Use Of Animals In Testing Research Articles

Animal use in the safety evaluation of chemicals: harmonization and emerging needs.

An overview is provided of the legal framework in Europe for the use of experimental animals set by European Union (EU) Directive 86/609/EEC and of the activities of EU member states to implement this directive for regulatory testing in animals. The use of animal data in the safety assessment of chemicals by services of the EU Commission and by EU member states is described. Specific examples are given for the current use of animal tests in Europe for the safety evaluation of industrial chemicals, pesticides and biocides, cosmetics, endocrine disrupters, and existing chemicals. Recent progress in implementing the 3Rs concept of Russell and Burch into regulatory guidelines of the EU are described, with particular reference to acute systemic and local toxicity testing. Progress in implementing the 3Rs concept in the EU is compared with the situation outside Europe, particularly with the incentive initiated by the Organisation for Economic Co-operation and Development, to reduce regulatory testing in animals. The harmonization of toxicity test guidelines initiated by the Organisation for Economic Co-operation and Development in 1982 has been the most successful measure to reduce pain and distress of laboratory animals in regulatory testing. From the animal welfare perspective, the international harmonization of test guidelines and the mutual acceptance of data are, therefore, the way forward for all areas of chemicals testing.

Perspectives on the manufacture of combination vaccines.

Evolving regulatory requirements in the United States and Europe create major challenges for manufacturers tasked with production of vaccines that contain > or =9 separate antigens capable of protecting against infectious diseases, such as diphtheria, tetanus, pertussis, polio, hepatitis B, and Haemophilus influenza b, in a single shot. This article describes 10 steps that can facilitate the process of licensing these complex vaccines. It also points out problems associated with the use of animal tests for the crucial step of potency testing for batch release caused by the inherent variability of such tests and the difficulties of interpreting their results.

Pasteurella multocida Toxin Type D Serological Assay as an Alternative to the Toxin Neutralisation Lethality Test in Mice

The objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Pasteurella multocida toxin type D, that correlated to a mouse lethality test. Currently, the mouse lethality test is one of several tests used world-wide to evaluate serological responses in animals immunised with vaccines containing toxoids. The mouse lethality test involves injecting mice with a mixture of toxin and test serum sample (from animals that have been vaccinated with a toxoid), and then determining antibody titre of the test serum from the number of mice that survive. Thus, the titre calculated is based on the neutralising activity of the test serum. The mouse lethality test requires large numbers of animals and causes severe distress to the animals. Organisations world-wide are working towards alternatives to animals in the development and control of biological products for human and veterinary use. Additionally, the mouse lethality test is labour-intensive, costly and lacks robustness and may be difficult to reproduce between different technicians. We have developed a double sandwich ELISA to measure anti- P. multocida toxoid type D antibodies in swine serum. Sera from swine immunised with vaccines containing type D toxoid showed good correlation to the mouse lethality assay (Spearman analysis=0·94 and Pearson analysis=0·84). When compared to the mouse lethality test, titres obtained using the ELISA format had higher correlation with protective immunity (i.e., lower turbinate atrophy) following challenge with virulent P. multocida. The ELISA assay is more robust, reproducible and costs less than the mouse lethality assay; and it complements efforts to reduce the use of animals in testing.

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100–1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay.

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100-1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.

Animal Models and Ethological Strategies for Early Drug-Testing in Humans

The preclinical evaluation of psychotropic drugs relies on the use of animal tests which are thought to generate clinically relevant information concerning the psychiatric condition for which the drug can be used. Despite their importance, views on the usefulness of animal “models” vary widely, ranging from outright negation on the grounds that human minds are unique, to the more biological view that because of a partially common phylogenetic history, animals share many behavioural features with humans, e.g. learning, attention, aggression or sociality, which are altered in psychiatric disorders. The widespread use of ethology with its emphasis upon the function and origin of behaviour, particularly social behaviour, is based upon this evolutionary view. Many ethopharmacological tests in animals use situations which bias the animal's behaviour towards flight or sociality but their validation requires clinical feedback from humans. Unfortunately, information relating to human social mechanisms is rarely obtainable from conventional clinical-assessment schemes. To help overcome this problem, we have applied ethological principles to a new Challenge Interview Situation which is designed to bias the behaviour of healthy subjects towards flight. Analysis shows that by staring, moving closer or asking the subjects to relate emotional experiences, shifts in non-verbal behaviour towards avoidance and mild social withdrawal are achieved which resemble behavioural strategies recorded ethologically in depressed patients as well as animals in an arrested flight situation. The Challenge Interview is intended for early drug studies in humans where sociality is a target and can be adapted for concomitant endocrine and biochemical studies if required.

