Nonhuman primates and teratological research

Nonhuman primates were first recognized as models for the study of developmental toxicity (teratology) following the thalidomide tragedy. Since that time they have played important roles in both testing of drugs for human safety and as models for studying specific malformations commonly seen in children. Although in vitro and alternative test systems using lower animal forms or simplified test systems have been incorporated into developmental toxicity studies, whole animal testing will be required for the foreseeable future because of the complex relationship of the maternal/embryofetal/placental unit. The nonhuman primate will be particularly valuable where equivocal results are experienced in other commonly used laboratory species, when the drug/chemical is likely to be used during pregnancy, and for human-derived biotechnical products which often are not bioactive in nonprimate species.     

GLP原则在体外毒理学评价实验室的应用

在动物福利运动的推动下,以减少、优化和替代动物试验为核心内容的体外试验系统已成为安全评价不可或缺的组成部分,在药品、化学品、化妆品毒理学评价中起到重要作用。体外试验系统不同于体内动物实验,体外毒理学实验室GLP原则的建立和运行应充分考虑体外试验系统的特殊性。目前我国专业的体外安全评价实验室的建设刚刚起步,还没有可供借鉴的成熟经验。本文从实验室组织、试验系统维护、人员职责、质量管理和运行几个方面,介绍了GLP原则在化妆品体外毒理学检验和评价实验室的应用。     

Strategy for genotoxicity testing--metabolic considerations

The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens "missed" in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance. The working group established consensus principles to guide strategy development. Thus, a human metabolite of interest should be represented in genotoxicity and carcinogenicity testing, including evaluation of alternative genotoxicity in vitro metabolic activation or test systems, and the selection of a carcinogenicity test species showing appropriate biotransformation. Appropriate action triggers need to be defined based on the extent of human exposure, considering any structural knowledge of the metabolite, and when genotoxicity is observed upon in vitro testing in the presence of metabolic activation. These triggers also need to be considered in defining the timing of human pharmaceutical ADME assessments. The working group proposed two strategies to consider; a more proactive approach, which emphasizes early metabolism predictions to drive appropriate hazard assessment; and a retroactive approach to manage safety risks of a unique or "major" metabolite once identified and quantitated from human clinical ADME studies. In both strategies, the assessment of the genotoxic potential of a metabolite could include the use of an alternative or optimized in vitro metabolic activation system, or direct testing of an isolated or synthesized metabolite. The working group also identified specific areas where more data or experiences need to be gained to reach consensus. These included defining a discrete exposure action trigger for safety assessment and when direct testing of a metabolite of interest is warranted versus the use of an alternative in vitro activation system, a universal recommendation for the timing of human ADME studies for drug candidates and the positioning of metabolite structural knowledge (through in silico systems, literature, expert analysis) in supporting metabolite safety qualification. Lastly, the working group outlined future considerations for refining the initially proposed strategies. These included the need for further evaluation of the current in vitro genotoxicity testing protocols that can potentially perturb or reduce the level of metabolic activity (potential alterations in metabolism associated with both the use of some solvents to solubilize test chemicals and testing to the guidance limit dose), and proposing broader evaluations of alternative metabolic activation sources or engineered test systems to further challenge the suitability of (or replace) the current induced liver S9 activation source.     

Behavior and neurobehavioral teratology using the ferret

A behavioral profile of the ferret is presented for those who would like to use this animal in behavioral teratology and toxicology, or other disciplines involving behavior. We have reviewed neurobehavioral teratology of lisencephalic ferrets and neuropsychology of ferrets sustaining frontal lesions, as well as most of the studies of "normal" ferret behavior that have appeared in the research literature. Emphasis is placed on discussion of the tests used and how ferrets behaved on them. The behaviors discussed include spatial (maze) learning, delayed response, visual discrimination learning, discrimination learning sets, schedule maintained behavior, shock avoidance learning and spontaneously occurring behaviors, such as ambulation in open field, spontaneous alternation and species specific behaviors. Although the use of the ferret in behavioral experiments is not yet extensive and large gaps exist in our knowledge about the basic functional capacities of this animal, the ferret is unquestionably well suited for behavioral studies.     

