The impact of the introduction of the ethical review process for research using animals in the UK: attitudes to alternatives among those working With experimental animals.

A postal questionnaire survey was carried out in late 1999 of those involved in working under the Animals (Scientific Procedures) Act 1986, namely the Certificate Holders, Project Licence Holders, Personal Licence Holders, Named Veterinary Surgeons, and Named Animal Care and Welfare Officers. The aim of the survey was to elicit views on the effectiveness of the introduction of the Ethical Review Process (ERP), introduced in April 1999. This report covers issues related to the use of alternatives, which were incorporated into the questionnaires. The number of returned questionnaires (45% of 1636 questionnaires) was sufficient for a meaningful analysis to be made of attitudes to the use of alternatives. In response to questions about the reason for the use of alternatives, more than 80% answered that alternatives should be used on moral or ethical grounds. Only about 50% of Certificate Holders and Licence Holders answered that alternatives were used because of legal requirements. Most respondents believed that replacement alternatives did not provide scientific information of equivalent value to that obtained from animal experiments. However, the majority also believed that it was possible to carry out valid scientific experiments by using replacement alternatives. The majority of Named Animal Care and Welfare Officers believed that the ERP had improved many aspects of refinement alternatives. In particular the "culture of care" had improved. Most establishments had a formal mechanism for discussing alternatives, although it was noteworthy that relatively few Personal Licence Holders believed this to be the case. In general, the majority of those working under the 1986 Act and most establishments seem to have a positive approach to the use of alternatives.

Scientific analysis of the proposed uses of the T25 dose descriptor in chemical carcinogen regulation.

The uncertainties that surround the methods used for risk assessment of exposure to carcinogens have been highlighted by a recent document advocating an approach based on the T25 dose (the dose giving a 25% incidence of cancer in an appropriately designed animal experiment). This method relies on derivation of the T25 dose then assesses risk at the exposure dose using proportionality provided by a linear extrapolation (T25/linear). To promote discussion of the scientific issues underlying methods for the risk assessment of chemical carcinogens, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) hosted a one-day workshop in Brussels on 10 November 2000. Several invited presentations were made to participants, including scientists from regulatory authorities, industry and academia. In general, it was felt that there was sufficient basis for using the T25 dose as an index of carcinogenic potency and hence as part of the hazard assessment process. However, the use of the T25 in risk assessment has not been validated. The T25/linear and other extrapolation methods based on metrics such as LED 10 assume linearity which may be invalid. Any risk calculated using the T25/linear method would provide a precise risk figure similar to the output obtained from the Linearised Multistage (LMS) method formerly used by the Environmental Protection Agency (EPA) in the United States of America. Similarity of output does not provide validation but rather reflects their reliance on similar mathematical approaches. In addition to the T25 issue, evidence was provided that using two separate methods (linearised non-threshold model for genotoxic carcinogens; no-observable-effect level with a safety factor (NOEL/SF) method for all other toxicity including non-genotoxic carcinogens) is not justified. Since the ultimate purpose of risk assessment is to provide reliable information to risk managers and the public, there was strong support at the workshop for harmonisation of approaches to risk assessment for all genotoxic and nongenotoxic carcinogens. In summary, the T25 method has utility for ranking potency to focus efforts in risk reduction. However, uncertainties such as the false assumption of precision and non-linearity in the dose-response curve for tumour induction raise serious concerns that caution against the use of T25/linear method for predicting human cancer risk.

Ethical review of regulatory toxicology guidelines involving experiments on animals: the example of endocrine disrupters.

