Chapter 4. A Timetable for Replacing, Reducing and Refining Animal use with the Help of in Vitro Tests: The Limulus Amebocyte Lysate Test (LAL) as an Example
- Dr. Christoph A Reinhardt
Published Online: 8 OCT 2008
Copyright © 1994 VCH Verlagsgesellschaft mbH
Alternatives to Animal Testing: New Ways in the Biomedical Sciences, Trends and Progress, Second Edition
How to Cite
Flint, O. (1994) A Timetable for Replacing, Reducing and Refining Animal use with the Help of in Vitro Tests: The Limulus Amebocyte Lysate Test (LAL) as an Example, in Alternatives to Animal Testing: New Ways in the Biomedical Sciences, Trends and Progress, Second Edition (ed C. A. Reinhardt), Wiley-VCH Verlag GmbH, Weinheim, Germany. doi: 10.1002/9783527616053.ch4
SIAT Swiss Institute for Alternatives to Animal Testing, Technopark, Pfingstweidstr. 30, CH-8005 Zürich
- Published Online: 8 OCT 2008
- Published Print: 24 FEB 1994
Print ISBN: 9783527300433
Online ISBN: 9783527616053
- mechanisms and behaviors;
- vidual components;
- rectal temperature;
- proclotting enzyme;
- underlying nature
Concern about progress in validating in vitro tests that might refine, reduce or replace animal use has, in recent years, generated vigorous debate about the process of test validation and acceptance. The debate has proceeded largely in the abstract without reference to actual cases. One in vitro test, the Limulus amebocyte lysate test (LAL), addresses the problem of identifying agents that cause endotoxic shock, a complex pattern of systemic toxicity. The LAL test has achieved the status of largely replacing the rabbit pyrogenicity test, formerly used in the detection of endotoxins. There are useful lessons to be learnt about the process of in vitro test development from the LAL test. The test is an extremely sensitive measure of coagulation (gelling) of Limulus blood, in the presence of endotoxin. Coagulation of Limulus (horseshoe crab) and mammalian blood is analogous in several respects. Disseminated intravascular coagulation (DIC) is one of the possible symptoms of endotoxic shock in Limulus and in mammals, depending on the amount of endotoxin present. Thus, the LAL test can be understood in terms of the mechanism of in vivo toxicity. However, the LAL test isolates only one of the in vivo mechanisms of toxicity. Hypotension and pyrogenicity, among other effects, are ignored.
In vitro tests, by definition, isolate specific mechanisms and behaviors of cell systems in the whole animal. This is a limitation as well as an advantage that defines how successful the test will be in its application. If the in vivo toxicity to be addressed by the in vitro test is well defined (for example DIC as a key feature of endotoxic shock) then the in vitro test may well come to reduce our reliance on animal testing methods. If the in vivo toxicity is ill defined, as is acute toxicity versus simple cytotoxicity tests, then it is unlikely that the in vitro test will ever reduce reliance on animals.
The main features of the LAL test were first described in 1964. Almost 20 years passed between first conception of this test and its final recognition by the FDA as a replacement for the rabbit pyrogenicity test. This intervening time has involved many validation studies and the development of standard reference samples of endotoxin. Most important has been the ongoing collaboration between regulatory authorities and industrial laboratories interested in applying the in vitro test. We must promote better collaboration between industrial, academic and regulatory scientists to achieve the goal of reducing animal use through the application of in vitro tests. We must also use practical examples, like the LAL test, as guides.