Further information and literature

This book is aimed at research workers using laboratory animals. Although it is not specifically about multi-strain experiments, it discusses the basic principles of the design of animal experiments, choice of animals (including the use of isogenic strains and outbred stocks), understanding and controlling variation, experimental designs (including factorial designs), the determination of sample size, presenting and interpreting results and making the appropriate decisions. It is published by the Royal Society of Medicine Press Ltd. and can be ordered directly from them at www.rsmpress.co.uk.

The following peer-reviewed papers are relevant to the theme of this web site.

Festing MF. 2004. The choice of animal model and reduction. Altern Lab Anim 32 Suppl 2:59-64.

Careful choice of the animal model is essential, if research is to be conducted efficiently, by using the minimum number of animals in order to provide the maximum amount of information. Inbred strains of rodents provide an excellent way of controlling and investigating genetic variation in characters of interest and in response to experimental treatments. Outbred stocks, in which genetic and non-genetic factors are inextricably mixed, are much less suitable, because random and uncontrolled genetic variation tends to obscure any treatment responses. In some cases, the use of inbred strains has led to major advances in scientific understanding. The specific example given here is in the understanding of host-parasite relationships but, more generally, inbred strains have been of critical importance in research which has resulted in the award of at least 17 Nobel prizes. And yet, despite the extensive literature on the properties and scientific value of inbred strains, many scientists continue to use outbred stocks in the mistaken belief that the use of such animals will, in some mysterious way, make their research more applicable to humans. There is really no evidence that this is so, and there is much evidence that the use of inbred strains has been highly successful in many disciplines

Festing MF, Altman DG. 2002. Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR J 43:244-258. (Full text available on ILAR web site)

For ethical and economic reasons, it is important to design animal experiments well, to analyze the data correctly, and to use the minimum number of animals necessary to achieve the scientific objectives—but not so few as to miss biologically important effects or require unnecessary repetition of experiments. Investigators are urged to consult a statistician at the design stage and are reminded that no experiment should ever be started without a clear idea of how the resulting data are to be analyzed. These guidelines are provided to help biomedical research workers perform their experiments efficiently and analyze their results so that they can extract all useful information from the resulting data. Among the topics discussed are the varying purposes of experiments (e.g., exploratory vs. confirmatory); the experimental unit; the necessity of recording full experimental details (e.g., species, sex, age, microbiological status, strain and source of animals, and husbandry conditions); assigning experimental units to treatments using randomization; other aspects of the experiment (e.g., timing of measurements); using formal experimental designs (e.g., completely randomized and randomized block); estimating the size of the experiment using power and sample size calculations; screening raw data for obvious errors; using the t-test or analysis of variance for parametric analysis; and effective design of graphical data

Shaw R, Festing MF, Peers I, Furlong L. 2002. Use of factorial designs to optimize animal experiments and reduce animal use. ILAR J 43:223-232. (Full text available on ILAR web site)

Optimization of experiments, such as those used in drug discovery, can lead to useful savings of scientific resources. Factors such as sex, strain, and age of the animals and protocol-specific factors such as timing and methods of administering treatments can have an important influence on the response of animals to experimental treatments. Factorial experimental designs can be used to explore which factors and what levels of these factors will maximize the difference between a vehicle control and a known positive control treatment. This information can then be used to design more efficient experiments, either by reducing the numbers of animals used or by increasing the sensitivity so that smaller biological effects can be detected. A factorial experimental design approach is more effective and efficient than the older approach of varying one factor at a time. Two examples of real factorial experiments reveal how using this approach can potentially lead to a reduction in animal use and savings in financial and scientific resources without loss of scientific validity

Festing MFW. 2001b. Guidelines for the Design and Statistical Analysis of Experiments in Papers Submitted to ATLA. ATLA (Alternatives to Laboratory Animals) 29:427-446.

