Rabbits were used for the pyrogen testing from 1940’s and were replaced with LAL testing from 1977. For LAL testing [Bacterial endotoxins], the blood of horse shoe crabs are used to till date. LAL testing continues to serve as an efficient and reliable safety test for human and animal injectable drugs. LAL test prevails in the pharmaceutical industry as a release test for Bacterial Endotoxin for the Pharmaceutical drug products which enters the blood stream. Due to the shrinking population of the horse shoe crab, necessity arises for the advancement in the testing and moving away from animals. Future will depend upon synthetic sources. The journey started with rabbits and then to Horse shoe crab and what will be the next?
Bacterial Endotoxins-What are they?
1.They are pyrogenic substances which rises temperature following intra-venous administration / injection.
2.There are many biological and chemical pyrogenic substances.
3.Most Significant pyrogen encountered in routine production of parenteral and medical devices is Gram-negative bacterial endotoxin.
4.The terms pyrogen and bacterial endotoxin are still often interchanged. Pyrogens are substances that can cause fever, shock and even death if high levels are introduced into a person’s body.
5.All Bacterial Endotoxins are Pyrogens but all Pyrogens are not Bacterial Endotoxins.
What is Gram-negative bacteria?
In 1884, Christian Gram was first to group the bacteria in two categories based on Gram staining reaction for the Peptidoglycan layer.
Gram-Positive Bacteria appear as violet / purple.
Gram-Negative Bacteria appear as pink.
Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria.
They are Lipopolysaccharide complex associated with the outer membrane of Gram-negative bacteria such as Escherichia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, and other leading pathogens.

Existence of Gram-negative bacteria?
Gram negative bacteria naturally occurs in air and water we consume. They have existed for hundred of millions of years. They are our host in intestine and helps in digestion.
When they are existing in our body, what makes them inactive to cause disease?
We have regulated mechanism by the liver that prevents absorption of these bacteria from gastrointestinal tract into the blood stream. However, if they enter, they cause high, fatal fevers and disease.
Sources of Bacterial Endotoxin during Pharmaceutical preparations
1.Water for Pharmaceutical Use.
2.Raw Material.
3.Primary Packaging Material.
How to control Endotoxin
Water for Pharmaceutical Use
Water represents major portion of pharmaceuticals.
1.In water, by preliminary treatment to reduce the initial level.
2.By processing to yield low endotoxin water [RO & Ultra-Filtration System].
3.By final processing to yield endotoxin free water [WFI System].
Raw Material
1.Manufacturing steps involving endotoxin controls [Acid hydrolysis, solvent rinsing, treatment with activated carbon].
2.Manufacturing involving Less Water activity.
Primary Packaging Material
1.Containers / glass vials shall be rinsed with endotoxin free water to dislodge the surface binding endotoxin.
2.Containers / glass vials shall be depyrogenated using heat to destroy the residual endotoxin of surface and matrix bounded endotoxin.
3.Rubber Stoppers [Closures] LIMITATION: Cannot be depyrogenated.
4.Control is to get closures from reliable sources manufacturing the stoppers in controlled condition using quality raw material and to have best cleaning & rinsing procedures.
5.Rubber closures made of the elastomer [Synthetic polymer, halobuty, chloro or Bromo butyl] has got high degree of impermeability.
How bacterial endotoxin can be detected?
- By primitive Rabbit Test.
- By in-vitro LAL test.
Rabbit Pyrogen test
1.In the 1940s, the Rabbit Pyrogen Test was introduced as a way to monitor water and other injectable solutions for the presence of harmful levels of endotoxin.
2.Rabbit Test involved the injection of the test solution into the bloodstreams of several rabbits and the subsequent monitoring of the rabbits’ temperatures over a period of three hours.
3.Rabbit was considered suitable for test because the rabbit, like the human, responds to Pyrogens by developing a fever, shock, or complications resulting in death depending on the endotoxin dose.
Test Principle: Involves measurement of rise in body temperature following Intra Venous administration of test specimen.
Administration volume should not exceed 10 mL/Kg body weight of rabbit with injection time of NMT 10 minutes and temperature sensor shall be inserted NLT 7.5 cm in rectum. Healthy rabbits housed in 20-23 Deg.C and temperature should not vary by +/- 3 Deg.C. Any rabbit used one time should not be used for further 48 hours. If the tested rabbit shows higher temperature than desired, it should not be used for 2 weeks. Rabbit shall be withhold by food and water during testing. Control temperature shall be monitored at least 30 minutes prior to injection. A group of 3 healthy rabbit shall be used. Control temperature between these rabbit should not be more than 1 Deg.C. Rabbit having control temperature of 39.8 or more should not be used. Test solution shall be warmed to 35-39 deg.C ( 37 +/- 2 Deg.C) and the test incubation is 3 hours. Temperature to be monitored for every 30 minutes. Temperature variation: Individually NMT 0.5 Deg.C. If any one reached above 0.5, test shall be repeated using 5 more rabbits. Out of 8 Rabbits, NMT 3 rabbits should have MT 0.5 Deg.C variation from control temperature, individually. The sum of variation should not be more than 3.3 Deg.C. If > 3.3 Deg.C, material fails to meet the requirement.
In-vitro LAL [Limulus Amoebocyte Test]
In the 1970’s, in vitro test has replaced the rabbit pyrogen test in most applications and is also being used in areas where the rabbit test is not feasible.