Exploring Extra-Binomial Variation in Teratology Data Using Continuous Mixtures

Discrete data from animal teratology experiments are known to exhibit extra-binomial variation. For example, we discuss a dominant lethal assay experiment in which male mice are exposed to various levels of radiation and are then mated to females. The response of interest is the number of resorptions out of the number of implantations. Most statistical work on analyzing such data has focused on modeling response rates as a function of dose of a suspected teratogen (radiation in this case) while accounting for the extra-binomial variability when calculating standard errors of the regression coefficients. Sometimes, however, when an unobserved genetic or exposure variable is suspected, the shape of the mixing distribution is of interest. We propose a mixture of beta-binomials (MBB) family of distributions that includes the non-parametric mixture of binomials model of Laird (1978) as a special case. The MBB family can accommodate a mixing distribution with one or more modes, and we develop a bootstrap test for multimodality. We apply the method to data from a dominant lethal teratology experiment.     

Regulatory requirements for licensing medicinal products of biotechnology

The recent and rapidly developing application of biotechnology which leads to the discovery of new therapeutic substances has raised a new set of safety issues for consideration by industry and regulatory bodies. The experience which already exists in the assessment of the safety and quality of biological products can contribute significantly to the approaches which are evolving with this new range of products. Industry and regulatory bodies should both resist the temptation to introduce testing programmes and requirements without a sound scientific rationale. This paper reviews some of the issues which should be considered when embarking on the safety evaluation of products derived from biotechnology.     

Guinea pigs in hypersensitivity testing

The aim of the present paper is to mention steps of development of predictive guinea pig animal models for contact sensitization evaluation in parallel with the development of understanding the mechanism of contact sensitization. The guinea pigs methods are reviewed and the presently accepted methods (Buehler test, Maximization test) are discussed in details. Influences on the outcome of guinea pig sensitization assays are mentioned too. The predictability of the guinea pig methods is compared with predictive methods in mice, with human tests (human repeated insult patch test, HRIPT) and epidemiological data. In the last part, a testing strategy and steps for risk assessment of contact sensitizing potential is proposed.     

Metabolic and functional studies on post-mortem human brain

The evidence that samples of human brain tissue obtained at autopsy may be used as starting material for the isolation of cellular and subcellular preparations which exhibit metabolic and functional activity when incubated in vitro has been reviewed. Supporting evidence has been found in data from model experiments which used animal brain as the source material. Active preparations have been obtained after considerable (up to 24 h) post mortem delays. Such findings are less surprising when the post mortem stability of key tissue components (enzymes, receptors, nucleic acids) and the retention of cellular integrity are examined. The data from these fields have been reviewed and their relevance to functional studies assessed. Studies which use human autopsy material must consider many additional sources of variation not found in experiments with animal brain and the major problems are briefly discussed. It is argued that functional experiments present few, if any, difficulties not already inherent in static analyses of autopsy material and some procedures which help to minimise these difficulties are outlined. Experimentation in this area is greatly aided by the finding that metabolically and functionally active preparations may be obtained from frozen tissue pieces. Dynamic studies provide a new approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of neuroactive drugs in man.     

The role of a clinical veterinarian in a safety assessment testing facility

A clinical veterinarian working in a safety assessment testing facility must balance animal welfare concerns with the attainment of sound, reproducible data necessary for regulatory studies. Drawing on her experience as a clinical veterinarian with specialty training in the field of toxicology, and past experience as a Study Director on safety assessment studies, the author describes practices that facilitate the veterinarian's role to ensure animal welfare on safety assessment studies.     

