Validation: A highly charged concept

In order that a proposal for an alternative to an animal test be developed as an internationally accepted guideline, there needs to be consensus on the validity of the method proposed. Over the years, considerable attempts have been made to ‘validate’ promising alternatives. Probably without exception, these validation programmes demanded considerable budgets whereas the high expectations as to the output, which would justify the costs involved, were hardly ever met. What went wrong? Obviously, as for each new animal test, each new alternative to an animal test should be subjected to a critical appraisal procedure involving its scientific justification, its sensitivity and its reproducibility, before it could be internationally acceptable. Although there may be differences of opinion on the extent of this exercise, there is considerable agreement that validation in one way or another is essential. None the less, validation programmes so far have not resulted in the broad acceptance of any alternative test method. There may be two reasons for this failure. First, the results of the validation studies may have been unsatisfactory, which could mean that either the method subjected to validation failed to show the desired relevance and reliability, or the validation study as such yielded inconclusive results. Secondly, despite clear-cut (supporting) results from the validation exercise, toxicologists/regulators appear reluctant actually to use the data provided for hazard and risk assessment procedures because of a lack of confidence with the (types of) endpoints of the new test. The latter in particular can be considered a major hurdle in the process of acceptance of alternative tests. Therefore, an independent and objective review of any new test, with a view to its usefulness as a contribution to the set of data essential for hazard characterization and risk assessment, should be considered the first step of any comprehensive validation project. Further, the establishment of international centres such as the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and the European Centre for the Validation of Alternative Methods (ECVAM) where scientists, including regulators, can meet and discuss strategies not only for validation but also for the use of alternative methods in risk assessment, is considered essential for a good understanding of the relevance of the new in vitro, toxicity tests.     

Evaluation of research activities and research needs to increase the impact and applicability of alternative testing strategies in risk assessment practice

The present paper aims at identifying strategies to increase the impact and applicability of alternative testing strategies in risk assessment. To this end, a quantitative and qualitative literature evaluation was performed on (a) current research efforts in the development of in vitro methods aiming for alternatives to animal testing, (b) the possibilities and limitations of in vitro methods for regulatory purposes and (c) the potential of physiologically-based kinetic (PBK) modeling to improve the impact and applicability of in vitro methods in risk assessment practice. Overall, the evaluation showed that the focus of state-of-the-art research activities does not seem to be optimally directed at developing in vitro alternatives for those endpoints that are most animal-demanding, such as reproductive and developmental toxicity, and carcinogenicity. A key limitation in the application of in vitro alternatives to such systemic endpoints is that in vitro methods do not provide so-called points of departure, necessary for regulators to set safe exposure limits. PBK-modeling could contribute to overcoming this limitation by providing a method that allows extrapolation of in vitro concentration-response curves to in vivo dose-response curves. However, more proofs of principle are required.     

Toxicology: a science and an art

Toxicology is both a science and an art. The science of toxicology characterizes the adverse effects of a chemical by integrating all available scientific information. Toxicologists must ask whether current protocols truly characterize the toxicity of a chemical in terms of identifying all the potential adverse effects. The art of toxicology involves the use of a database to predict toxic risk. A key question for the future of toxicology is “can alternative models contribute to progress or are they useless?” This paper concludes in favour of progress if such alternative models help to improve the science of toxicology. As a practical approach to improve the art of toxicology it proposes reinforcement of flexibility of current regulations, encouragement of the production and submission of data of alternative tests, and evaluation of these data on a strictly scientific basis. ECVAM, the European Centre for the Validation of Alternative Methods, will play a major role in implementing this new and more scientific approach to toxicology.     

Assessment of reproductive toxicity under REACH

The European REACH regulation requires the evaluation of reproductive toxicity in screening tests according to OECD TG 421 and 422 for substances above the tonnage level of 10 tons/year. The overall aim of this paper is to increase flexibility in combination with a reduced number of experimental animals. Therefore, in contrast to the existing approach the registrant should have the possibility to file a dossier for a substance at the level of 10 tons/year and above also on the basis of data from a developmental toxicity study (OECD TG 414) plus a full-scale subacute toxicity study (OECD TG 407 according to the 1995 protocol). The proposed new test strategy takes into account overall considerations of duty of care and animal welfare. It enables an assessment of developmental toxicity on a definitive instead of a screening level. Registrants should be allowed to select between these two options, either the existing approach (OECD TG 421/407 and alternatively TG 422) or the approach proposed in this paper (OECD TG 407 plus TG 414).     

