The three Rs of Russell & Burch and the testing of biological products.

The principles of humane animal experimentation proposed by Russell & Burch (1959), namely, replacement, reduction, and refinement, are now commonly known as the Three Rs. These principles are clearly embodied in Article 7 of Directive 86/609/EEC. It is instructive, therefore, to consider the priority currently attached to compliance with these principles and to the Directive in the development and control of biological products. Specific comments are made on the need for the application of the Three Rs in relation to the testing of human diphtheria and tetanus vaccines, human pertussis vaccine, inactivated veterinary Gumboro vaccine, veterinary Newcastle disease vaccine, veterinary clostridial vaccines and botulinum toxin. We pose three questions: a. Are the minimum numbers of animals already being used in this area? b. Is any unnecessary pain, suffering, distress or lasting harm being inflicted on the animals used? and c. What could and should be done about any shortcomings in current practice? Finally, the role of ECVAM in the promotion of the Three Rs within the European Union will be reviewed.

The UK Animals (Scientific Procedures) Act-Implications for the future of alternative toxicity tests.

The UK Animals (Scientific Procedures) Act 1986 requires a project licence for all potentially harmful animal tests in regulatory toxicology. Full consideration has to be given to alternatives, to the use of hierarchical strategies and to adherence to published guidelines. Advice on these matters is available from the Inspectorate. The mechanisms for developing and implementing policies on hierarchical strategies and alternatives are discussed. The paper notes some of the problems in the general context of toxicology, and uses as an example the Home Office guidelines on eye irritation tests published in 1987. These areas of regulatory toxicology tests are being considered currently by the Animal Procedures Committee.

Influence of 5-hydroxytryptamine uptake on the apparent 5-hydroxytryptamine antagonist potency of metoclopramide in the rat isolated superior cervical ganglion.

Metoclopramide, 1 X 10(-6) -1 X 10(-4) M, was found to behave as a reversible, competitive antagonist of 5-hydroxytryptamine (5-HT)-induced depolarization of the rat isolated vagus nerve (VN) and superior cervical ganglion (SCG). The pKB values were 6.60 (+/- 0.04) and 5.74 (+/- 0.07), respectively. The possibility that this apparent difference in potency was due to saturable 5-HT uptake was investigated. The SCG, but not the VN, accumulated tritium-labelled 5-HT via a saturable, sodium- and temperature-dependent mechanism. Ganglionic 5-HT uptake was blocked by desmethylimipramine (IC50 1.4 X 10(-6)M), chlorimipramine (8.7 X 10(-9) M), zimelidine (1.5 X 10(-7) M), paroxetine (4.3 X 10(-8) M) and citalopram (6.2 X 10(-8) M). The 5-HT uptake inhibitor paroxetine, 1 X 10(-6) M, did not modify the apparent 5-HT antagonist potency of metoclopramide on the VN, but raised the pKB obtained against 5-HT on the SCG from 5.74 (+/- 0.07) to 6.25 (+/- 0.03). It is suggested that the observed difference in the potency of metoclopramide as a 5-HT antagonist on the rat VN and SCG was due to saturable 5-HT uptake in the latter preparation. The results do not support a difference in the 5-HT receptors mediating depolarization on the VN and SCG.

Third Report on the Organization of Pharmacology in Great Britain.

The results presented in this Report on the Organization of Pharmacology in the United Kingdom are based on replies to questionnaires sent out in 1983. In addition to academic and industrial research and development (R&D) departments we also surveyed industrial departments of toxicology, sales, clinical liaison and medical information, as well as specialized research units with no formal teaching function. The overall response rate was 87%. Since the last Report in 1971, the total number of pharmacologists has doubled to 2220, and they are now almost equally distributed between Universities and Industry. The most striking feature has been a major (3 fold) increase in the total number of industrial pharmacologists to 1014. In contrast, there was only a 1.3 fold increase in the number (99) of pharmacologists in established academic posts, over half of which are in the new departments of clinical pharmacology. Of the established pharmacologists in academic departments only 8% were less than 30 years of age and 22% over 50, whereas in industrial R&D departments the corresponding figures were 44% and 6% respectively. Since 1971 the proportion of pharmacy graduates in established academic or industrial R&D posts has fallen from 30% to 13% but the proportion with a medical qualification is almost unchanged. In academic departments, there was a net loss of established staff during 1982 and 1983 compared with a net gain of almost 100 appointments in industrial departments. The major single cause of academic pharmacologists leaving posts was early retirement, whilst in the industrial sector the major single destination was another industrial department. In 1983, 227 students graduated with a special B.Sc. degree in Pharmacology compared with 67 in 1971. The same period saw a decline in pharmacy students specializing in pharmacology (293 to 204) and the emergence of Joint Honours courses that include pharmacology. Pharmacology students who graduated in 1983 were also sent a questionnaire. From those responding (57%) over 90% were satisfied with their course and over 80% considered it adequate preparation for their future occupation. At the time of the survey in 1983, only 8% of students graduating in 1982 were unemployed, but all of the 1982 postgraduates were employed.