The use of animal tests in risk assessment for immunotoxicology

The design and content of a screening battery using a ‘tier’ approach for detecting potential immunotoxic compounds in mice has been described (M. I. Luster et al., Fundamental and Applied Toxicology 1988, 10, 2–19). The database generated from these studies, which consists of over 50 selected compounds, has been analysed in an attempt to improve future testing strategies and provide information to aid in developing future quantitative risk assessment for immunotoxicity. In a recent study it was shown that as few as two or three immune parameters were needed to predict immunotoxicants in mice (M. I. Luster et al., Fundamental and Applied Toxicology 1992, 18, 200–210). The analyses described here focus on the use of this database to develop statistical models that examine the qualitative and quantitative relationship(s) between the immune function and host resistance tests. The conclusions derived from these analyses are as follows: (1) A good correlation exists between changes in the immune tests and altered host resistance, in that there were no instances where host resistance was altered without affecting one or more immune test(s). However, in some instances immune changes occurred without corresponding changes in host resistance. (2) No single immune test could be identified that was fully predictive for altered host resistance, although most assays were relatively good indicators (i.e. 70%). Several others, such as proliferative response to lipopolysaccharide and leucocyte counts, were found to be relatively poor indicators for host resistance changes. (3) The ability to resist infectious agent challenge is dependent on the degrees of immunosuppression and the quantity of infectious agent administered. (4) Logistic and standard regression modelling using one extensive chemical data set from the immunosuppressive agent, cyclophosphamide, indicated that most immune function-host resistance relationships followed linear rather than linear-quadratic (threshold-like) models. For most of the relationships this could not be confirmed using a large chemical data set and, thus, a more mechanistically based approach for modelling will need to be developed.

Multivariate QSAR analysis of a skin sensitization database.

There is a regulatory requirement for the potential of a new chemical to cause skin sensitization to be assessed. This requirement is presently fulfilled by the use of animal tests. In this study a data base of heterogeneous organic compounds from the guinea pig maximization test has been subjected to multivariate QSAR analysis. The compounds were described both by whole molecule parameters and structural features associated with likely sites of reactivity. Principal component analysis was applied to the data set and although it functions reasonably well to reduce the dimensionality of a large data matrix, it is only moderately useful as a predictive tool when descriptors were chosen rationally. Stepwise discriminant analysis produces a fourteen parameter model, of which twelve were structural features associated with reactivity. This however predicts only 82.6% of compounds correctly after cross validation. There is trend for the linear discriminant analysis model to predict compounds as non sensitizers, suggesting that the parameters incorporated were not wholly suitable for discriminating between the two classes. Another criticism of linear discriminant analysis is that it may be unable to cope with the likely embedded data structure. With this in mind, the structural alerts may be better employed in an expert system, to identify potential hazard, where they will not suffer the limitations of a statistical model.

The use of animals in testing: A focal point for controversy

The use of animals in testing: A focal point for controversy

In vitro Methods for the Assessment of Primary Local Effects of Topically Applied Preparations

Three 'alternative' methods for the screening of the primary irritation potentials of topically applied preparations and raw materials are presented as a test battery and their in vitro endpoints are compared with in vivo data. The first method is the intermediate test on the chorioallantoic membrane of hens' fertilized eggs, which is generally proposed for the prediction of the irritation potential of chemicals. Relevant test parameters, such as the time-dependent appearance of hemorrhages, vascular lysis and coagulation, are used to classify the test substances. Secondly, cytotoxicity tests with Balb/c 3T3 fibroblasts are used to characterize the influence of test materials on cellular homeostasis and viability, which is quantified by the dose-dependent uptake of Neutral red. As a second parameter changes in the cellular protein level can be easily measured by subsequent staining with Kenacid blue. Finally, the third approach to in vitro evaluation is presented in the form of the red blood cell assay, a rapid photometric assay which permits distinguishing basically between damage to the membrane and to proteins as endpoints which correlate with lesions observed on the conjunctiva, iris and cornea in the eye and the acute inflammatory responses evoked during epicutaneous irritancy testing. Changes in the optical behavior of oxyhemoglobin are used as the inherent indicator for monitoring both endpoints and their interrelations. These methods are proposed and can be used advantageously for testing newly developed topical preparations and their ingredients in view of their local irritation potential without further use of animal testing. Moreover, they can also be used for screening chemicals.