The safety assessment of new chemicals (including medicines, pesticides, food additives, and industrial chemicals) relies on the results of animal experiments. Because the safety of those exposed to these products and the welfare of the experimental animals used are considered critically important, both testing requirements and the welfare of experimental animals are controlled by law. In the U.K., projects that propose to use animals for experimental purposes, including for the testing of chemicals, have been controlled by law for over a century, with the most recent legislation (Animals [Scientific Procedures] Act of 1986) requiring a cost/benefit assessment before it may proceed. New regulations introduced in 1998 will require an ethical review process for all projects from April 1999. Such ethical review will have to take account of the toxicity testing methods and schemes that are required by the legislation aimed at protecting human health. Neither national nor international proposals for toxicity testing methods and schemes are generally subjected to ethical review from the point of protecting animal welfare. The international nature of the chemical and pharmaceutical industry means that testing requirements from one of the major national regulatory agencies (USA, EU, or Japan) or the international organizations (Organization for Economic Co-operation and Development [OECD]or the International Conference on Harmonization [ICH]) have an impact on the testing carried out by industrial organizations in all countries. The recent proposals for screening and testing chemicals to identify endocrine disrupters (ED) from the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) of the U.S. Environmental Protection Agency (EPA) are used as an example of the interaction between regulatory proposals and animal welfare issues. The current proposals are the most extravagant in the use of animals. Between 0.6 and 1.2 million animals would be required for each 1000 chemicals tested. The EPA, before incorporating them into regulation, is subjecting the recommendations to further review. This will undoubtedly moderate the number of animals actually used from the worst-case calculation. The variables that have the greatest impact on the number of animals required for testing are the prevalence of ED chemicals in the chemicals to be tested, and the sensitivity and specificity of the testing methods. The modeling demonstrates, for example, that increasing the prevalence from 10 to 50% reduces the number of animals used to detect one ED from 10,000 to 2700. Knowledge of the prevalence of EDs in the chemicals to be tested would allow rational selection of tier one screening based on the sensitivity and specificity of the screening tests. The EDSTAC proposals are difficult to justify from an ethical perspective, as equally effective detection rates may be achieved with fewer animals. National and international regulatory testing proposals should be subjected to formal independent ethical review before they are finalized, with a view to improving animal welfare.

Judgments of chemical risks: comparisons among senior managers, toxicologists, and the public.

Nineteen Senior Managers of a major chemical company in the United Kingdom participated in a survey to determine their attitudes, beliefs, and perceptions regarding risks from chemicals. Similar surveys had previously been conducted with toxicologists and members of the general public in the United States and Canada. In general, the Senior Managers tended to judge risks to be quite small for most chemicals. Moreover, they had lower risk perceptions than did members of the British Toxicological Society and even far lower perceptions of risk than a comparison group of members of the Canadian public. The managers held views that were similar to British toxicologists working in industry and government and dissimilar to the views of toxicologists working in academia. The observed differences between views of managers, toxicologists, and the public must be recognized and understood in order to facilitate communication and constructive efforts to manage chemical risks.

Workshop overview: scientific and regulatory challenges for the reduction, refinement, and replacement of animals in toxicity testing.

Public concern for animal welfare has been expressed through legislative control of animal use for experimental purposes since the first legislation was introduced in 1876 in the United Kingdom. Legislative control of animal use has been introduced in virtually every developed country, with major initiatives in Europe (1986) and the United States (1966 and 1985). Advances in scientific thinking resulted in the development of the concept of the three Rs--refinement, reduction, and replacement--by Russell and Burch in 1959. The field has expanded substantially since, with specialist scientific journals dedicated to alternatives, World Congresses organized to discuss the scientific and philosophical issues, and European and U.S. validation organizations being launched. Current scientific attention is focused on validation of alternative methods. The underlying scientific principles of chemical toxicity are complicated and insufficiently understood for alternative methods for all toxicity endpoints of importance in protecting human health to be available. Important lessons have been learned about how to validate methods, including the need to have prediction models available before the validation is undertaken, the need to understand the variability of the animal-based data which is to be used as the validation standard, and the need to have well-managed validation programs. Future progress will depend on the development of novel methods, which can now be validated through international collaborative efforts.

Evaluating chemical risks: results of a survey of the British Toxicology Society.