Science based guidance for the assessment of endocrine disrupting properties of chemicals

The European legislation on plant protection products (Regulation (EC) No. 1107/2009) and biocides (Directive 98/8/EC), as well as the regulation concerning chemicals (Regulation (EC) No. 1907/2006 ‘REACH’) only support the marketing and use of chemical products on the basis that they do not induce endocrine disruption in humans or non-target species. However, there is currently no agreed guidance on how to identify and evaluate endocrine activity and disruption. Consequently, an ECETOC task force was formed to provide scientific criteria that may be used within the context of these three legislative documents. Specific scientific criteria for the determination of endocrine disrupting properties that integrate information from both regulatory (eco)toxicity studies and mechanistic/screening studies are proposed. These criteria combine the nature of the adverse effects detected in studies which give concern for endocrine toxicity with an understanding of the mode of action of toxicity so that adverse effects can be explained scientifically. The criteria developed are presented in the form of flow charts for assessing relevant effects for both humans and wildlife species. In addition, since not all chemicals with endocrine disrupting properties are of equal hazard, assessment of potency is also proposed to discriminate chemicals of high concern from those of lower concern. The guidance presented in this paper includes refinements made to an initial proposal following discussion of the criteria at a workshop of invited regulatory, academic and industry scientists.     

欧洲官方药品质量控制实验室联盟的运行和管理

目的：介绍欧洲官方药品质量控制实验室联盟的日常运行和管理,为发挥我国各级药检所协同合作、资源互补提供制度和管理上的借鉴。方法：通过调研和查阅欧洲药品健康管理局发布的系列关于欧洲官方药品质量控制实验室联盟的章程、制度和技术指导原则,以及与欧洲药品质量控制实验室同行面对面的交流,全面详细了解欧洲官方药品质量控制实验室联盟的日常工作、运行机制和管理方式。结果与结论：在秘书处的统一协调领导下,欧洲官方药品质量控制实验室联盟有健全的管理体系,在实验室质量管理体系、能力验证、打击假药、上市后药品质量监督检验等方面发挥了重要的作用。     

Defining the role of ECVAM in the development, validation and acceptance of alternative tests and testing strategies

The background to the establishment of the European Centre for the Validation of Alternative Methods (ECVAM) is reviewed, and the main events at the opening of the Centre and an ECVAM symposium on practical aspects of validation are summarized. Finally, recommendations made to ECVAM for consideration in developing the Centre's strategy are listed.     

Comparison of the mouse Embryonic Stem cell Test, the rat Whole Embryo Culture and the Zebrafish Embryotoxicity Test as alternative methods for developmental toxicity testing of six 1,2,4-triazoles

The relatively high experimental animal use in developmental toxicity testing has stimulated the search for alternatives that are less animal intensive. Three widely studied alternative assays are the mouse Embryonic Stem cell Test (EST), the Zebrafish Embryotoxicity Test (ZET) and the rat postimplantation Whole Embryo Culture (WEC). The goal of this study was to determine their efficacy in assessing the relative developmental toxicity of six 1,2,4-triazole compounds,¹ flusilazole, hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole. For this purpose, we analyzed effects and relative potencies of the compounds in and among the alternative assays and compared the findings to their known in vivo developmental toxicity. Triazoles are antifungal agents used in agriculture and medicine, some of which are known to induce craniofacial and limb abnormalities in rodents. The WEC showed a general pattern of teratogenic effects, typical of exposure to triazoles, mainly consisting of reduction and fusion of the first and second branchial arches, which are in accordance with the craniofacial malformations reported after in vivo exposure. In the EST all triazole compounds inhibited cardiomyocyte differentiation concentration-dependently. Overall, the ZET gave the best correlation with the relative in vivo developmental toxicities of the tested compounds, closely followed by the EST. The relative potencies observed in the WEC showed the lowest correlation with the in vivo developmental toxicity data. These differences in the efficacy between the test systems might be due to differences in compound kinetics, in developmental stages represented and in the relative complexity of the alternative assays.     

