WHAT IS ENDOTOXIN?
Endotoxin is a component of the cell wall in Gram negative bacteria. Specifically, endotoxin is a major component of the outside portion of the outer membrane of the Gram negative cell wall. Endotoxin is a very strong pyrogen and is considered a bacterial toxin.

Endotoxin is a lipopolysaccharide (LPS). There are two major parts to this molecule, the polysaccharide and lipid A. The polysaccharide is not toxic. It is hydrophilic and faces outward from the cell membrane and into the medium in which the bacteria is growing. It contains major antigens of the bacteria and is the most important target of Gram-negative bacteria for the immune response. Each species of Gram negative bacteria makes its own unique LPS molecule. The polysaccharide portion of the molecule is highly variable.

Lipid A is the toxic component of LPS. Lipid A is composed of a disaccharide (glucosamine) containing phosphate groups and fatty acids. The fatty acid portion of the molecule is very hydrophobic and faces inward toward the middle of the outer membrane. This causes LPS to behave in a similar manner as phospolipids, which make up most membranes. There is much less variability among bacteria in the composition of Lipid A and it is mainly found in the fatty acid chains.

Why test for endotoxin? Endotoxin causes adverse reactions in humans. If endotoxins are present in drugs, devices, or other items placed inside the body, they can cause illness. Drugs taken orally do not need to be tested for endotoxin. Bacteria do not need to be alive for endotoxin to be toxic. In fact, endotoxin in live bacteria is less toxic than when the bacteria are dead because endotoxin from dead cells is released, thereby increasing the apparent dose.

Endotoxin is relatively heat stable. It will survive common heat-based disinfection and sterilization procedures. Hence, sterile items are not necessarily free from endotoxin.

RELATED INFORMATION 

Pyrogen: A pyrogen is any compound that gives rise to a pyrogenic reaction, which is characterized by fever. Endotoxin is the most potent natural pyrogen and a potential health risk to humans. In large doses, endotoxins can cause shock and death.

Bacterial Toxins: There are three classes of bacterial toxins. Endotoxins are named “endo” because they are part of the cell wall and not released in large quantities unless the cell is destroyed. Endotoxins are made by only Gram-negative bacteria. Exotoxins are actively secreted from bacteria while they are alive and growing. Enterotoxins are secreted in the intestine and cause diarrhea and dysentery.

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REGULATORY ASPECTS OF ENDOTOXIN TESTING
The FDA approves limits for exposure and testing methods for endotoxin. The USP, in conjunction with the EP and JP, refine testing methods for endotoxin testing.

There are several major regulatory documents that describe how drugs, devices, dialysate, water, and other substances are to be tested for endotoxin.

• Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices, FDA, December, 1987.
• Interim Guidance for Human and Veterinary Drug Products and Biologicals, Kinetic LAL Techniques, FDA, July 1991.
• Bacterial Endotoxins Test, United States Pharmacopoeia <85>.
• Transfusion and Infusion Assemblies and Similar Medical Devices, United Sates Pharmacopoeia <161>.
• Bacterial Endotoxins Test, European Pharmacopoeia <2.6.14>.
• Bacterial Endotoxins Test, Japanese Pharmacopoeia XIV, <6>.

BASIC LIMITS

The limits apply for drugs and devices administered to humans or animals.

A limit listed for a compendial drug takes precedence over calculated values.

• Limit for parenteral drugs: 5.0 EU/kg. Assume a 70 kg human adult, unless administering drugs to children.
• Limit for drugs being injected intrathecally: 0.2 EU/kg.
• Limit for devices: 20 EU/device
• Limit for devices being used intrathecally: 2.15 EU/device

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WHAT IS GLUCAN?
Glucans are a class of polysaccharides. Specifically, they are chains of glucose that are usually branched and sometimes cross-linked. They are part of the cell wall in fungi (yeasts and molds), algae, some bacteria, and plants, where they contribute mechanical strength and integrity to the wall.