Crossing the kingdom border: Human diseases caused by plant pathogens

Interactions between pathogenic microorganisms and their hosts are varied and complex, encompassing open-field scale interactions to interactions at the molecular level. The capacity of plant pathogenic bacteria and fungi to cause diseases in human and animal systems was, until recently, considered of minor importance. However, recent evidence suggests that animal and human infections caused by plant pathogenic fungi, bacteria and viruses may have critical impacts on human and animal health and safety. This review analyses previous research on plant pathogens as causal factors of animal illness. In addition, a case study involving disruption of type III effector-mediated phagocytosis in a human cell line upon infection with an opportunistic phytopathogen, Pseudomonas syringae pv. tomato, is discussed. Further knowledge regarding the molecular interactions between plant pathogens and human and animal hosts is needed to understand the extent of disease incidence and determine mechanisms for disease prevention.     

Human teratogens update 2011: can we ensure safety during pregnancy?

Anniversaries of the identification of three human teratogens (i.e., rubella virus in 1941, thalidomide in 1961, and diethylstilbestrol in 1971) occurred in 2011. These experiences highlight the critical role that scientists with an interest in teratology play in the identification of teratogenic exposures as the basis for developing strategies for prevention of those exposures and the adverse outcomes associated with them. However, an equally important responsibility for teratologists is to evaluate whether medications and vaccines are safe for use during pregnancy so informed decisions about disease treatment and prevention during pregnancy can be made. Several recent studies have examined the safety of medications during pregnancy, including antiviral medications used to treat herpes simplex and zoster, proton pump inhibitors used to treat gastroesophageal reflux, and newer-generation antiepileptic medications used to treat seizures and other conditions. Despite the large numbers of pregnant women included in these studies and the relatively reassuring results, the question of whether these medications are teratogens remains. In addition, certain vaccines are recommended during pregnancy to prevent infections in mothers and infants, but clinical trials to test these vaccines typically exclude pregnant women; thus, evaluation of their safety depends on observational studies. For pregnant women to receive optimal care, we need to define the data needed to determine whether a medication or vaccine is "safe" for use during pregnancy. In the absence of adequate, well-controlled data, it will often be necessary to weigh the benefits of medications or vaccines with potential risks to the embryo or fetus.     

Eating nanomaterials: cruelty-free and safe? the EFSA guidance on risk assessment of nanomaterials in food and feed

Nanomaterials are increasingly being added to food handling and packaging materials, or directly, to human food and animal feed. To ensure the safety of such engineered nanomaterials (ENMs), in May 2011, the European Food Safety Authority (EFSA) published a guidance document on Risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain. It states that risk assessment should be performed by following a step-wise procedure. Whenever human or animal exposure to nanomaterials is expected, the general hazard characterisation scheme requests information from in vitro genotoxicity, toxicokinetic and repeated dose 90-day oral toxicity studies in rodents. Numerous prevailing uncertainties with regard to nanomaterial characterisation and their hazard and risk assessment are addressed in the guidance document. This article discusses the impact of these knowledge gaps on meeting the goal of ensuring human safety. The EFSA's guidance on the risk assessment of ENMs in food and animal feed is taken as an example for discussion, from the point of view of animal welfare, on what level of uncertainty should be considered acceptable for human safety assessment of products with non-medical applications, and whether animal testing should be considered ethically acceptable for such products.     

Experience of two teratology information services in Europe

Teratology Information Services (TIS) are started in different countries in Europe in order to gather available data on exogenous agents, to evaluate their pertinence to human subjects, and to apply this knowledge to specific cases. Most European centers can only be consulted by medical professionals. The experience of two such services (Lyon, France, and Bilthoven, The Netherlands) is described. Attention is given to the task of TIS, risk evaluation, operational methods, and functioning and future developments.     

Proteomic analysis and in vitro developmental toxicity tests for mechanism-based safety evaluation of chemicals

Mechanism-based safety evaluation and reduction of animal use are important issues in recent developmental toxicology. In vitro developmental toxicity tests with proteomic analysis are the most promising solution to these issues. Groebe et al. systematically applied proteomic analysis to the embryonic stem cell test, a validated in vitro developmental toxicity test, and found protein-expression changes induced by model test chemicals selected from various categories of toxicity. Cluster analysis of all the proteins with expression changes classified the test chemicals into two groups: highly embryotoxic chemicals and non- or weakly embryotoxic chemicals. In addition, some protein biomarker candidates that were known to be involved in normal development were identified. Although further mechanistic investigations are needed, the use of in vitro developmental toxicity tests with proteomic analysis will contribute to mechanism-based safety evaluation with minimal use of animals.     

