Normal temperature variation in New Zealand white rabbits during restraint for preliminary pyrogen test.

The in vivo pyrogen test is the main toxicological assay used in the quality control of injectable products, especially immunobiologicals. Pharmacopoeias state that, before the main test, a preliminary test must be conducted on all animals, and must follow the same conditions as the main test. The aim of this study was to determine the normal temperature variation in New Zealand white rabbits during restraint and propose a limit value for considering an animal as suitable for testing. Results of the temperature variation in 4,689 rabbits during preliminary tests were obtained from the routine database of the Pharmacology and Toxicology Department of the National Institute of Quality Control in Health (INCQS/FIOCRUZ), Brazil. From these preliminary tests, 3,364 rabbits were considered suitable for testing according to the Brazilian Pharmacopoeia criteria (temperature variation < 0.5 °C). Results showed that about 92 per cent of the rabbits presented a normal individual temperature variation equal to or below 0.30 °C. Animals presenting a temperature variation close to the fever temperature must not be included in the main test, since they can be stressed or sick. Consequently, the temperature variation of 0.30 °C could be adopted by pharmacopoeias as a limit temperature to be considered in the preliminary test to determine which animals can be used in the main rabbit pyrogen test. Animals can be pre-tested until presenting this safe variation, in order to ensure they are healthy and minimise interference in the result.

The views and policy of the Japanese control authorities on the three Rs.

NIH Japan has tested and regulated the three Rs of animal experiments in the development and control of biological products in a stepwise manner. (1) The number of monkeys was reduced from 108 to 72 for the neurovirulence test of OPV in each type, since paralysed monkeys inoculated intraspinally revealed a linear relationship between average scores of the lumbar lesion and cumulated paralysis occurrence ratio (%). (2) Rabbits for the pyrogen test were replaced by the endotoxin test for PPF, albumin and interferon products. The endotoxin is measured by the parallel line assay method using both turbidimetric kinetic and colorimetric methods. (3) Histopathological examination was introduced to the abnormal toxicity test as a refinement. Mean body weight loss of two guinea pigs inoculated with five ml. of an albumin product in each was far below the mean weight of pooled guinea pigs used (P < or = 0.01) and appeared repeatedly. The histopathological examination showed focal necrosis in the liver. This finding was suggestive of the presence of endotoxin in the product. The product contained 0.1 EU/ml of endotoxin. The same amount of the reference endotoxin produced a similar change in guinea pigs. The mechanism of the liver cell damage by endotoxin has been investigated by an in vitro method.

Studies with the International Pyrogen Standard on the sensitivity and reproducibility of pharmacopoeial pyrogen testing.

Rabbits, 27 or 36 in each experiment, were injected with the International Pyrogen Standard (I.P.St.) in different seasons. The maximum temperature rises were registered, randomized and interpreted according to the requirements of the B.P. (1973), U.S.P. (1970), P. Hung. (1970) and P. Nord. (1962). Although the dose of 3.5 ng kg(-1) I.P.St. proved to be non-pyrogenic as tested in summer, when tested in winter the same dose was qualified pyrogenic (to be rejected) by up to one third of the combinations by the criteria of the four Pharmacopoeias. In the spring experiment "to be rejected" qualifications predominated as based on the response of large groups of rabbits. Exclusion of the rabbits showing low sensitivity (before randomization) barely influenced the results with 3.5 ng kg(-1) I.P.St. in the experiment in which the mean temperature rise was 0.49 degrees. If, however, the mean temperature rise was higher (0.57 or 0.69 degrees), such a selection practically resulted in the disappearance of "passable" qualifications in the triplet groups and a great predominance of "to be rejected" qualifications in the larger groups. The dose 7.0 ng kg(-1) consistently proved to be pyrogenic in large groups of rabbits.