Evaluation of modified methods for determining skin irritation

The premarket testing of household cleaning products for dermal irritancy is best achieved via human testing. Animal dermal irritation testing is generally limited to screening for possible dermal hazard of totally new or unique products or ingredients prior to human testing or to meeting regulatory requirements of government bodies. Alternatives to animal tests are being sought; however, until such time that these alternatives are identified, validated, and accepted by government bodies, the judicious use of animal testing remains a necessity. Modifications to standard animal skin irritation test procedures have been evaluated against human skin irritation results with the objective of defining one method that could be used in place of current standard procedures that differ slightly from one another, and thereby avoid excessive and redundant use of animals. Hill Top Chambers (19 mm) and standard gauze patches (U.S. Department of Transportation procedure) were used to obtain comparative irritation responses for 24 cleaning products, common caustics, and acids in rabbits and humans. Exposure times were 1 or 4 hr, and responses were graded over a 72-hr period. Results indicate that use of the Chamber offers the potential to (1) reduce the number of animals used for skin irritation screening (smaller group size and up to eight test substances/concentrations per animal); (2) eliminate the need for conducting multiple tests to satisfy different governmental requirements; and (3) reduce animal stress by reducing exposure times without compromising the value of the irritancy patch test as a screening tool. When animal data are required, it is suggested that the use of a Chamber and other modifications of traditional test procedures offers advantages that could result in using fewer animals and/or have less potential for producing unnecessarily severe responses in animals.

Recent developments in replacing, reducing, and refining animal use in toxicologic research and testing

Significant progress has been made in replacing animals in toxicology/safety assessment with in vitro systems, in reducing the number of animals used, and in refining how they are used. Review of annual reports of the numbers of animals used in testing in the United States, the United Kingdom, and Japan shows a continuing reduction in the numbers for all species. Multiple in vitro systems have been developed for screening/testing for eye and skin irritation, skin sensitization, teratology, and other endpoints and a scientific consensus has been formed on requirements and process for validation. However, the use of these test systems in place of existing in vivo tests is minimal. At the same time, innovative designs have been developed (and are in wide use) for in vivo tests which reduce both the numbers and the pain and distress of animals used in testing. Progress and dialogue continue on modification of both U.S. and international requirements and guidelines for testing, and for defining an “approval” process for alternatives and innovations.

Animals in Research: The American Medical Association's Position

To the Editor.— I am a full-time academic physician and I basically support the use of animals in research to benefit both other animals and humans. However, I think that the American Medical Association has gone too far in supporting all forms of animal experimentation and testing. An example of this extreme position that the American Medical Association seems to be taking is the article by Loeb et al. 1 There are many forms of animal experimentation and use of animals for testing that are a needless waste of life and cause of animal suffering. I think the interests of bona fide medical research using animals would be better served if the American Medical Association did not indiscriminately support all uses of animals, however cruel and useless. I can see an analogy between the American Medical Association's position on animal testing and the National Rifle Association's position on gun

Revlon, others end tests of products on animals

Revlon announced late last month that it will eliminate all use of animal testing in research, development, and production of its products. The New York City firm thus becomes the fourth U.S. cosmetics company to end such testing in recent weeks. Avon Products and Amway Corp. had made similar announcements last month, whereas Mary Kay Cosmetics declared its decision to halt such testing in May. The companies will use a combination of in-vitro tests, clinical testing with humans, and their databases of previous testing on formulations and ingredients to evaluate product safety and irritant potential. Development and validation of such test programs resulted from an eight-year, $3 million effort funded by the Cosmetic Toiletry & Fragrance Association and its member companies at Johns Hopkins University Center for Alternatives to Animal Testing (CAAT) in Baltimore. Perhaps the most emotionally charged animal test for cosmetics irritation has been the Draize test. In that test, technicians apply a ...