欧洲官方药品质量控制实验室联盟管理机制介绍

目的：介绍欧洲官方药品质量控制实验室联盟的管理机制,为我国药检系统的管理和能力建设提供借鉴。方法：通过调研和查阅欧洲药品健康管理局（EDQM）发布的系列关于欧洲官方药品质量控制实验室联盟的章程、制度和技术指导原则,以及与欧洲药品质量控制实验室同行面对面的交流,全面详细地了解欧洲官方药品质量控制实验室联盟的资质管理、状态评估和成员申请等各项组织管理工作。结果与结论：在秘书处的统一协调领导下,欧洲官方药品质量控制实验室联盟在资质管理、成员申请等方面具有一套严格完整的工作程序和评估办法,确保各成员实验室能够按照联盟的要求高质量地履职尽责。     

Safety assessment of myristic acid as a food ingredient.

Myristic acid is used in the food industry as a flavor ingredient. It is found widely distributed in fats throughout the plant and animal kingdom, including common human foodstuffs, such as nutmeg. Myristic acid (a 14-carbon, straight-chain saturated fatty acid) has been shown to have a low order of acute oral toxicity in rodents. It may be irritating in pure form to skin and eyes under exaggerated exposure conditions, but is not known or predicted to induce sensitization responses. Myristic acid did not induce a mutagenic response in either bacterial or mammalian systems in vitro. Relevant subchronic toxicity data are available on closely related fatty acid analogs. In particular, a NOEL of >6000mg/kg was reported for lauric acid (a 12-carbon, straight-chain saturated fatty acid) following dietary exposure to male rats for 18 weeks and a NOEL of >5000mg/kg was reported for palmitic acid (a 16-carbon, straight-chain saturated fatty acid) following dietary exposure to rats for 150 days. The data and information that are available indicate that at the current level of intake, food flavoring use of myristic acid does not pose a health risk to humans.     

The intra- and inter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro: Results of a ring-study in five laboratories

Due to regulatory constraints and ethical considerations, research on alternatives to animal testing to predict the skin sensitization potential of novel chemicals has gained a high priority. Accordingly, different in vitro, in silico and in chemico approaches have been described in the scientific literature to achieve this goal. To replace regulatory approved animal tests, these alternatives need to be transferable to other labs, their within and between laboratory reproducibility must be assured, and their predictivity should be high. The KeratinoSens assay is a cell-based reporter gene assay to screen substances with a full dose-response assessment. It is based on a stable transgenic keratinocyte cell line. The induction of a luciferase gene under the control of the antioxidant response element (ARE) derived from the human AKR1C2 gene is determined. Here we report on the results of a ring-study with five laboratories performing the KeratinoSens assay on a set of 28 test substances. The assay was found to be easily transferable to all laboratories. Overall both the qualitative (sensitizer/non-sensitizer categorization) and the quantitative (concentration for significant gene induction) results were reproducible between laboratories. A detailed analysis of the transferability, the within- and between laboratory reproducibility and the predictivity is presented.     

The urgent need for in vitro tests in ecotoxicology

Ecotoxicological risk assessment should be regarded as consisting of several elements including molecular biology, cell toxicology, toxicology, ecology, chemistry and computer modelling [including quantitative structure-activity relationship (QSAR) considerations]. A successful risk assessment requires an approach that integrates data and knowledge from all the separate elements. The relative importance of a certain element will vary, depending on the situation or the chemical in question. One important, perhaps the most important, piece of information needed in risk assessment is the information about the concentration range at which a chemical exerts adverse effects on the organisms living in the aquatic environment. Without such information no prediction can be made or safety factors established. A combination of good in vitro tests could provide that information. A proper choice of endpoints in in vitro testing could also provide information on sublethal effects and toxic mechanisms involved, enabling even a more efficient approach than whole animal testing. For aquatic in vitro toxicology to progress further, there is a need for a more integrated, a more comparative and a more mechanistic approach and for large-scale evaluations. It would be beneficial if researchers could agree on using a common set or a few sets of reference chemicals in their studies and if some organization would take the scientific responsibility for aiding in the development of a more integrated strategy.     