In vitro experiments need to be well designed and correctly analysed if they are to achieve their full potential to replace the use of animals in research. An “experiment” is a procedure for collecting scientific data in order to answer a hypothesis, or to provide material for generating new hypotheses, and differs from a survey because the scientist has control over the treatments that can be applied. Most experiments can be classified into one of a few formal designs, the most common being completely randomised, and randomised block designs. These are quite common with in vitro experiments, which are often replicated in time. Some experiments involve a single independent (treatment) variable, while other “factorial” designs simultaneously vary two or more independent variables, such as drug treatment and cell line. Factorial designs often provide additional information at little extra cost. Experiments need to be carefully planned to avoid bias, be powerful yet simple, provide for a valid statistical analysis and, in some cases, have a wide range of applicability. Virtually all experiments need some sort of statistical analysis in order to take account of biological variation among the experimental subjects. Parametric methods using the t test or analysis of variance are usually more powerful than non-parametric methods, provided the underlying assumptions of normality of the residuals and equal variances are approximately valid. The statistical analyses of data from a completely randomised design, and from a randomised-block design are demonstrated in Appendices 1 and 2, and methods of determining sample size are discussed in Appendix 3. Appendix 4 gives a checklist for authors submitting papers to ATLA

Festing MF. 2001a. Experimental approaches to the determination of genetic variability. Toxicol Lett 120:293-300.

Toxicology is concerned with the interaction between xenobiotics and biological molecules directly or indirectly coded in the DNA, and can be regarded as a branch of genetics. There is genetic variation in these interactions, which has important implications for risk assessment and because it can be used as a tool in studying toxic mechanisms. The genetics of susceptibility can be studied by forward or reverse genetics. Forward genetics involves working from an observed phenotype such as susceptibility to a particular xenobiotic and identifying the susceptibility genes. Often, this involves mapping and identifying quantitative trait loci, as most toxic responses have a polygenic mode of inheritance. The use of inbred strains is almost essential. Reverse genetics involves starting with a known genetic polymorphism and determining its effects on the response to xenobiotics. Studies of ‘knockout’ animals are a good example, although there are many naturally occurring polymorphisms that may affect toxic responses. In both cases, care has to be taken to ensure that the genetic background is carefully controlled in any comparison between animals thought to be carrying susceptible and resistant alleles

Festing MFW, Diamanti P, Turton JA. 2001. Strain differences in haematological response to chloramphenicol succinate in mice: implications for toxicological research. Food and Chemical Toxicology 39:375-383.

Much toxicological research continues to be done using genetically undefined “outbred” stocks of mice and rats, although the case for using isogenic strains has been made repeatedly in the literature over a period of more than two decades. Also, very few studies are conducted using more than one strain, with the result that genetic variation in response is seldom apparent to the investigator. Here we report qualitative and quantitative strain differences in the haematological response to chloramphenicol succinate (CAPS) when administered by gavage at 500-2500 mg/kg for 7 days, to four inbred strains of mouse (C3H/He, CBA/Ca, BALB/c and C57BL/6) and one outbred stock (CD-1). CAPS caused anaemia and reticulocytopenia in all mouse strains, and leucopenia in the inbred strains but not in the outbred CD-1 stock. All four inbred strains showed significant (P<0.01) responses to CAPS at lower dose levels than in CD-1 mice, which were phenotypically more variable than the inbred animals. A simulated experiment, using a sample of records from the present study, showed that the use of two mice at each dose level using CD-1, CBA, BALB/c and C57BL/6 (48 total mice), would have given a more sensitive experiment than the use of 47 CD-1 mice alone, and would also have shown that the response is partly strain dependent. These studies provide additional evidence that inbred strains, because of their greater sensitivity and other valuable properties, should be more widely used in toxicology

Beck JA, Lloyd S, Hafezparast M, Lennon-Pierce M, Eppig JT, Festing MF, Fisher EM. 2000. Genealogies of mouse inbred strains. Nat Genet 24:23-25.

The mouse is a prime organism of choice for modelling human disease. Over 450 inbred strains of mice have been described, providing a wealth of different genotypes and phenotypes for genetic and other studies. As new strains are generated and others become extinct, it is useful to review periodically what strains are available and how they are related to each other, particularly in the light of available DNA polymorphism data from microsatellite and other markers. We describe the origins and relationships of inbred mouse strains, 90 years after the generation of the first inbred strain. Given the large collection of inbred strains available, and that published information on these strains is incomplete, we propose that all genealogical and genetic data on inbred strains be submitted to a common electronic database to ensure this valuable information resource is preserved and used efficiently

Festing MF. 1999a. Reduction in animal use in the production and testing of biologicals. Dev Biol Stand 101:195-200.

