Safety testing of acellular pertussis vaccines: Use of animals and 3Rs alternatives.

The current test of acellular Bordetella pertussis (aP) vaccines for residual pertussis toxin (PTx) is the Histamine Sensitization test (HIST), based on the empirical finding that PTx sensitizes mice to histamine. Although HIST has ensured the safety of aP vaccines for years, it is criticized for the limited understanding of how it works, its technical difficulty, and for animal welfare reasons. To estimate the number of mice used worldwide for HIST, we surveyed major aP manufacturers and organizations performing, requiring, or recommending the test. The survey revealed marked regional differences in regulatory guidelines, including the number of animals used for a single test. Based on information provided by the parties surveyed, we estimated the worldwide number of mice used for testing to be 65,000 per year: ∼48,000 by manufacturers and ∼17,000 by national control laboratories, although the latter number is more affected by uncertainty, due to confidentiality policies. These animals covered the release of approximately 850 final lots and 250 in-process lots of aP vaccines yearly. Although there are several approaches for HIST refinement and reduction, we discuss why the efforts needed for validation and implementation of these interim alternatives may not be worthwhile, when there are several in vitro alternatives in various stages of development, some already fairly advanced. Upon implementation, one or more of these replacement alternatives can substantially reduce the number of animals currently used for the HIST, although careful evaluation of each alternative's mechanism and its suitable validation will be necessary in the path to implementation.

La reducción del número de animales de experimentación y el cálculo del tamaño muestral: una mesa con cinco patas / Reduction of the number of animals in experiments and sample size calculation: a five-legged table

El cálculo del tamaño muestral necesario para la consecución de los objetivos de un experimento está basado en cuatro factores interdependientes: tamaño de efecto, nivel de significación, potencia estadística, y variabilidad de la muestra. El trabajo de planificación previo a la ejecución del estudio es fundamental para obtener el máximo de información con el número mínimo de animales (AU)

Sample size calculation to accomplish with the objectives of an experiment is based in four interdependent factors: effect size, significance level, statistical power and sample variability. The planning of the study, prior to execution, is fundamental to obtain the maximum of information from the minimum number of animals (AU)

Perceived barriers to the adoption of alternatives to laboratory animal use for rabies diagnosis.

The use of laboratory animals is still common practice, but some uses can be replaced by alternative methods, such as Virus Isolation in Cell Culture (VICC) instead of the Mouse Inoculation Test (MIT) for rabies diagnosis. The objective of this work was to describe current rabies diagnosis methods in Brazil and other countries, and the constraints associated with replacing this use of mice with alternative methods. Nine out of 12 Brazilian and 14 out of 43 non-Brazilian respondents reported that they currently used the MIT. Respondents in countries other than Brazil, male respondents, and those already employing in vitro methods for rabies diagnosis, expressed higher levels of support for the use of alternatives. The most frequently reported constraints associated with the use of alternatives were lack of laboratory facilities, equipment and materials (cited 17 times by respondents), and lack of financial resources (cited 15 times). The results indicate that many laboratories continue to use mice for rabies diagnosis. The proportion of laboratories that use mice appears to be especially high in Brazil, despite animal protection laws and technical guidelines that favour the use of alternatives. The barriers to the adoption of alternative methods identified in the current study provide a basis for facilitating changes in Brazil and elsewhere.

Prolonged pain research in mice: trends in reference to the 3Rs.

This literature review documents trends in the use of mice in prolonged pain research, defined herein as research that subjects mice to a source of pain for at least 14 days. The total amount of prolonged pain research on mice has increased dramatically in the past decade for the 3 pain categories examined: neuropathic, inflammatory, and chronic pain. There has also been a significant rise in the number of prolonged mouse pain studies as a proportion of all mouse studies and of all mouse pain studies. The use of transgenic mice has also risen significantly in prolonged pain research, though not as a proportion of all mice used in prolonged pain research. There has not been significant overall change in the number of mice being used per study for any of the 3 pain categories or for any of 3 common pain inducement models: chronic constriction injury, partial sciatic nerve ligation, and complete Freund's adjuvant. Finally, although most authors referred to approval of experiments by an institutional nonhuman animal use committee, there were no references to the "3Rs" in a random selection of 55 papers examined. Given the proportionally high volume of mice used in invasive research and the gravity of studies that inflict lasting pain, these trends raise serious questions about whether the 3Rs principles of Replacement, Reduction, and Refinement are being appropriately implemented by researchers and institutions.

Assessment of diurnal systemic dose of agrochemicals in regulatory toxicity testing--an integrated approach without additional animal use.

Integrated toxicokinetics (TK) data provide information on the rate, extent and duration of systemic exposure across doses, species, strains, gender, and life stages within a toxicology program. While routine for pharmaceuticals, TK assessments of non-pharmaceuticals are still relatively rare, and have never before been included in a full range of guideline studies for a new agrochemical. In order to better understand the relationship between diurnal systemic dose (AUC(24h)) and toxicity of agrochemicals, TK analyses in the study animals is now included in all short- (excluding acute), medium- and long-term guideline mammalian toxicity studies including reproduction/developmental tests. This paper describes a detailed procedure for the implementation of TK in short-, medium- and long-term regulatory toxicity studies, without the use of satellite animals, conducted on three agrochemicals (X11422208, 2,4-D and X574175). In these studies, kinetically-derived maximum doses (KMD) from short-term studies instead of, or along with, maximum tolerated doses (MTD) were used for the selection of the high dose in subsequent longer-term studies. In addition to leveraging TK data to guide dose level selection, the integrated program was also used to select the most appropriate method of oral administration (i.e., gavage versus dietary) of test materials for rat and rabbit developmental toxicity studies. The integrated TK data obtained across toxicity studies (without the use of additional/satellite animals) provided data critical to understanding differences in response across doses, species, strains, sexes, and life stages. Such data should also be useful in mode of action studies and to improve human risk assessments.

