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For scientific, ethical and economic reasons, experiments involving animals should be appropriately designed, correctly analysed and transparently reported. This increases the scientific validity of the results, and maximises the knowledge gained from each experiment. A minimum amount of relevant information must be included in scientific publications to ensure that the methods and results of a study can be reviewed, analysed and repeated. Omitting essential information can raise scientific and ethical concerns. We report the findings of a systematic survey of reporting, experimental design and statistical analysis in published biomedical research using laboratory animals. Medline and EMBASE were searched for studies reporting research on live rats, mice and non-human primates carried out in UK and US publicly funded research establishments. Detailed information was collected from 271 publications, about the objective or hypothesis of the study, the number, sex, age and/or weight of animals used, and experimental and statistical methods. Only 59% of the studies stated the hypothesis or objective of the study and the number and characteristics of the animals used. Appropriate and efficient experimental design is a critical component of high-quality science. Most of the papers surveyed did not use randomisation (87%) or blinding (86%), to reduce bias in animal selection and outcome assessment. Only 70% of the publications that used statistical methods described their methods and presented the results with a measure of error or variability. This survey has identified a number of issues that need to be addressed in order to improve experimental design and reporting in publications describing research using animals. Scientific publication is a powerful and important source of information; the authors of scientific publications therefore have a responsibility to describe their methods and results comprehensively, accurately and transparently, and peer reviewers and journal editors share the responsibility to ensure that published studies fulfil these criteria.

The safety of chemicals, drugs, novel foods and genetically modified crops is often tested using repeat-dose sub-acute toxicity tests in rats or mice. It is important to avoid misinterpretations of the results as these tests are used to help determine safe exposure levels in humans. Treated and control groups are compared for a range of haematological, biochemical and other biomarkers which may indicate tissue damage or other adverse effects. However, the statistical analysis and presentation of such data poses problems due to the large number of statistical tests which are involved. Often, it is not clear whether a \"statistically significant\" effect is real or a false positive (type I error) due to sampling variation. The author's conclusions appear to be reached somewhat subjectively by the pattern of statistical significances, discounting those which they judge to be type I errors and ignoring any biomarker where the p-value is greater than p = 0.05. However, by using standardised effect sizes (SESs) a range of graphical methods and an over-all assessment of the mean absolute response can be made. The approach is an extension, not a replacement of existing methods. It is intended to assist toxicologists and regulators in the interpretation of the results. Here, the SES analysis has been applied to data from nine published sub-acute toxicity tests in order to compare the findings with those of the author's. Line plots, box plots and bar plots show the pattern of response. Dose-response relationships are easily seen. A \"bootstrap\" test compares the mean absolute differences across dose groups. In four out of seven papers where the no observed adverse effect level (NOAEL) was estimated by the authors, it was set too high according to the bootstrap test, suggesting that possible toxicity is under-estimated.

Too many pre-clinical experiments are giving results which cannot be reproduced. This may be because the experiments are incorrectly designed. In “Completely randomized” (CR) and “Randomised block” (RB) experimental designs, both the assignment of treatments to experimental subjects and the order in which the experiment is done, are randomly determined. These designs have been used successfully in agricultural and industrial research and in clinical trials for nearly a century without excessive levels of irreproducibility. They must also be used in pre-clinical research if the excessive level of irreproducibility is to be eliminated. A survey of 100 papers involving mice and rats was used to determine whether scientists had used the CR or RB designs. The papers were assigned to three categories “Design acceptable”, “Randomised to treatment groups”, so of doubtful validity, or “Room for improvement”. Only 32 ± 4.7% of the papers fell into the first group, although none of them actually named either the CR or RB design. If the current high level of irreproducibility is to be eliminated, it is essential that scientists engaged in pre-clinical research use “Completely randomised” (CR), “Randomised block” (RB), or one of the more specialised named experimental designs described in textbooks on the subject.

Outbred mouse stocks, often used in genetics, toxicology and pharmacology research, have been generated in rather haphazard ways. Understanding the characteristics of these stocks and their advantages and disadvantages is important for experimental design. In many studies these mice are used inappropriately, wasting animals' lives and resources on suboptimal experiments. Recently, however, researchers from the field of complex trait analysis have capitalized on the genetics of outbred stocks to refine the identification of quantitative trait loci. Here we assess the most widely used outbred stocks of mice and present guidelines for their use.

There are often large strain differences in the response of laboratory animals to toxic chemicals and carcinogens, with some strains being totally resistant to dose levels that cause acute toxicity and/or cancer in other strains. The current National Toxicology Program carcinogenesis bioassay (NTP-CB) uses only a single isogenic strain of mice and rats and may therefore miss some carcinogens. New short-term tests to predict mutagenesis and possible carcinogenesis are validated using data from the NTP-CB. If the animal data are inaccurate, it may hinder this validation. The accuracy of the NTP-CB could be improved by using two or more strains of each species without increasing the total number of animals. It would be possible to continue to use sample sizes of 48-50 animals, but subdivide these into groups of 12 animals of 4 different strains (48 animals total) per dose/sex group, for example, instead of 48 identical animals. This would quadruple the number of genotypes without any substantial increase in cost. Such a multistrain \"factorial\" design would, on average, be statistically more powerful then the present design and should increase the chance of detecting carcinogens that currently may give equivocal results or go undetected because the test animal strains happen to be specifically resistant. When strains differ in response, studies of differences in metabolism, pharmacokinetics, DNA damage/ repair, cellular responses, and in some cases identification of genetic loci governing sensitivity may provide biological information on toxic mechanisms that would help in assessing human risk and setting permissible exposure limits. The NTP may have made the world a safer place for F344 rats and B6 C3 F1mice. The challenge now is to show that the results can be generalized to other genotypes and that the database can be used to validate short-term tests. A first step could be to explore the importance of strain differences in the carcinogenesis bioassay.

Many laboratory rats used in testing the carcinogenic safety of chemicals are strains which are 'fat, frail and dying young' in badly designed and inefficient experiments, in which the rodents sometimes die before tests for long-term toxicity are completed. They are also costly and unethical because greater numbers of animals are needed. It is recommended that suitable long-lived test panels of 'isogenic' strains be developed for use in future, until eventually sufficiently accurate \\+i\\in vitro\\-i\\ tests might be developed, rendering animal testing unnecessary. (Abstract quotes from original text)

More than 50 million animals are used in biomedical research in the world each year. It is highly desirable that this number is reduced both for ethical and for economic reasons. Better experimental design could lead to the use of fewer animals and improve the repeatability of animal experiments so that alternative methods would be easier to validate. Screening experiments aimed at identifying rodent carcinogens would be more powerful if more than one strain of mice and/or rats were used. Attempts to validate alternative test methods by using chemicals already tested in the Draize test for eye irritation are complicated by limited information on the interexperiment variability of the whole animal test. In academic toxicological research, surveys suggest that many experiments are poorly designed, and some seem to be unnecessarily large.

Festing discusses the inbred or isogenic line of mice developed by Clarence Little. Little's mice and his research laboratory have been responsible for a great deal of scientific knowledge in the past millennium.

Animal Research and Ethical Conflict. By M. T. Philips and J. A. Sechzer. Springer-Verlag: 1989. Pp.251. DM132, £43.50.Animal Experimentation: The Consensus Changes. Edited by Gill Langley. Macmillan: 1989. Pp.260. Hbk £27.50; pbk £10.95. Distributed by Chapman and Hall in the United States $45, $15.95.