Consensus Report on the Future of Animal-Free Systemic Toxicity Testing

Since March 2013, animal use for cosmetics testing for the European market has been banned. This requires a renewed view on risk assessment in this field. However, in other fields as well, traditional animal experimentation does not always satisfy requirements in safety testing, as the need for human-relevant information is ever increasing. A general strategy for animal-free test approaches was outlined by the US National Research Council’s vision document for Toxicity Testing in the 21st Century in 2007. It is now possible to provide a more defined roadmap on how to implement this vision for the four principal areas of systemic toxicity evaluation: repeat dose organ toxicity, carcinogenicity, reproductive toxicity and allergy induction (skin sensitization), as well as for the evaluation of toxicant metabolism (toxicokinetics) (Fig. 1). CAAT-Europe assembled experts from Europe, America and Asia to design a scientific roadmap for future risk assessment approaches and the outcome was then further discussed and refined in two consensus meetings with over 200 stakeholders. The key recommendations include: focusing on improving existing methods rather than favoring de novo design; combining hazard testing with toxicokinetics predictions; developing integrated test strategies; incorporating new high content endpoints to classical assays; evolving test validation procedures; promoting collaboration and data-sharing of different industrial sectors; integrating new disciplines, such as systems biology and high throughput screening; and involving regulators early on in the test development process. A focus on data quality, combined with increased attention to the scientific background of a test method, will be important drivers. Information from each test system should be mapped along adverse outcome pathways. Finally, quantitative information on all factors and key events will be fed into systems biology models that allow a probabilistic risk assessment with flexible adaptation to exposure scenarios and individual risk factors

t4 Workshop Report: Integrated Testing Strategies (ITS) for Safety Assessment

Integrated testing strategies (ITS), as opposed to single definitive tests or fixed batteries of tests, are expected to efficiently combine different information sources in a quantifiable fashion to satisfy an information need, in this case for regulatory safety assessments. With increasing awareness of the limitations of each individual tool and the development of highly targeted tests and predictions, the need for combining pieces of evidence increases. The discussions that took place during this workshop, which brought together a group of experts coming from different related areas, illustrate the current state of the art of ITS, as well as promising developments and identifiable challenges. The case of skin sensitization was taken as an example to understand how possible ITS can be constructed, optimized and validated. This will require embracing and developing new concepts such as adverse outcome pathways (AOP), advanced statistical learning algorithms and machine learning, mechanistic validation and “Good ITS Practices”.JRC.I.5-Systems Toxicolog

No alternatives? : Animal experimentation and the future of research

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Detektion und Charakterisierung der immunstimulatorischen Eigenschaften luftgetragener Pyrogene