What is Horseshoe Crab?

1.Horseshoe Crabs are considered to be living fossils. Horseshoe crab is large, primitive marine arthropod, similar to Spider. They are also called as King Crabs. Their appearance has not changed for over 300 million years.
2.Despite their name, they are not true crabs They are not actually a crab but more closely related to spiders. True crabs have 10 legs but Horseshoe crabs have 12. The first pincer-like pair are used for feeding; the next 4 pairs are walking-5th pair for feeding legs; and the sixth pair are walking legs used to clear away mud and silt during burrowing.
3.Crabs have gills, whereas horseshoe crabs have book lungs as in spider.
Clotting mechanism In-Vivo.
Bangs studies on horseshoe crab revealed that the amoebocyte cells in horseshoe crab blood act as primitive immune system. When crab is wounded, the amoebocytes swarm to the area and coagulate, forming viscous gel surrounding the invading bacteria. Unable to escape, the bacteria are soon engulfed by defense molecules such as antimicrobial proteins and peptides. This blood clotting mechanism prevents infection being transmitted in horseshoe crabs body or blood.
Significance of horseshoe
1.They are harvested to extract clotting agent of their blood called as Limulus amoebocyte lysate(LAL)
2.The crabs are transported to LAL manufacturing facility, bled and then transported back to harvesting facility.
3.Ecologically, horseshoe crab eggs are primary source of fat for at least 20 species of migratory shore birds, hence maintains food chain. Larval and juvenile crabs are also food for many species of fish and invertebrates. It normally habitats in shallow water and offshore.
4.They first appeared in Upper Silurian period, a number of fossil species are described. Five species are still alive.
5.Four of these are found along the pacific coast of Asia. The American species is common called Limulus Polyphemus.
6.The circulatory fluid, or hemolymph, of Horse shoe crab is analogous to human blood. Blood is blue in colour due to reaction between Copper and Oxygen carrying protein called hemocyanin.
LAL Production
Crabs are caught and examined for good health. They are bled with SS needle by inserting in Circulatory system, blood is centrifuged. Amoebocytes are separated from blood plasma. Amoebocytes are freeze-dried and processed.
Bleeding of Limulus Polyphemus

World wide production
Four United States Company produce almost all LAL products used world wide:
1.Associates of Cape Cord-ACC.
2.Lonza [Formerly known as Cambrex].
3.Charles River Endosafe.
4.Heamachem.
Advantages Over Rabbit Test
- Fast incubation- 60 minutes versus 180 minutes.
- Greater sensitivity.
- Less variability.
- Much less false positives.
- Less Expensive.
Types of LAL test.
Qualitative test- Gel clot test.
Will know pass or fail at a defined concentration.
Sample must have pH 6.0-8.0 and must be tested at < MVD [Maximum Valid Dilution].
MVD = ERL( EU/mg) x Concentration( mg/mL)
Sensitivity of LAL( EU/mL)
Where ERL: Endotoxin Release Limit
- Incubation: 37 +/- 1 Deg.C

Quantitative test- Will be able to quantify the concentration
1.Kinetic Turbidimetric Assay [KTA]
Measures increase in turbidity based on the concentration of endotoxin or on-Set OD.

2.Kinetic- Chromogenic Assay [KCA].
Measurement of chromophore released from suitable chromogen. A suitable chromogen after reaction with endotoxin and LAL gives yellow colour.

Development of LAL
- As early as 1885 W.H. Howell of Johns Hopkins University described the clotting of Limulus [Horseshoe crab] blood.
- In 1950s, it was by Dr. Frederick Bang identified Bacterial endotoxin as causative agent for clotting.
- The gel-clot technique was pioneered in 1960s by Bang, Levin & James F. Cooper.
- 1969-1971, Cooper demonstrated that LAL test is more sensitive than Rabbit test.
- In 1975, James F. Cooper demonstrated that the drug passed by rabbit test has shown positive test with LAL.

Test History
- Initially pyrogen were detected by means of Rabbit test, originally developed by Horst & Perfold in 1912.
- Later introduced as Compendial test in 1942, in USP-XII.
- Later developed by Pharmacopoeial authorities including BP & EP.
- Rabbit test remained for 40 years as compendial test for detection of pyrogen.
- In 1964, Levin & Bang developed LAL test.
- Regulatory acknowledgement was given in 1973. FDA announced LAL as Licensed biological product.
- 1977, First appeared in USP.
- 1987, FDA published guideline on LAL testing.
- 1988, first appeared in EP, other official issue 1998, supplement to EP.
- The Limulus amoebocyte lysate [LAL] test is an alternative method to the rabbit pyrogen test focused on detection of pyrogenic substances in sterile parenteral drugs.
- In a notice published in the Federal Register on November 4, 1977, the FDA described conditions for the use of LAL as an end-product test for endotoxin in human biological products and medical devices.
Conclusion
LAL testing will continue to serve as an efficient and reliable safety test for human and animal injectable drugs. LAL test prevails in the pharmaceutical industry by the careful avoidance of the LAL manufacturers of bringing harm to live animals during both production and testing. However, the need for endotoxin testing is not in decreasing trend and the demands are certainly going to increase and there are concerns related to the sustainability of the horseshoe crab population. Future will be to protect the living fossil and finding out alternative animal free testing.
Note-The images given for representation in this blog are taken from Google Images. Many thanks for Google.