Alternatives to Laboratory Animals

An interactive computer-assisted learning program is described, aimed at teaching students, on a range of undergraduate/diploma level courses, the basic pharmacology of transmission at autonomic ganglia and sympathetically innervated smooth muscle. The program, which is menu-driven, simulates experiments which can be conducted on the superior cervical ganglion nictitating membrane preparation (in vivo) of the anaesthetised cat, and is written for IBM-compatible microcomputers. Students “design” experiments by selecting certain experimental parameters such as: an appropriate drug to investigate from a comprehensive list; the site of administration; and whether to stimulate pre-ganglionic nerves. Simulated contractions of the nictitating membrane, derived from actual experimental data, are presented on a scrolling display on the monitor in a form comparable to that of a chart recorder. The learning package includes background information and teacher-designed help screens, and is supported by tutors’ notes and suggested assignments. The use of the program in teaching pharmacology and as an alternative to animal experiments, is discussed.

Acute alveolar injury commonly occurs as a result of exposure to a variety of toxic stimuli and is characterised by widespread necrosis of the epithelium, followed by regeneration and hyperplasia of type II alveolar epithelial cells. We studied epithelial proliferation in rabbit lung resulting from a chemical stimulus by isolating the mitotically active cells and establishing them in continuous culture.

Acute alveolar injury was induced in rabbits with two daily injections of N-nitroso-N-methylurethane (NNNMU). Four to six days after treatment, the lungs were enzymatically digested and a homogeneous monolayer of epithelial cells was established in culture. The cells displayed a typical cobblestone-like arrangement and cuboidal shape, and represented 95% of the cells isolated. The cultures exhibited contact inhibition and achieved a population doubling level of 11 ± 2. Their epithelial origin was confirmed by positive reactions for immunofluorescent cytokeratin, alkaline phosphatase, and the Papanicolaou stain. Ultrastructurally, the cells showed abundant rough endoplasmic reticulum with dilated cisternae, desmosomes, cytokeratin filaments, and lamellar-like structures early in culture. In addition, the cells accumulated paraquat in a manner similar to that shown by established epithelial cell lines. The results demonstrate that regenerating epithelial cells can be isolated and propagated in culture as a relatively homogeneous population. This system, which is designed to further understanding of the metabolic role of epithelial cells following acute injury, may serve as an in vitro model for pulmonary toxicity studies using a minimal number of animals.

The CTFA Evaluation of Alternatives Program is a multi-year effort organised by a scientific advisory committee (the CTFA Animal Welfare Task Force), and is designed to evaluate (in Phase I) approximately twenty-five potential alternative methods to the Draize eye irritancy test. Coordination, management of logistics, collection and statistical analysis of data, are being conducted by the Columbus Division of the Battelle Memorial Institute. The US Food and Drug Administration has been aware of the Program since its inception. The intention of the Program is to provide industry with information on the performance of a series of potential alternatives to the Draize test, so as to aid individual companies to identify those methods which seem best suited to their own particular testing needs. The participants are either CTFA member companies who are already using or developing alternative tests, or those independent investigators whose development work is being sponsored by CTFA members. The purpose of the Program is to determine the effectiveness and limitations of a variety of tests for a range of different product types. Specifically, the Program will evaluate the capacity of the tests under investigation to rank and discriminate between the ocular irritation potential of a range of prototype cosmetic and personal care products. The Program is designed as a multi-year effort with a different product type evaluated each year. In Phase I, ten different ethanol-based substances are being tested; oil/water emulsions will be evaluated in Phase II.

The neutral red uptake inhibition assay and the EYTEX™ system were investigated as alternative methods for the assessment of eye irritation, determined according to the EEC protocol. The 17 test chemicals used were mainly organic solvents. The xenobiotics were applied to Hep G2 cells for 24 hours at different concentrations. Neutral red uptake inhibition was then measured. The results are expressed as the NI50 value, which is the concentration of test compound required to induce a 50% reduction in neutral red uptake. The same chemicals were also tested as coded samples by the EYTEX™ test according to the manufacturer's directions. A nearly identical quantitative correlation was found for both in vitro methods with corneal opacity scores: r = 0.84 for EYTEX™ scores and r = 0.83 for log NI50, expressed in μg/ml. Whilst these correlations are certainly not perfect, it is clear that both in vitro methods can be used as valuable prescreening methods.

A literature-based survey of the use of non-human primates as laboratory animals in Great Britain in 1984–1988 was carried out as a background to extending debate about the ethical and practical issues involved. The 289 publications considered were grouped in 15 subject areas and reviewed in terms of scientific purpose, methods employed, numbers and species of animals used, and their source, care and ultimate fate. In addition, the Association of the British Pharmaceutical Industry provided a comment on the use of non-human primates by pharmaceutical companies. Specific causes for concern were identified, and future prospects considered.

Twenty-six chemicals supplied coded by FRAME, were tested on HEp-2 cells. Three endpoints were used to measure the cytotoxicity of these compounds: total protein content, LDH release and total acid phosphatase content.

The sensitivity of these parameters is discussed. The results are compared with those obtained by other groups participating in the validation scheme organised by FRAME.