The benzodiazepine receptor ligand CL218,872 has both anxiolytic and sedative properties in rodents.

The triazolopyridazine, CL218,872, inhibited the binding of [3H]flunitrazepam in the cerebellum more potently than in cortex, both in vitro and in vivo. The relationship between this phenomenon and the selectivity of benzodiazepine receptor subtypes is considered. In a range of behavioural tests the overall profile of CL218,872 was similar to that of diazepam in the rat and mouse, although CL218,872 was half as potent as diazepam in the rat and some 20-fold weaker in the mouse. In particular contrast to published observations, CL218,872 reduced locomotor activity in rats and mice at doses only slightly larger than those required to inhibit conflict in rats and footshock-induced fighting in mice. The only qualitative difference between the compounds that could be detected was in the mouse where large doses of CL218,872 did not produce the marked degree of motor incoordination seen after diazepam. However, no such difference was observed in the rat. The presently held view that CL 218,872 is a non-sedative anxiolytic agent needs to be revised in the light of these observations.

Responses of olfactory bulb neurones to the dipeptide carnosine.

Carnosine has been applied by microiontophoresis ot identified neurones in the olfactory bulb of the rat from solutions of different pH. Although mainly without effect when compared with conventional excitatory and inhibitory amino acids, the dipeptide tended to be depressive when ejected as a cation and excitant when ejected as an anion. The results obtained are not in favour of this substance being an excitatory transmitter in the primary olfactory pathway.

Antagonism of gamma-aminobutyric acid and glycine by convulsants in the cuneate nucleus of cat.

1. Some convulsant substances have been applied to single neurones in the cat cuneate nucleus by microiontophoresis. Numerical values were derived for the effectiveness and selectivity of the substances as antagonists of gamma-aminobutyric acid (GABA) and glycine. 2. (+)-Bicuculline methochloride was the most effective GABA antagonist and it also excited many neurones. It antagonized GABA in 93% of experiments but also antagonized glycine in 41% of experiments. In most experiments the antagonism of GABA was greater than the antagonism of glycine resulting in an overall selective antagonism of GABA that was statistically significant. Nevertheless, in only about one quarter of the individual experiments was the GABA antagonism substantial and the selectivity clearcut. 3. (+)-Bicuculline and picrotoxin were less easily applied to neurones by microiontophoresis and were found to antagonize GABA in 30% and 35% of experiments, respectively. They also antagonized glycine in 25% and 30% of experiments, respectively. Overall, neither substance could be shown to be selective, statistically, although in the few individual experiments where the GABA antagonism was substantial the antagonism was clearly selective. 4. (+)-Tubocurarine antagonized GABA in 59% and glycine in 32% of experiments and it also excited many neurones. Penicillin antagonized GABA in 33% of experiments without antagonizing glycine. Neither antagonist caused any substantial antagonisms of GABA and neither showed significant selectivity overall. (-)-Bicuculline methochloride, leptazol and bemegride antagonized GABA or glycine in less than 10% of experiments, although (-)-bicuculline methochloride excited most neurones. 5. Strychnine antagonized glycine in every experiment while antagonizing GABA in only 5% of experiments. In each individual experiment the antagonism of glycine was substantial and clearly selective, resulting in a statistically significant selectivity overall. 6. It is concluded that the selective glycine antagonist strychnine is considerably better than the presently available GABA antagonists for distinguishing between responses to GABA and glycine, when the antagonists are applied by microiontophoresis.