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EFFECTS OF GLUCAN
(1,3)-ß-D-Glucans are immunomodulators. Folk remedies from many cultures use mushrooms to aid healing. It is believed that the healing effect is attributable to the glucans in the mushrooms. There are many types of (1,3)-ß-D-Glucans, and physical and chemical properties of the molecule affect its biological activities. (1,3)-ß-D-Glucans are known to bind to and activate macrophages, neutrophils, monocytes, and NK cells. They are believed to bind to a variety of cells, eg, endothelial cells and fibroblasts.
Among the biological effects caused by glucans is the production of cytokines, which are important components in inflammation. Other activities associated with glucans are nitric oxide synthesis, activation of the complement cascade, and activation of lymphocytes and macrophages.
There is evidence from animal studies that glucan and endotoxin can act synergistically to increase the inflammation response. In some studies, this synergy has been used to treat cancer.
For manufacturers of drugs and devices, (1,3)-ß-D-glucans is considered a contaminant. Although the FDA does not regulate this immunostimulatory contaminant, it can be of concern because of two reasons. One, the presence of (1,3)-ß-D-glucans can give a falsely higher reading in the LAL assay for endotoxins. This could produce and Out-Of-Specification result. Two, (1,3)-ß-D-glucans can contribute to adverse reactions in patients.

Common sources of glucans experienced by drug and device manufacturers are filters made from cellulose materials, plant-derived raw materials, cotton-containing enclosures, sugars, naturally-derived raw materials, cellulose products (eg, sponges), etc.

Examples of products found to contain glucan contamination include blood products (albumin, plasma protein, immunoglobulin preparations, coagulation factors), oligonucleotide drugs, collagen products, saline preparations, glucose preparations, water for injection,

There are two approaches to testing for glucan. One, the subtraction method, uses two assays for endotoxin, one of which has a blocking substance to reduce the response of the assay to glucan in the sample. The difference between the two results is proportional to the amount of glucan present. Two, a direct measurement of (1,3)-ß-D-glucans. A direct measurement assay is specific for (1,3)-ß-D-Glucans. It is quantitative, specific, and much more accurate than the subtraction method. The only available versions of this assay are called Glucatell® and Fungitell® and both are available only from Associates of Cape Cod, Inc. For more information click on the links below.

Link to Glucatell for drugs and devices

Link to Fungitell

Link to Beacon

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COMMON TESTING PROBLEMS
INHIBITION

Inhibition is characterized by a Positive Product Control (PPC) being recovered at less than 50% in a chromogenic or turbidimetric assay or a failure of the 2? PPC to clot. Inhibition can also cause a result to be lower than it should be. There are several possible causes for this:

• The pH. The LAL assay works best between pH 6.0 to 8.0. The further the pH of the testing solution is from this range, the more inhibition will be seen. Check the pH of the sample to make sure it is within the proper range. If the sample is close, addition of Pyrosol, a buffer agent, will help bring the sample into the right range. Other samples will require neutralization.
• Organic solvents. Organic solvents inhibit the assay by inhibiting the activity of enzymes. Dilute the sample with water.
• Precipitates. Precipitates will adversely affect the chromogenic assay because the light path will be blocked. Dilute the sample.
• Salt. If the concentration is too high, the enzymes of the assay will be unable to work efficiently. Dilute the sample.

ENHANCEMENT 

Enhancement is characterized by a Positive Product Control (PPC) being recovered at more than 200% in a chromogenic or turbidimetric assay. Most gel-clot assays cannot detect enhancement. In the Inhibition-Enhancement assay, enhancement in a gel-clot assay is assumed when the 0.25 ? Product Standard clots. Enhancement can also cause results to be reported as higher than they should be. There are several possible causes for this:

• Proteolytic enzymes. The LAL assay is a series of serine protease enzymes. Serum, plasma, and other biological samples may have enzymes that can cause the reaction to proceed, even when no endotoxin is present. An example is Trypsin. Heat the sample to denature the enzymes is an easy way to remove this interference.
• Glucans. (1,3)-ß-D-glucans may enhance the assay by activating an alternative pathway in the LAL assay. Glucans are biologically active compounds that can cause effects similar to endotoxin. There are two ways to determine if glucans are causing enhancement. One is to use an agent that block glucan. If the endotoxin result is lower, then glucan was causing enhancement. Two is to use GlucatellTM which is specific for (1,3)-ß-D-glucans and can quantify the amount of glucans present in the sample.
• Surfactants. Surfactants can reduce the size of endotoxin aggregates, thereby making the endotoxin standard in the PPC tube appear as if there were a higher level of endotoxin present. Conversely, too much surfactant can inhibit the ability of enzymes to work efficiently, which will appear as inhibition. Dilute the sample.

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