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Genetic and Phenotypic Definition of Laboratory Mice and Rats / What Constitutes an Acceptable Genetic-Phenotypic Definition Joseph DeGeorge Associate Director, Pharmacology and Toxicology ORM, CDER/FDA Rockville, Maryland NECESSITY FOR GLOBALLY STANDARDIZED OUTBRED RATS FOR CARCINOGENICITY BIOASSAY â¢ Why is the issue of outbred strains of current importance from a regula- tory perspective? â¢ Why was there virtually no interest in the issue several (for example, 10) years ago? â¢ What makes outbred strains important now? I believe the answer to all three questions can be summed up in one simple sentence: There has really been a large change in the paradigm of pharmaceutical development. And that is the basis for my concern today. CHANGE IN LABORATORY ANIMAL SCIENCE The change has occurred over the last 10 to 15 years and mainly over the last 5 to 10 years. I address below some specific aspects of the changes that have led to my concern about standardization of the animal models on which we rely. One additional point to keep in mind is that as a regulatory agency, the US Food and Drug Administration (FDA) is the end user of the data from all of the pharmaceu- tical testing that goes on. We have to rely on it to make judgments about potential human health risks. The outcome is not a research paper but rather, marketing to millions of people around the world of a product that has gone through a particu- lar testing process. 71
72 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE GLOBALIZATION OF DRUG DEVELOPMENT The first of the major changes that have occurred recently in pharmaceutical development is the globalization of the drug development process. It has been mentioned that there is an International Conference on Harmonization of Techni- cal Requirements for International Registration for Pharmaceuticals for Human Use (ICH), where we have harmonized and in fact have agreed on certain stan- dards. However, beyond that is the fact that pharmaceutical companies are almost no longer national. There are very few national pharmaceutical companies, and most market worldwide. Most also develop their drugs as a worldwide activity. Worldwide marketing, in fact, has reached the point where we are developing a common technical document so that the test studiesâthe same studiesâare being submitted simultaneously around the world for marketing approval. This is an attempt to achieve drug approval in Europe, the United States, and Japan at roughly the same time. The extent of globalization is very large, and âglobalâ companies are head- quartered throughout the world. There is a perceived need for global standards that companies can follow to make certain that if they do a study for one country, it is acceptable in another country. This standardization is actually the basis for Office of Economic Cooperation and Development (OECD) guidances, which is familiar to most of you as providing information on various test paradigms and specifically for pharmaceuticals. ICH guidance refers to specific aspects of carci- nogenicity testing, genotoxicity testing, and reproductive toxicity testing for phar- maceuticals. If companies follow these guides, no matter where in the world they do the study, that study is accepted internationallyâat least within the United States, Europe, Japan, Australia, Canada, Taiwan, and wherever else drugs are manufactured. Companies are looking to these standards to try to establish test systems and hopefully market a pharmaceutical. One of the accomplishments of this harmonization of standards has been the elimination of duplicate testing. Thus, when an institution or company proposes a drug development plan for Japan, it is not necessary to complete another set of tests for the United States. In the past, that necessity might have been the case; or they would have made certain that the studies were done in both the United States and Japan. Furthermore, this harmonization has actually eliminated many spe- cific national test requirements. The US test requirements do not differ from the Japanese or the European under this ICH process, at least for pharmaceuticals. Such standardization is one aspect of this global drug development plan that has an impact on the use of animals. SEGMENTATION OF TOXICOLOGY TESTING Another aspect of globalized pharmaceutical development is the segmenta- tion of toxicology testing. International companies have, in fact, often changed
JOSEPH DEGEORGE 73 their development processes. It is often no longer an all in-house operation done in a particular facility where all the data are generated on the same colony of animals, which then undergo every test. There is an increasing use of contract facilities, a blend of contract facilities with sponsor facilities, and an increase of multisourcing outsourcing, going to the lowest bidder. In other words, if it is cheaper to do a certain study in the southwestern United States and another study more cheaply in Japan, they will do those studies in those two places, presumably using the same animal models and the same strain. One real example is a company that conducts its chronic toxicity studies in their European facility, dose-ranging studies for carcinogenicity studies in their US facility, and then contracts out the carcinogenicity studies. Looking at the results from all those studies from different sources, we have to make a decision about how those results from the chronic study and the dose-ranging study apply in the interpretation of the carcinogenicity study. If the animals appear to be responding