Human whole blood is an accessible source of primary immune cells and plasma components involved in host defense against infection. It can be used both to study the human inflammatory reaction to microbes and their pyrogenic components and to detect their presence in samples by measuring the production of a representative inflammatory factor upon challenge. The latter was exploited by developing a method to evaluate the pyrogenic burden in air samples collected on filters. As fungal spores represent a large portion of airborne microbia, their contribution to the pyrogenic burden in the air was evaluated by characterizing their inflammatory activity and studying the mechanisms and molecules involved. The strategy to collect pyrogens from the air on a solid phase was further developed to enable detection of pyrogens in toxic or immunomodulatory drugs and to improve sensitivity for testing of the pyrogenic load in dialysis fluids and large volume parenterals by collecting pyrogens on albumin-linked beads. 1. An air sampling method to collect pyrogens from the air on a filter and assess cytokine release by blood incubated with the filter was established and an adequate reference material was developed, enabling field studies. These showed a good correlation between the inflammatory activity in the air and the live germ count. Donor comparison revealed that the immune response of allergic people differed from that of non-allergic individuals. Standardized, cryopreserved blood proved a suitable substitute for fresh blood that could exclude such donor variances. Comparison of this new method with the Limulus amoebocyte lysate test, which is limited to the detection of endotoxins, proved as expected that the latter underestimates the total inflammatory burden in the air. 2. The immunostimulatory capacity of fungal spores was assessed by characterization of the cytokine pattern they induced in human whole blood. The 44 species tested induced a highly homogenous pattern of cytokine release with similar kinetics, indicating that fungal spores share a surface structure that is targeted by the innate immune system. Analysis of donor variance revealed that the cytokine amounts released in human blood, both in response to fungal spores and to other stimuli employed, is determined mainly by the individual monocyte count. The relative response to the different stimuli was highly consistent. Comparison of the cytokine response of human blood with an alveolar macrophage cell line revealed a high correlation, suggesting that the blood monocytes and lung macrophages respond similarly. 3. Analysis of the role of fungal surface glycans by chemical and enzymatic detachment of sugar chains strongly questioned their proposed role as the immunostimulatory principle of fungal spores. Examination of TLR dependency of immune recognition of fungal spores using cells from respective knock-out mice revealed that the presence of TLR-2 is necessary for full cytokine response to fungal spores. Interestingly, the presence of TLR-4 appeared to suppress cytokine release. 4. The role of TLR-2 in the recognition of fungal spores suggested that the immunostimulatory principle might be structurally related to that of other TLR-2 agonists. Conidia of Cladosporium cladosporioides were subjected to butanol extraction and separation by FPLC and HPLC. This resulted in the purification of an immunostimulatory, amphiphilic compound that consists mainly of alkyl chains probably bearing sugar moieties as revealed by 1HNMR spectroscopic analysis. This compound induces a cytokine pattern consistent with that induced by the whole spores and showed the same TLR dependency. Its activity was not affected by coincubation with the LPS inhibitors Polymyxin B and LAL-F, but the LTA inhibitor PPG 1200 reduced its inflammatory capacity. 5. Developing the idea of collecting pyrogens onto a solid phase, a method was developed to collect pyrogens from liquid onto albumin-linked beads. This allowed the separation of the pyrogens from immunomodulatory or toxic drugs. The beads were washed and then brought into contact with human whole blood. It could be shown that the beads bind LPS, LTA or zymosan spikes and the sensitivity of detection was comparable to that of the standard tests for parenterals. The ability of the test to detect pyrogens could be shown in drugs known to interfere with other pyrogen tests, i.e. paclitaxel, gentamicin, prednisolone, liposomal amphotericin B and liposomal daunorubicin. 6. Further modifications of the setting aimed to accumulate very low concentrations of pyrogens in large samples on the albumin-linked beads. This increased the sensitivity of the assay by a factor of 250, which is useful for measurement of pyrogens in dialysis fluids and large volume parenterals. The tests developed in this thesis may help to evaluate the health risk of working and living environments posed by the burden of air-borne pyrogens and to improve drug safety and chronic problems in dialysis patients caused by exposure to pyrogens. The studies on the immunostimulatory qualities of fungal spores revealed that, like Gram-negative and Gram-positive bacteria, fungi also share common surface structures that are targeted by the human innate immune system. In contrast to previous suggestions, these appear to be amphiphilic molecules. A better understanding of the interactions between fungi and the immune system may provide new therapeutic targets

The Center for Alternatives to Animal Testing - Europe (CAAT-EU) : a transatlantic bridge for the paradigm shift in toxicology

The Center for Alternatives to Animal Testing - Europe (CAAT-EU) was founded based collaboration between the Johns Hopkins Bloomberg School of Public Health and the University of Konstanz. CAAT-EU, housed at the University of Konstanz, will coordinate transatlantic activities to promote humane science in research and education, and participate, as partner or coordinator, in publicly and privately funded European projects. Thomas Hartung will serve as program liaison representing Johns Hopkins University and Marcel Leist as the University of Konstanz liaison. CAAT-EU aims to: 1) Set up transatlantic consortia for international research projects on alternative methods. 2) Establish a CAAT Europe faculty and advisory board composed of sponsor representatives and prominent academics from Europe . 3) Participate in the Transatlantic Think Tank for Toxicology (t4) devoted to conceptual work for the paradigm shift in toxicology. 4) Coordinate a series of information days in Europe on relevant developments in the US, similar to the 2009 series CAAT held in the US on EU issues (one on the 7th Amendment to the EU Cosmetics Directive and one on EU and US chemical regulation). 5) Support ALTEX as the official journal of CAAT and CAAT-EU. 6) Develop strategic projects with sponsors to promote humane science and new toxicology, especially with CAAT faculty members. 7) Develop a joint education program between Johns Hopkins and the University of Konstanz, such as e-courses and the existing Humane Science Certificate program developed by CAAT, a student exchange program, and collaboration with the International Graduate School "Cell-based Characterization of De- and Regeneration" in Konstanz.publishe