1. Members of the British Toxicology Society participated in a survey to determine their attitudes, beliefs, and perceptions regarding risks from chemicals. Similar surveys had previously been conducted with toxicologists and members of the general public in the United States and Canada. Data from 312 completed questionnaires were analyzed. 2. In general, the British toxicologists judged risks to be quite low for most hazards, with the exception of cigarette smoking and asbestos. They tended to have quite favorable attitudes toward the use of chemicals and were confident about the adequacy of chemical regulations. 3. As in previous studies of toxicologists, women expressed higher perceptions of risk than did men and had consistently stronger anti-chemical attitudes. 4. Toxicologists working in industry had more favorable attitudes towards chemicals and their use than did those working in academic settings. 5. When asked to evaluate chemical technical summaries of various animals studies there was considerable disagreement among the respondents about the toxicity of the chemicals involved. 6. In general, British toxicologists were equivocal about the reliability of animal studies in predicting human effects (particularly carcinogenicity) probably because of the belief that animal studies overestimate risk. However, they were rather confident that human health risks could be assessed reasonably accurately.

Prospects for reduction and replacement alternatives in regulatory toxicology.

There is a considerable impetus to developing alternative methods in regulatory toxicity testing from the introduction of legislation requiring alternative methods to be used whenever possible. Some progress has been made in the search for and validation of alternative methods in regulatory toxicology. When replacement and reduction alternatives are considered, the absolute reduction in the number of animals used in testing is a good indicator of the progress that is being made. Attempts at replacing existing animal methods have focused on acute studies where the endpoints are simple and the mechanism of toxic action understood. The majority of animal studies rely on non-specific endpoints from chronic studies and where there is little understanding of the mechanism of toxic action; these studies are much more difficult to replace. The conclusion is that the methods that are currently under validation to replace regulatory toxicology studies will only reduce the number of animals used to test a chemical (such as a pesticide) by about 3%. The process of validation is likened to technology, where additional resources can improve and hasten the process; however, the rate-limiting step in the overall objective of reducing animal use is the development of suitable tests for validation, a scientific process which draws on the full range of biomedical research and hence will be slow to reveal the necessary data for test development.

Thresholds in chemical carcinogenesis.

It is common practice to base carcinogenic risk assessment on the view that there is no threshold for chemical carcinogenesis. In this context, the threshold is defined as a dose below which no effects are observed. Analysis of epidemiological and experimental data on chemical carcinogenesis confirms that no thresholds have been demonstrated in human or animal studies, including the ED01 study which used very large group sizes. Indeed, demonstrating a threshold by experiment is as difficult as proving a negative. Mechanistic data provide the justification for the assertion that thresholds do not exist in chemical carcinogeneses. This is commonly thought of as a dose-response relationship which is linear at low dose. It is noted that primary sites of action for other forms of toxicity--for example, inhibition of enzymes or occupation of receptors--also have a dose-response relationship which is linear at low dose. It is concluded that the use of differing methods of low-dose risk assessment for different toxicological endpoints is not justified. The same method should be used in order to provide symmetry in assessment of different risks; the influence of assumptions, such as the magnitude of safety factors or the mathematical model selected, should be clearly stated so that risk managers can make balanced judgments.

Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis.

In this review we have evaluated the relationship between peroxisome proliferation and hepatocarcinogenesis. To do so, we identified all chemicals known to produce peroxisome proliferation and selected those for which there are data (on peroxisome proliferation and hepatocarcinogenesis) which meet certain criteria chosen to facilitate comparison of these phenomena. The summarised data and definition of the methodology used has been collected in appendices. These comparisons enabled us to evaluate the relationship between these phenomena using reliable data. As there is a good correlation between them, we further explored the mechanisms of action that have been proposed (direct genotoxic activity, production of hydrogen peroxide, cell proliferation and receptor activation). The relationship between these events in other species, including humans, was also reviewed and finally an overview of the assessment of human hazard is presented in section IX. Some of the first chemicals which were shown to produce peroxisome proliferation were also hepatocarcinogens whose carcinogenicity could not be readily explained by genotoxic activity. This raised the suggestion that the unusual phenomenon of peroxisome proliferation was intricately linked to the carcinogenic activity of these agents. Three questions have exercised the attention of regulatory, industrial and academic toxicology since then; are chemicals which elicit peroxisome proliferation in the liver actually a coherent class of chemical carcinogens?; does the early biological phenomenon of peroxisome proliferation have real predictive value for and mechanistic association with rodent carcinogenesis?; and what hazard/risk do these agents pose to humans that may be exposed to them? Whether peroxisome proliferators are indeed a discrete class of rodent carcinogens would appear to be the single, most important question. If so, then the assumptions and procedures relevant to human hazard and risk assessment should be applied to the class and should be essentially generic; if not, each chemical should be considered independently. Our critical analysis of the published data for over 70 agents which have been shown to possess intrinsic ability to induce peroxisome proliferation in the livers of rodents has led to the conclusion that there exists a strong correlation between peroxisome proliferation as n early effect in the liver and hepatocarcinogenicity in chronic exposure studies. An almost perfect correlation was observed between the induction of peroxisomes in the rodent liver and the eventual appearance of tumours following chronic exposure The few exceptions to this were largely explainable (section II).(ABSTRACT TRUNCATED AT 400 WORDS)