differently, we have a big problem. That, then, is what I mean by segmentation of toxicology testing and how it is a major issue in terms of inter- national drug development. A major concern in terms of carcinogenicity testingâand one reason that we are focusing now on rats rather than miceâis because (as Dr. Usui mentioned, according to the ICH guidance) our long-term carcinogenicity studies are now generally performed using rats rather than in mice. We are using rats mainly because transgenic models are available primarily in mice; so to have two-species testing, the standard 2-year bioassay tends to be done in rats. Another reason is that most pharmaceutical development strategy uses rats as the rodents and dogs as the nonrodents. Because these companies develop a large database on the effects of the pharmaceutical on the rat during their testing, they want to be able to use that information in test approach selection, dose selection, and interpreta- tion of results. The preceding observations lead to a couple of concerns in terms of carcino- genicity assessment, the first of which are differences in stock survival. Dr. Usui mentioned that these differences might be related to diet. In some cases, they may be related to housing conditions; some companies let their rats get very fat because they put the food in wire baskets and allow them to feed ad libitum. Other companies and facilities put their food in jars and as the rats get fat, they can no longer reach the bottom of the jar. The rats thus undergo a kind of spontaneous dietary restriction, which maintains their weight at a lower level; and those differences, although not necessarily outbred related, can affect the outcomes and interpretations of studies. Diet and body weight are two factors that can clearly affect survival characteristics of the strain and the species. If they are overlaid with differences in survival within a strain of the outbred animals, then you end up with a very difficult problem for interpretation of carcinogenicity data. Diet, weight, and survival characteristics can also result in differences in
74 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE responsiveness to various pharmaceutical products. A set of similar studies with outbred animals may show a difference in response to treatment at some dose level. For example, if a chronic toxicity study is performed in a contract facility by gavage, and a second study for dose ranging is done in the in-house facility by dietary administration using dietary restriction (because it is much less expensive to plan the carcinogenicity study by dietary administration), and the studies result in different outcomes regarding the toxicity of the product, one will not know whether those differences are related to the modalities of the ingestion of the drug, the husbandry of the stock being treated, outbred differences from different sources, dietary restriction, or some other factor. NEED FOR INTEGRATED FINDINGS When we interpret carcinogenicity studies, we no longer can simply state, âIt was positive in this site, and it was negative in all of the other sites.â As a part of the ICH guidance for pharmaceuticals (with which the US Environmental Protec- tion Agency [EPA] agrees), we now perform integrated assessments of carcino- genicityâweight of the evidence. It is necessary to consider all of the data collected on the product and try to make a determination of whether or not those findings are important for human risk. In so doing, it is often necessary to rely on historic data to interpret the findings. When looking at historic data, it is impor- tant to note its relevance to the particular strain of animal and, unfortunately, to the animal stock. MANAGING CHANGES OVER TIME For some facilities that have been testing for 20 years, changes over time can clearly be seen in the historic response rate for spontaneous tumor incidence. Other facilities have no historic data but instead, rely on published data. How relevant is that published data to that in-house-contained and bred outbred or inbred strain? The answer to such a question is critical for the assessment and determination of the carcinogenic risk for humans. It is imperative for us to understand and manage test data accurately. One approach to managing test results is to try to control as many variables as possible. That method is the best solutionâto try to control the dietary supplies, the strain of animals, the stock of the animals, the dosing regimens, and the like so that one can say that the corresponding data are reliable for interpreta- tion. If a second person repeats the experiment or study, the person can be reasonably confident of obtaining the same result. However, controlling all of the variables is probably not feasible in the global development arena. How can you ensure that all animals have the same diet, no matter where the test facilities are located? I believe we can at least begin in this direction by controlling stocks of animals.
JOSEPH DEGEORGE 75 Nevertheless, we must be very careful not to select a single stock of a single strain of animal and proceed with that alone. It is known that some animal strains metabolize and respond to pharmaceuticals more like humans than do other strains. Having those strains for testing to find the most relevant response is important so that resulting data from a well-controlled strain is relevant to humans. If we pick only one stock and standardize it, we will loose a great deal because even though we will have a reproducible result with that strain, we may not want to use that strain for much of our testing.