Preclinical and pharmacology and toxicology of hematopoietic growth factors

Initial evaluation of the safety of biological products, whether manufactured by biotechnology or not, is dictated by the extent of knowledge of the biological effects in vitro, in animals, and in man at the proposed dose and duration of administration. The quantity of useful information that will be gained from an animal study is considered for each product on a case-by-case basis. This requires consideration of the availability of a relevant species. Relevance is determined by presence and relative affinity and distribution of receptors for the biological product, relative potency in induction of biological activity related to the putative mechanism of action and pharmacokinetic considerations. The impurities and immunogenic potential of the product are also considered in designing the preclinical studies. After evaluating all these factors, the residual lack of knowledge is FDA's basis for recommending specific preclinical testing. The considerations inherent in the preclinical evaluation of factors acting primarily on hematopoietic cells, erythropoietin (EPO), interleukins (IL's) and colony stimulating factors (CSF) (1) will be discussed using case examples.     

Human teratogens update 2011: Can we ensure safety during pregnancy?

Anniversaries of the identification of three human teratogens (i.e., rubella virus in 1941, thalidomide in 1961, and diethylstilbestrol in 1971) occurred in 2011. These experiences highlight the critical role that scientists with an interest in teratology play in the identification of teratogenic exposures as the basis for developing strategies for prevention of those exposures and the adverse outcomes associated with them. However, an equally important responsibility for teratologists is to evaluate whether medications and vaccines are safe for use during pregnancy so informed decisions about disease treatment and prevention during pregnancy can be made. Several recent studies have examined the safety of medications during pregnancy, including antiviral medications used to treat herpes simplex and zoster, proton pump inhibitors used to treat gastroesophageal reflux, and newer‐generation antiepileptic medications used to treat seizures and other conditions. Despite the large numbers of pregnant women included in these studies and the relatively reassuring results, the question of whether these medications are teratogens remains. In addition, certain vaccines are recommended during pregnancy to prevent infections in mothers and infants, but clinical trials to test these vaccines typically exclude pregnant women; thus, evaluation of their safety depends on observational studies. For pregnant women to receive optimal care, we need to define the data needed to determine whether a medication or vaccine is “safe” for use during pregnancy. In the absence of adequate, well‐controlled data, it will often be necessary to weigh the benefits of medications or vaccines with potential risks to the embryo or fetus. Birth Defects Research (Part A), 2012. © Published 2012 Wiley Periodicals, Inc.     

Reduction strategies in animal research: a review of scientific approaches at the intra-experimental, supra-experimental and extra-experimental levels

When discussing animal use and considering alternatives to animals in biomedical research and testing, the number of animals required gets to the root of the matter on ethics and justification. In this paper, some reduction strategies are reviewed. Many articles and reports on reduction of animal use focus mostly on the experimental level, but other approaches are also possible. Reduction at the intraexperimental level probably offers the greatest scope for reduction, as the design and statistical analysis of individual experiments can often be improved. Supra-experimental reduction aims to reduce the number of animals by a change in the setting in which a series of experiments take place--for example, by improved education and training, reduction of breeding surpluses, critical analysis of test specifications, and re-use of animals. At the extra-experimental level, reduction is a spin-off of other developments, rather than the direct goal. Through improved research or production strategies, aimed at better quality, consistency and safety, reduction in the number of animals used can be substantial. A revised definition of reduction is proposed, which does not include the level of information needed, as in some cases reduction in the number of animals resulting in less information or data, is still acceptable.     