In the control of biologicals, animals are used largely to measure the concentration of a specific substance, rather than as a “model” of humans, so there is considerable scope for the development of replacement alternatives. When animals continue to be used, a critical analysis of guidelines and regulations has suggested many ways in which the use of animals could be reduced [1]. However, the widespread failure to use genetically and microbiologically defined animals is scientifically questionable and almost certainly results in the use of excessive numbers. International standardisation on a small number of genetically defined F1 hybrid mice should lead to greater precision in individual tests as well as greater comparability among different laboratories

Festing MFW. 1999b. Warning: the use of genetically heterogeneous mice may seriously damage your research. Neurobiology of Aging 20:237-244.

Genetically heterogeneous (GH) mice and rats continue to be widely used in research even though the case for using isogenic strains has been argued repeatedly. The paper by Miller et al. in this issue appears to be the only one in the last 22y to attempt a scientific justification for the continued use of (a limited subset of) GH stocks. However, although they are to be commended for bravery, they fail to make their case. GH stocks represent poor material for controlled studies because genetic heterogeneity normally leads to phenotypic variability and a decline in experimental sensitivity. To counter this argument, Miller et al. claim that phenotypic variability may actually be smaller in GH animals than in their isogenic parents. Were this so (e.g., all mice being short lived, small, and aggressive), it is difficult to see how the use of such a stock could increase the generality of research results based on it, as claimed by Miller et al. Isogenic strains are a vital, proven, and powerful resource for biomedical research, and should be used in preference to GH stocks by all scientists who use laboratory rodents

Festing MFW. 1997. Experimental design and husbandry. Experimental Gerontology 32:39-47.

Rodent gerontology experiments should be carefully designed and correctly analyzed so as to provide the maximum amount of information for the minimum amount of work. There are five criteria for a “good” experimental design. These are applicable both to in vivo and in vitro experiments: (1) The experiment should be unbiased so that it is possible to make a true comparison between treatment groups in the knowledge that no one group has a more favourable “environment.” (2) The experiment should have high precision so that if there is a true treatment effect there will be a good chance of detecting it. This is obtained by selecting uniform material such as isogenic strains, which are free of pathogenic micro organisms, and by using randomized block experimental designs. It can also be increased by increasing the number of observations. However, increasing the size of the experiment beyond a certain point will only marginally increase precision. (3) The experiment should have a wide range of applicability so it should be designed to explore the sensitivity of the observed experimental treatment effect to other variables such as the strain, sex, diet, husbandry, and age of the animals. With in vitro data, variables such as media composition and incubation times may also be important. The importance of such variables can often be evaluated efficiently using “factorial” experimental designs, without any substantial increase in the overall number of animals. (4) The experiment should be simple so that there is little chance of groups becoming muddled. Generally, formal experimental designs that are planned before the work starts should be used. (5) The experiment should provide the ability to calculate uncertainty. In other words, it should be capable of being statistically analyzed so that the level of confidence in the results can be quantified

Festing, MF 1997. Fat rats and carcinogenesis screening. Nature 388:321-322

No abstract available

Kacew S, Festing MF. 1996. Role of rat strain in the differential sensitivity to pharmaceutical agents and naturally occurring substances. J Toxicol Environ Health 47:1-30.

The development of drugs to combat diseases, chemicals to improve food production, or compounds to enhance the quality of life necessitates, by law, the use of laboratory animals to test their safety. In order to simulate the human condition it is necessary to choose a species in which pharmacokinetic and toxicokinetic mechanisms are established and resemble those of humans. The advantages of the use of the rat in drug and chemical toxicity testing include (a) metabolic pathway similarities to humans; (b) numerous similar anatomical and physiological characteristics; (c) a large database, which is extremely important for comparative purposes; and (d) the ease of breeding and maintenance of animals at relatively low cost. However, the choice of rat can be complicated, especially when over 200 different strains of rat are known to exist. The aim of this review is to summarize genetically determined differences in the responsiveness of rat strains to drugs and naturally occurring chemicals and to show that susceptibility is dependent on the target organ sensitivities, which may also be strain dependent. It is suggested that detailed studies of strain differences may help to clarify toxic mechanisms. Such studies are usually best conducted using inbred strains in which the genetic characteristics have been fixed, rather than in outbred stocks in which individual samples of animals may differ, the phenotype is variable, and the stocks are subject to substantial genetic drift. The fact that strains may differ also needs to be taken into account in assessing the potential hazard of the chemical, particularly when a study involves only a single strain and therefore provides no assessment of likely strain variation