Effects of T-2 mycotoxin on gastrointestinal tissues: a review of in vivo and in vitro models.

T-2 mycotoxin, a trichothecene, is the principal toxic component ofFusarium sp. Agricultural products and food are frequently contaminated with this toxin. Various animal models have been used to determine its metabolic fate, rate of excretion, and distribution. A modulation effect on cell-mediated immunity and alterations in gastrointestinal propulsion have been demonstrated. The toxin has been shown to produce some similar pathologic alterations in various animal species studied. The consistent alteration appears to mainly affect mitotic cells of the gastrointestinal tract and the lymphoid system. A host of bioassay systems are now being used as alternative methods to the use of animals for testing of the mycotoxin. These tests may accurately assess and define the role of the subject-toxin interactions following consumption of T-2 mycotoxin contaminated food sources.T-2 mycotoxin, as observed above within vivo andin vitro models, promotes a chemically-induced change in structure and function of affected gastrointestinal cells from a transient and reversible aberration in a single enzymatic reaction to cell death. Regardless of the end point measured, the toxic response brought about in cells appears to involve the interactions of virtually all subcellular processes—membrane transport and permeability, chemical metabolism, DNA function, and energy production/expenditure—as cells attempt to maintain their functional integrity while disposing of the toxicant. The variation in the quality of the toxic response with dose suggests that more cellular processes are perturbed as the chemical dose is increased.

The Use of Animals in Testing

The Use of Animals in Testing

Stakeholder analysis for effective issues management

For years the pharmaceutical industry has been aware of public sentiment against the use of animals for testing and experimentation. Most companies assumed that they were dealing with ineffectual fringe groups who were unlikely to force them to change their experimental practices.

Further characterization of a simple, automated exploratory model for the anxiolytic effects of benzodiazepines

Anxiolytics specifically increase the number of exploratory transitions in a two-chambered model system for anxiety in mice. Characterization of parameters to optimize and standardize this model required analysis of multiple use of test animals, intertrial interval, and circadean variability. Time of day did not affect exploratory activity in mice treated with vehicle or diazepam between 10 a.m. and 11 p.m. Intertrial intervals of 1, 3, 5, or 7 days were equally effective. The diazepam-induced increase in exploratory activity was significant over the first three uses of test animals. These data recommend reuse of mice to a maximum of three trials, throughout the daytime or evening hours of their lighting schedule.

ADRENOTROPHIC ACTIVITY OF HUMAN BLOOD1

The presence of adrenocorticotrophic hormone, or of a substance or substances possessing adrenotrophic activity, has not been convincingly demonstrated in human urine (Locke, Albert and Kepler, unpublished). We have therefore turned our attention to the blood, in the hope that adrenotrophic material may be present in a concentration sufficiently high to be measured by current assay procedures. A review of the literature pertinent to this subject is summarized in table 1. It appears that the results obtained are conflicting, and that much of this confusion has resulted from the use of test animals with intact pituitaries. Such a procedure does not, of course, permit specific detection of adrenotrophic activity in the material tested. Thus, there is considerable doubt as to the validity of all results obtained with human blood, with the sole exception of those obtained by Cooke, Graetzer and Reiss (1948).

Recent Advances in Materia Medica: being a Description of the Methods of Preparing and Testing Sera and Vaccines, Hormones and Vitamins, with an Account of their Properties and Medicinal Uses

IN this volume of the Recent Advances Series, the author has confined himself to a consideration of some of those substances of value in therapeutics which are obtained from animal sources or require the use of animal tests of toxicity and potency before issue. The work is intended chiefly for students of pharmacy, and therefore includes certain elementary physiological details. It covers a field, however, of increasing importance; and its pages make available in small compass much information which ordinarily could only be obtained by a search of the original literature or a larger book dealing with biological assays. For this reason it can be recommended to physiologists and pharmacologists, as well as to medical practitioners who may wish to have some knowledge of the methods of preparation and test of biological products. The specialist may also find it useful for information on the subject of the biological assay of substances with which he may have had no recent experience.