Summary and Validation of New Animal-Free Toxicity Tests

Alternatives to animal testing have been developed mainly in the fields of toxicology and vaccine testing. Typical examples are the evaluation of phototoxicity, eye irritation, or skin corrosion resulting from cosmetics and industrial chemicals. Examples also can be found in other biomedical areas, however, including the control of the quality of drug preparations or for the control of the production process of biologics. For regulatory purposes, the quality, transferability, and predictivity of an alternative method need to be evaluated. This procedure often is called the “validation process” of a new method. It follows defined rules, and several governmental institutions have been established to perform, supervise, or advise on this process. As this often results in a delay of method implementation, different alternatives for the evaluation of a method’s suitability and quality are subject to discussion. We describe here the principles of model development and quality control. We also give an overview of methods that have undergone validation. Strengths and shortcomings of traditional approaches are discussed, and new developments and challenges are outlined

Integrated Testing Strategies (ITS) for safety assessment

Integrated testing strategies (ITS), as opposed to a single definitive test or fixed batteries of tests, are expected to efficiently combine different information sources in a quantifiable fashion to satisfy an information need, in this case for regulatory safety assessments. With increasing awareness of the limitations of each individual tool and the development of highly targeted tests and predictions, the need for combining pieces of evidence increases. The discussions that took place during this workshop, which brought together a group of experts coming from different related areas, illustrate the current state of the art of ITS, as well as promising developments and identifiable challenges. The case of skin sensitization was taken as an example to understand how possible ITS can be constructed, optimized and validated. This will require embracing and developing new concepts such as adverse outcome pathways (AOP), advanced statistical learning algorithms and machine learning, mechanistic validation and “Good ITS Practices”

Validation and quality control of replacement alternatives : current status and future challenges

Alternatives to animal testing have been developed mainly in the fields of toxicology and vaccine testing. Typical examples are the evaluation of phototoxicity, eye irritation or skin corrosion/irritation of cosmetics and industrial chemicals. However, examples can also be found in other biomedical areas, such the control of the quality of drug preparations for pyrogens or for the control of the production process of biologics, such as botulinum neurotoxin. For regulatory purposes, the quality, transferability and predictivity of an alternative method needs to be evaluated. This procedure is called the “validation process” of a new method. It follows defined rules, and several governmental institutions have been established to perform, supervise or advise on this process. As this often results in a delay of method implementation, different alternatives for the evaluation of a method's suitability and quality are under discussion. We describe here the principles of model development and quality control. We also give an overview on methods that have undergone validation. Strengths and shortcomings of traditional approaches are discussed, and new developments and challenges are outlined

Animal use for science in Europe

To investigate long-term trends of animal use, the EU animal use statistics from the 15 countries that have been in the EU since 1995 plus respective data from Switzerland were analyzed. The overall number of animals used for scientific purposes in these countries, i.e., about 11 million/year, remained relatively constant between 1995 and 2011, with net increases in Germany and the UK and net decreases in Belgium, Denmark, Italy, Finland, the Netherlands and Sweden. The relatively low and constant numbers of experimental animals used for safety assessment (toxicology, 8%) may be due to the particularly intensive research on alternative methods in this area. The many efficiently working NGOs, multiple initiatives of the European Parliament, and coordinated activities of industry and the European Commission may have contributed to keeping the animal numbers in this field in check. Basic biological science, and research and development for medicine, veterinary and dentistry together currently make up 65% of animal use in science. Although the total numbers have remained relatively constant, consumption of transgenic animals has increased drastically; in Germany transgenic animals accounted for 30% of total animal use in 2011. Therefore, more focus on alternatives to the use of animals in biomedical research, in particular on transgenic animals, will be important in the future. One initiative designed to provide inter-sector information exchange for future actions is the "MEP - 3Rs scientists pairing scheme" initiated in 2015 by CAAT-Europe and MEP Pietikäinen.publishe

Assessment of pyrogenic contaminations with validated human whole-blood assay

We present an internationally validated protocol for the evaluation of pyrogenic contaminations using human whole blood. In the in vitro pyrogen test (IPT) the sample is incubated with fresh or cryopreserved human whole blood, and the proinflammatory cytokine interleukin-1 beta (IL-1 beta) is detected by enzyme-linked immunosorbent assay (ELISA). In addition to detecting pyrogenic contaminations in aqueous samples, e.g., parenteral drugs; adaptations allow the assessment of lipidic, toxic or immunomodulatory substances; detection of low-grade contaminations in large-volume parenterals, e.g., dialysis water and fluids; pyrogenicity assessment of solid materials, e.g., medical devices; and evaluation of airborne pyrogenic burden. In contrast to the rabbit pyrogen test and the limulus amoebocyte lysate (LAL) test, it requires no components of animal origin. In comparison with the LAL, it also detects nonlipopolysaccharide pyrogens. In comparison with other monocyte activation tests it requires no cell preparation steps or cell culture facilities. The procedure takes 21-35 h to complete