Festing MFW. 1995. Use of a multi-strain assay could improve the NTP carcinogenesis bioassay program. Environmental Health Perspectives 103:44-52.

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 B6C3F1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Festing MF. 1994. Reduction of animal use: experimental design and quality of experiments. Lab Anim 28:212-221.

Poorly designed and analysed experiments can lead to a waste of scientific resources, and may even reach the wrong conclusions. Surveys of published papers by a number of authors have shown that many experiments are poorly analysed statistically, and one survey suggested that about a third of experiments may be unnecessarily large. Few toxicologists attempted to control variability using blocking or covariance analysis. In this study experimental design and statistical methods in 3 papers published in toxicological journals were used as case studies and were examined in detail. The first used dogs to study the effects of ethanol on blood and hepatic parameters following chronic alcohol consumption in a 2 x 4 factorial experimental design. However, the authors used mongrel dogs of both sexes and different ages with a wide range of body weights without any attempt to control the variation. They had also attempted to analyse a factorial design using Student’s t-test rather than the analysis of variance. Means of 2 blood parameters presented with one decimal place had apparently been rounded to the nearest 5 units. It is suggested that this experiment could equally well have been done in 3 blocks using 24 instead of 46 dogs. The second case study was an investigation of the response of 2 strains of mice to a toxic agent causing bladder injury. The first experiment involved 40 treatment combinations (2 strains x 4 doses x 5 days) with 3-6 mice per combination. There was no explanation of how the experiment involving approximately 180 mice had actually been done, but unequal subclass numbers suggest that the experiment may have been done on an ad hoc basis rather than being properly designed. It is suggested that the experiment could have been done as 2 blocks involving 80 instead of about 180 mice. The third study again involved a factorial design with 4 dose levels of a compound and 2 sexes, with a total of 80 mice. Open field behaviour was examined. The author incorrectly used the t-test to analyse the data, and concluded that there was no dose effect, when a correct analysis showed this to be highly significant. In all case studies the scientists presented means +/- standard deviations or standard errors involving only the animals contributing to that mean, rather than the much better estimates that would be obtained with a pooled estimate of error. This is virtually a universal practice.(ABSTRACT TRUNCATED AT 400 WORDS)

Festing MF. 1993. Genetic variation in outbred rats and mice and its implications for toxicological screening. J Exp Anim Sci 35:210-220.

There are two basic types of laboratory rodent used in toxicological screening. Isogenic (inbred) strains are rather like clones of genetically identical individuals whereas outbred stocks are usually more variable, though the amount of variability depends on the previous history of the colony. In some cases outbred stocks may be genetically quite uniform. Many different strains of both types are available. Both types and a variety of strains are used for toxicological screening. There is clear evidence of important genetic variation both in spontaneous disease and in response to toxic agents, yet little account is taken of this in choosing suitable animals. Three options appear to be available. The first is to ignore genetic variation and use a single isogenic strain. However, if the strain happens to be insensitive to the test chemical, a toxic chemical may be judged to be relatively safe. The second option would be to synthesize a genetically heterogeneous stock by crossing two or more strains. However, this could lead to both increased false positive and false negative results as experimental “noise” either obscures true treatment effects, or is mistaken for a treatment effect. The third option is to use more than one strain, but without increasing the total number of animals used. This would provide a broad range of genotypes, so reducing the chance that they are all insensitive, without increasing experimental noise. This appears to be the only sensible way of broadening the genetic base in toxicological screening. Where strain differences are found, they may provide a tool for studying toxic mechanisms, which may be helpful in extrapolating to human populations