EMA与我国不同用途制药用水质要求的对比分析

欧洲药品管理局（EMA）于2018年11月发布了"制药用水质量指导原则（草案）"，详细介绍了在人用、兽用制剂和原料药生产时，不同情况下注射用水的选择。介绍EMA该文件中对不同用途的水质要求，并与国内的相关要求进行对比，期望引起有关各方关注和思考，保障我国制药用水的质量，进而保证药品质量，保护用药者安全。     

Pulmonary toxicity after exposure to military-relevant heavy metal tungsten alloy particles

Significant controversy over the environmental and public health impact of depleted uranium use in the Gulf War and the war in the Balkans has prompted the investigation and use of other materials including heavy metal tungsten alloys (HMTAs) as nontoxic alternatives. Interest in the health effects of HMTAs has peaked since the recent discovery that rats intramuscularly implanted with pellets containing 91.1% tungsten/6% nickel/2.9% cobalt rapidly developed aggressive metastatic tumors at the implantation site. Very little is known, however, regarding the cellular and molecular mechanisms associated with the effects of inhalation exposure to HMTAs despite the recognized risk of this route of exposure to military personnel. In the current study military-relevant metal powder mixtures consisting of 92% tungsten/5% nickel/3% cobalt (WNiCo) and 92% tungsten/5% nickel/3% iron (WNiFe), pure metals, or vehicle (saline) were instilled intratracheally in rats. Pulmonary toxicity was assessed by cytologic analysis, lactate dehydrogenase activity, albumin content, and inflammatory cytokine levels in bronchoalveolar lavage fluid 24 h after instillation. The expression of 84 stress and toxicity-related genes was profiled in lung tissue and bronchoalveolar lavage cells using real-time quantitative PCR arrays, and in vitro assays were performed to measure the oxidative burst response and phagocytosis by lung macrophages. Results from this study determined that exposure to WNiCo and WNiFe induces pulmonary inflammation and altered expression of genes associated with oxidative and metabolic stress and toxicity. Inhalation exposure to both HMTAs likely causes lung injury by inducing macrophage activation, neutrophilia, and the generation of toxic oxygen radicals.     

Identification and assessment of the effects of chemicals on reproduction and development (reproductive toxicology)

Most studies for determining the reproductive toxicity of a chemical have to be conducted with whole animals. Test procedures used to investigate parts or the whole of the reproductive cycle are described in current guidelines. Other techniques, such as in vitro methods, and those for investigating specific events in the cycle, are under development. Epidemiological studies can give valuable information, although they are difficult to perform and interpret in practice. There is a need for more epidemiological studies of exposed populations and for recording and quantifying the concentrations of chemicals to which such populations are exposed. It is suggested that animal experiments should be programmed in a stepwise manner, and should take into account effects seen in previous toxicity studies. The programme of tests for determining reproductive toxic potential should be established on a case-by-case basis, since many factors will influence the choice of studies and the sequence in which they should be performed.     

An enhanced tiered toxicity testing framework with triggers for assessing hazards and risks of commodity chemicals

This paper presents an enhanced integrated testing framework based on tiered testing and endpoint-specific decision triggers envisioned for application to commodity chemical safety assessments. The framework has two tiers in which exposure information can be integrated with hazard data at each Tier. Tier 1 tests are used to screen chemicals for major toxic effects (i.e., acute toxicity potential, target organs of repeat dose toxicity, genotoxicity potential, neurotoxicity potential, reproductive toxicity potential, immunotoxicity potential, and developmental toxicity potential), and to direct planning for more complex and targeted testing in Tier 2. The proposed decision triggers coupled with information on use and potential for exposure allow for scientifically-based decisions to be made about further testing in Tier 2, indicating which specific endpoints and tests warrant further evaluation, and which do not. The testing framework addresses risks to humans during all stages of development and provides data relevant to assessing hazards to sensitive subpopulations, such as infants and children. The REACH program in Europe and TSCA in the United States have led to an increased focus on development of hazard and risk information for chemicals used in industrial processes and consumer products. This framework and its toxicity decision triggers will allow for scientifically justified evaluation of chemicals that is comprehensive in terms of hazard screening, focuses resources on the specific complex tests that are most important for hazard characterization, and minimizes the use of animals.     