Training strategies for IACUC members and the institutional official

Institutions are required by federal laws and regulations to oversee and evaluate their programs, facilities, and procedures for using animals in research, teaching, and/or testing activities. These responsibilities are specifically charged to an institutional official (IO) and an institutional animal care and use committee (IACUC). Initially, the individuals tasked with these responsibilities seldom have the requisite knowledge or experience to fulfill their charges effectively. Furthermore, simply reading the regulatory requirements does not prepare the novice IO and IACUC members to effectively monitor and guide the program. As a result, many new IOs and IACUC members are managing their responsibilities with insufficient understanding of the laws, regulations, standards, and policies. Specific training strategies for inexperienced IACUC members are needed to help them understand their responsibilities for ensuring animal welfare through an effective, high-quality, and compliant animal care and use program that supports the critical research needed to improve human and animal health. Likewise, most IOs would benefit from training to help them better understand their responsibility for enhancing or maintaining the quality of the institution's animal care and use program. Education and training should begin with an orientation to the laws, regulations, standards, and policies. Continuing training and education are also important to keep abreast of the changes in the interpretation of these laws and regulations as well as the changes in veterinary science. For both the IO and the IACUC, understanding and acceptance of their authority and responsibilities are significant factors in establishing and maintaining a quality animal care and use program.     

Assessment of the eye irritating properties of chemicals by applying alternatives to the Draize rabbit eye test: the use of QSARs and in vitro tests for the classification of eye irritation

Huggins has reported on the current situation relating to the development of alternatives to the Draize eye irritation test with rabbits, and an ECVAM Working Group have reviewed the efforts needed in order to replace this animal test within the next 10 years by using the results of non-animal assessment methods. Our report reviews regulatory experience gained over the last 20 years with the EU chemicals notification procedure with respect to the assessment of eye lesions observed in Draize tests. The nature of eye lesions and their importance for classification and labelling of possible hazards to human eyes are evaluated and discussed, with a view to promoting the development of specific in vitro assays which are able to discriminate between eye damage, moderate eye irritation, and minor irritation effects which are completely reversible within a few days. Structural alerts for the prediction of eye irritation/corrosion hazards to be classified and labelled according to international classification criteria, are presented, which should be validated in accordance with internationally agreed (OECD) principles for (Q)SAR system validation. Physicochemical limit values for prediction of the absence of any eye irritation potential relevant for human health can make available a definition of the applicability domains of alternative methods developed for the replacement of the Draize eye irritation test.     

Review: domestic animal forensic genetics – biological evidence,genetic markers,analytical approaches and challenges

This review highlights the importance of domestic animal genetic evidence sources, genetic testing, markers and analytical approaches as well as the challenges this field is facing in view of the de facto ‘gold standard’ human DNA identification. Because of the genetic similarity between humans and domestic animals, genetic analysis of domestic animal hair, saliva, urine, blood and other biological material has generated vital investigative leads that have been admitted into a variety of court proceedings, including criminal and civil litigation. Information on validated short tandem repeat, single nucleotide polymorphism and mitochondrial DNA markers and public access to genetic databases for forensic DNA analysis is becoming readily available. Although the fundamental aspects of animal forensic genetic testing may be reliable and acceptable, animal forensic testing still lacks the standardized testing protocols that human genetic profiling requires, probably because of the absence of monetary support from government agencies and the difficulty in promoting cooperation among competing laboratories. Moreover, there is a lack in consensus about how to best present the results and expert opinion to comply with court standards and bear judicial scrutiny. This has been the single most persistent challenge ever since the earliest use of domestic animal forensic genetic testing in a criminal case in the mid‐1990s. Crime laboratory accreditation ensures that genetic test results have the courts’ confidence. Because accreditation requires significant commitments of effort, time and resources, the vast majority of animal forensic genetic laboratories are not accredited nor are their analysts certified forensic examiners. The relevance of domestic animal forensic genetics in the criminal justice system is undeniable. However, further improvements are needed in a wide range of supporting resources, including standardized quality assurance and control protocols for sample handling, evidence testing, statistical analysis and reporting that meet the rules of scientific acceptance, reliability and human forensic identification standards.