic approach.