A Kenyan perspective on the use of animals in science education and scientific research in Africa and prospects for improvement.

INTRODUCTION: Animal experimentation is common in Africa, a region that accords little priority on animal protection in comparison to economic and social development. The current study aimed at investigating the prevalence of animal experimentation in Kenya, and to review shortfalls in policy, legislation, implementation and enforcement that result in inadequate animal care in Kenya and other African nations. METHODS: Data was collected using questionnaires, administered at 39 highly ranked academic and research institutions aiming to identify those that used animals, their sources of animals, and application of the three Rs. Perceived challenges to the use of non-animal alternatives and common methods of euthanasia were also queried. Data was analyzed using Epidata, SPSS 16.0 and Microsoft Excel. RESULTS: Thirty-eight (97.4%) of thirty-nine institutions reported using animals for education and/or research. Thirty (76.9%) institutions reported using analgesics or anesthetics on a regular basis. Thirteen (33.3%) institutions regularly used statistical methods to minimize the use of animals. Overall, sixteen (41.0%) institutions explored the use of alternatives to animals such as cell cultures and computer simulation techniques, with one (2.6%) academic institution having completely replaced animals with computer modeling, manikins and visual illustrations. The commonest form of euthanasia employed was chloroform administration, reportedly in fourteen (29.8%) of 47 total methods (some institutions used more than one method). Twenty-eight (71.8%) institutions had no designated ethics committee to review or monitor protocols using animals. CONCLUSION: Animals are commonly used in academic and research institutions in Kenya. The relative lack of ethical guidance and oversight regarding the use of animals in research and education presents significant concerns.

The current status of animal use and alternatives in Korean veterinary medical schools.

Two new Korean laws regulating animal welfare and the humane use of animals in science came into effect in 2008 and 2009. Both these laws impose ethical committee review prior to the performance of animal experiments in research, testing and education. This study briefly summarises the new Korean laws, and investigates the current status regarding the numbers of animals used, the alternatives to animals which are used, the curricula relating to the humane use of animals, and ethical review practices in Korean veterinary education. Approximately 4,845 animals, representing 20 different species, were used in veterinary medical education in Korea in 2007. Korea has begun to introduce formal courses on animal welfare for the humane treatment of animals used in experiments, and an ethical protocol review system prior to animal use in education. Korea is moving toward better animal welfare, by incorporating practices consistent with international standards. The information presented represents the first such data gathered in Korea, which should prove useful for monitoring the implementation of replacement, reduction, and refinement measures in animal use for education purposes.

European experimental animal use declines ever so slightly.

The latest report on experimental animal use in the EU indicates a slight decrease. However, the figures, which are for 2008, show that around 12 million animals are still used in laboratories throughout Europe. To date, only a summary of the data has been published, and so this report is limited to giving a comparison of trends and significant changes. The impact on the figures of the revision of Directive 86/609/EEC and the 7th Amendment to the Cosmetics Directive 76/768/EEC is discussed. In addition, consideration is given to which areas of research and regulation will need to be closely monitored, and the importance of continuing to push for the implementation of Three Rs initiatives is highlighted.

More is less: reducing animal use by raising awareness of the principles of efficient study design and analysis.

Good experimental design and the appropriate use of statistical tests form the corner stone of high-quality scientific research. This is especially important when the experiments involve the use of laboratory animals, to ensure that their use is appropriate and that the minimum number of animals will be used that will provide data which are sufficiently statistically-sound to meet the objectives of the study. One way to raise awareness of the importance of efficient study design and analysis is to provide training courses. This paper reports the views of participants at two such training schools, with reference to why they felt that attendance was necessary and how effective they felt the experience had been. The implications of the responses are discussed, and considerations for future training events are noted.

Planning for reduction.

Reduction is one of the Three Rs which can be readily achieved in practice. This can be done by careful consideration of the experimental strategy and the implementation of good experimental design. Moreover, strategic planning leads to 'best' scientific practice and can have a positive impact on both refinement and replacement. The FRAME Reduction Steering Committee has developed a flow chart for an overall strategy for planning and conducting biomedical research. This, and important planning considerations for each of the steps proposed, are discussed. The strategy involves taking an initial overview and undertaking related background research, then planning a sequence of experiments expected to give satisfactory results with the least animal use and minimal severity, choosing an efficient design for each experiment in the sequence, reviewing the results of one experiment before progressing to the next, and conducting an overall analysis at the end of the programme. This approach should minimise animal use and maximise the quality of the resultant scientific output.

Worldwide trends in the use of animals in research: the contribution of genetically-modified animal models.

The Three Rs--Reduction, Replacement and Refinement--which were first proposed in 1959 by Russell and Burch, have become widely accepted principles in the governance of humane animal research. However, there is substantial variation in the ways in which different countries document the numbers and types of research animals used, making it difficult to determine how effectively the Three Rs are being implemented. Here, we provide the first data illustrating worldwide trends in animal use for research purposes. To document global trends in animal use, we sampled 2691 articles from 24 countries, published between 1983 and 2007, in four scientific journals. We show that the percentage of articles reporting animal use has risen in the past 15 years. The rising popularity of genetic modification methods has contributed to this trend: reported genetically-modified animal use has more than doubled since 1997. We also show that mice are the most commonly-used species for genetic modification, and that, even in 2007, relatively inefficient random integration methods were still widely used to achieve genetic modification. These results illustrate shortcomings in the effort to implement the Three Rs in animal research.