2014年5月—2015年3月美国FDA批准的突破性治疗药物

FDA突破性治疗药物指定评审新政的实施填补了治疗某些疾病药物的空白,并得到制药企业的积极响应.药品评价与研究中心(CDER)截止到2015年3月,生物药品评价与研究中心(CBER)截止到2015年1月,FDA共收到299项突破性治疗药物申请,其中授权87项,拒绝161项,其余51项处在审查中,2014年5月—2015年3月获批了18项突破性治疗药物,从药物治疗类别、作用靶点或机制、获得批准上市情况以及不良反应等方面重点介绍其中的突出品种.     

Alternatives to animal testing: A review

The number of animals used in research has increased with the advancement of research and development in medical technology. Every year, millions of experimental animals are used all over the world. The pain, distress and death experienced by the animals during scientific experiments have been a debating issue for a long time. Besides the major concern of ethics, there are few more disadvantages of animal experimentation like requirement of skilled manpower, time consuming protocols and high cost. Various alternatives to animal testing were proposed to overcome the drawbacks associated with animal experiments and avoid the unethical procedures. A strategy of 3 Rs (i.e. reduction, refinement and replacement) is being applied for laboratory use of animals. Different methods and alternative organisms are applied to implement this strategy. These methods provide an alternative means for the drug and chemical testing, up to some levels. A brief account of these alternatives and advantages associated is discussed in this review with examples. An integrated application of these approaches would give an insight into minimum use of animals in scientific experiments.     

Exposure-driven risk assessment: Applying exposure-based waiving of toxicity tests under REACH

The REACH Regulation 1907/2006/EC aims to improve knowledge of the potential risks to humans and the environment of the large number of chemicals produced and used in the EU. The testing requirements are likely to trigger numerous toxicological studies, potentially involving millions of experimental animals, despite the professed goal of REACH to reduce vertebrate testing. It may be necessary therefore to shift emphasis away from animal studies towards more pragmatic strategies, reserving animal tests for the substances of greatest concern. One approach is to waive certain tests based on levels of exposure to the substance. This review explores application of ‘Exposure-Based Waiving’ (EBW) of toxicity studies, with a particular focus on inhalation where possible, considering the potential qualitative and quantitative supporting arguments that might be made, including the use of thresholds of toxicological concern. Incorporating EBW into intelligent testing strategies for substance registration could advance the goals of REACH and the 3Rs (reduction, replacement and refinement of animals in research) by reducing the usage of animals in toxicity tests, whilst maintaining appropriate protection of human health and the environment. However greater regulatory evaluation, acceptance and guidance are required for EBW to achieve its full impact.     

The challenge of reproductive and developmental toxicology under REACH.

The European Union's REACH regulation has explicit requirements for reproductive and developmental toxicity data on all substances manufactured in or imported into the European Union at > or = 10 metric tons/year. Meeting the data requirements with whole-animal testing could result in the use of almost 22 million vertebrate animals for the registration of existing chemicals and cost up to several hundred thousand dollars per registered substance. The requirement for financial and animal resources can be reduced by the use of in vitro testing, quantitative structure-activity relationship models, and grouping of related substances. Although REACH strongly encourages these methods of avoiding vertebrate animal testing, it does not appear that in vitro testing or quantitative structure-activity relationship analysis will be able to replace whole-animal reproductive and developmental toxicity testing. Grouping of related compounds offers the possibility, perhaps in conjunction with in vitro testing and structure-activity analysis, of reducing vertebrate animal testing provided there is sufficient information on the related compounds and sufficient reason to believe that the related compounds will have similar toxicological properties. The designation of a substance as a reproductive or developmental toxicant follows criteria that do not consider the dose level of the substance at which reproductive or developmental effects occur, as long as excessive generalized toxicity does not occur. This method of labeling substances without consideration of effective dose level does not provide information on the actual risk of the chemical. Designation of a substance as a reproductive or developmental toxicant may have important implications under REACH and can be expected to result in the need to obtain authorization for marketing of the substance in the European Union.