ROUTINE PYROGEN TESTING
1 The examination of preparations from large‐scale manufacture for freedom from pyrogens is described. 2 Typical results from the examination of 11. and 0·51. packs of water for injection, glucose‐saline and solution and normal saline solution are presented. 3 Evidence supporting the claim that reduction in the pyrogen levels take place on storage is provided.
SOME OBSERVATIONS ON THE B.P. AND U.S.P. TESTS FOR PYROGENS
THE B.P. and U.S.P. tests for pyrogens depend on the temperature response of rabbits to intravenous administration of the test material. The methods of interpreting the data differ in the two tests but, in each, the material fails to pass the test if it produces a temperature rise greater than that prescribed. The maximum permissible response, in both the B.P. and the U.S.P., is a specified temperature rise, which is not related to any comparison with a standard preparation. Therefore, any factor which affects the magnitude of the temperature response will affect the sensitivity of the tests. The writer has already shown that the degree of restraint imposed on the rabbits, the type of thermometer used, and the frequency of administration of pyrogen, all affect the magnitude of the rabbit’s temperature response to bacterial pyrogenl. Thus when the degree of restraint imposed on the rabbits is severe and clinical thermometers are used, the magnitude of the response to pyrogens is much less than when the degree of restraint is slight and electrical thermometers are used. Furthermore, there is a progressive reduction in response when pyrogen is given repeatedly. The B.P. and U.S.P. specify neither the degree of restraint nor the type of thermometer to be used. Both pharmacopceias permit the repeated use of the same animals. Two questions of practical importance arise from these considerations. The first and more obvious is; how far may the reliability of the tests be affected by failure to take these sources of variation into account? The second, which derives from the different methods of interpreting the data in the B.P. and U.S.P. tests, is; how do the relative efficiencies of the two tests compare when conducted in similar conditions? This paper seeks to provide an answer to these two questions.
SOME QUANTITATIVE STUDIES ON A BACTERIAL PYROGEN
THE quantitative estimation of bacterial pyrogens has not, until recent years, been seriously attempted. This is mainly because interest in bacterial pyrogens has hitherto centred in ensuring their absence from solutions for injection, rather than i n seeking to estimate the amount present. All the qualitative tests of any value are biological-inadequate knowledge of the chemistry of these substances having precluded the development of chemical tests. The work about to be described represents an attempt to establish a quantitative approach to the study of bacterial pyrogens, and is based upon the methods used in the older qualitative tests. Biological tests for the presence or absence of pyrogens depend on whether the test material, when administered intravenously to rabbits or dogs, produces a rise in t e m p e r a t ~ r e l . ~ . ~ ~ " ~ , ~ , or a change in the number of circulating l e u c o c y t e ~ ~ ~ ~ ~ ~ ~ ~ ~ . But only those tests dependent on the temperature response of the rabbit have come into general use. An essential prerequisite for quantitative studies on the temperature response of the rabbit to pyrogens is an active and stable pyrogen preparation. A number of impure solutions, and dry extracts, containing bacterial pyrogens have been described by previous ~ ~ r k e r ~ ~ , ~ , ~ ~ , ~ ~ , ~ ~ , ~ ~ , ~ ~ , ~ ~ , some of whom have attempted, with varying success, to make quantitative observations upon them. Some have claimed that impure solutions are table^?^^,^^, but others have shown that this is not always sol5. Others again have stated-but without submitting their evidence-that even highly purified preparations are unstable17. In the absence, therefore, of any generally accepted stable pyrogen preparation, it was decided to prepare and study, in the first instance, a crude, dry, pyrogen-containing extract. Part I of this paper describes the preparation of this extract, and the different methods used to measure the temperature response of rabbits to it. Part I1 gives an account of the various experimental studies subsequently undertaken. These were carried out over a period of 5 years and are described in the order in which they were undertaken. As the successive studies constitute a logical series the results of each are fully discussed before proceeding to the next.