Transgenic animals in toxicology.

Recent advances have been made in the characterization of a number of transgenic animal models. These animal models have provided a powerful toxicological tool for studying in vivo chemical effects and have increased our understanding of the role of specific genetic alterations as predisposing factors for chemical carcinogenesis. The goal of this symposium was to introduce the development of transgenic animals and the utilization of transgenics in toxicology research focusing on understanding tissue-specific mutation, chemical effects, and cancer. The production of transgenic animals, including gene insertions and gene knockouts, and the utilization of transgenic technology for studying multistage carcinogenesis and tumor suppressor genes are described. Data on the application and implications of transgenics as a genetic endpoint are also discussed. The use of transgenic animals in toxicology should improve our understanding of the role of specific genetic alterations in the carcinogenic process and lead to improved estimations of human health risks.

Nongenotoxic carcinogens: an extension of the perspective provided by Perera.

Perera recently discussed the very real problems that accompany any attempt to classify rodent carcinogens into two groups--genotoxic or nongenotoxic. Not the least of these problems is that no agreed definition of these two terms exist. Nonetheless, the current carcinogen databases, for example, that of the U.S. National Toxicology Program (NTP), clearly comprise two broad groups of carcinogens--DNA reactive, mutagenic and multiply carcinogenic chemicals, and others. The others appear to be nonreactive to DNA, are inactive in the primary mutagenicity assays, and usually elicit highly selective carcinogenic responses in animals. These two classes of carcinogen are illustrated by examples taken from the NTP database and are discussed within the possible context of the latter group not being active in humans or, if they are, only when a threshold dose has been exceeded, chronically.

Strategic considerations in industry's use of in vitro toxicology.

Industrial concerns have a legal and ethical responsibility to provide information that allows products to be used safely. Current toxicological practice and legal requirements rely on animal experiments to provide much of this information on safety. Nevertheless, there is a clear role for in vitro methods in the overall development and testing of chemicals. During the early stages of development of a new chemical, in vitro tests provide information used in the selection of appropriate candidates from among the many that may be available. Such use of in vitro tests for screening of chemicals must be preceded by adequate validation. The interpretation of results of screening chemicals requires a detailed knowledge of the sensitivity and specificity of the test and of the structures of the chemicals under test. Once in vivo toxicological data are available, in vitro tests may have a key role in providing an understanding of species differences in toxic responses. Examples of the use of in vitro techniques to improve the specificity of animal studies are given and these include studies of trichloroethylene, di(2-ethylhexyl) phthalate, hexachlorobutadiene and a substituted triazole. In the overall process of risk assessment, extrapolation of animal data to the assessment of human hazard is a prerequisite. In vitro techniques can be used to assess species differences in transdermal absorption. They may also help in assessing quantitative differences in metabolic conversion between species. Examples of the use of in vitro techniques to improve the sensitivity of animal studies include the study of the toxicity of methylene chloride. In vitro techniques have developed rapidly over the last decade. Nevertheless, there is only one category of testing within regulatory guidelines that specifies in vitro methods, namely mutagenicity. At the moment, in vivo methods are considered to provide the best general information for risk assessment, with in vitro methods contributing as screening techniques and as adjuncts to improve the sensitivity and specificity of animal studies.