Endotoxins limits

The preparation being examined passes the test if the endotoxin concentration of each of all tested samples is less than the endotoxin limit concentration specified in the specification and the test meet the following three conditions ... 

The endotoxin limits (measured by endotoxin units, or EU) apply for drugs and devices administered to humans or animals are listed below ... 

Pyrogenic contamination is detected using two tests. In the older method, rabbits are injected with product samples, and rectal temperature is measured. Compendial limits are established with respect to how much temperature increase is permitted before the product is judged to be free or contaminated with pyrogens. The newer method involves a relatively simple in vitro technique called the Limulus Amebocyte Lysate (LAL) test. It is based on the high senstivity of amebocytes of the horseshoe crab (Limulus) to the lipopolysaccharide component of endotoxins originating from Gramnegative bacteria. The LAL test is now the USP method of choice with endotoxin limits established for most SVIs. ... 

Endotoxin limits are therefore unique to each dosage form. Because K is fixed, it is usually only necessary to determine M from the maximum human dosage indicated in the product instructions. 

Weary, M.E. Understanding and setting endotoxin limits. J. Parenteral Sci. Technol. 1990, 44, 16-17. 

The first large-scale conversion of the rabbit test to the BET occurred in Supplement 5 to USP XXII when the BET became the official endotoxin test for 185 USP articles. The USP 24 has endotoxin limits for over 650 USP articles. 

This guideline has been the most influential document in LAL testing to date. Annual updates of the Appendix E, endotoxin limits for established parenteral products, were published by the FDA until 1994. With the upgrades in test procedures and endotoxin limits published in individual product monographs, the FDA no longer has a need to revise the 1987 guideline. 

The results of endotoxin tests for in-process solutions, bulk materials, and finished parenteral products should be reported in the same units as those assigned to the product. Two factors determine the sensitivity of a BET. For infusion solutions and device extracts, the gel-clot sensitivity or the lowest point on the standard curve (lambda for kinetic LAL) and the amount of dilution determine test sensitivity.For products that have an endotoxin limit in EU/mg, the choice of lambda and the concentration of the test material determine sensitivity. The formula for product-specific sensitivity (PSS) is a convenient way to calculate the sensitivity of a BET for this type of product, where ... 

Municipal water systems usually contain 5-50 EU/ml of endotoxin. Pharmaceutical waters are treated by ultrafiltration, distillation, or reverse osmosis to separate endotoxin from water.f The principal source of endotoxin is bacteria within a water system in the form of biofilm or colonies entrapped in resin beds, etc. A water system will be contaminated unless there is an ongoing sanitization program. The endotoxin limit for Water for Injection was set at 0.25 EU/ml because it is a critical vehicle and a major source of pyrogens. 

The limits for endotoxin are based on the dose of the product. Put simply, the endotoxin limit, EL, which represents the maximum amount of endotoxin that is allowed in a specific dose, is inversely related to the dose of the drug it may be assessed from the following equation (United States Pharmacopoeia, 2002) ... 

Another important consideration for excipients to be used in parenteral products is their quality, particularly in microbiological terms. Commonly used parenteral excipients can often be obtained in an injectable grade which will meet strict bioburden and endotoxin limits. Pharmacopoeial grades of other excipients may be acceptable, but it is prudent to apply in-house microbiological specification limits, where none are present in the pharmacopoeias. For non-pharmacopoeial excipients, the best approach is always to purchase the highest grade available and apply internal microbiological specification limits. 

Table 9.4.1. Products in the European Pharmacopeia with endotoxin limits...

The bacterial endotoxin limit for WFI does not exceed 0.25 USP endotoxin units per ml. The microbial action limit is not greater than 10CFU/100 ml. 

Microbiological testing should be conducted on each lot of API required to be free of objectionable microorganisms. Appropriate testing should also be conducted on each lot of API required to be pyrogen free or with a specified endotoxin limit (e.g., APIs intended for use in the preparation of parenteral drug products). 

Rylander et al. made some tentative recommendations based on fever and influenza-like symptoms observed to be experienced among workers at wastewater treatment plants. They suggested that until more precise data on the dose-response relationships became available, values up to 1000 Gram-negative bacteria/m, derived from an endotoxin limit of 0.1 [xg/m, was acceptable. 

However, the limit should be even lower a patient receives 60 L of water per dialysis treatment. A treatment lasts for 3 h. That means an intravenous administration of 20 L in 1 h. According to the Ph. Eur. the allowed maximum amount of bacterial endotoxins for parenteral use is 5 lU per kg bodyweight in 1 h. This means for a 60 kg patient that 300 lU bacterial endotoxins are allowed to be present in 20 L resulting in a requirement of <0.015 lU/mL. At this moment the assay technique allows an endotoxin limit of 0.025 lU/ mL to be detected and present water treatment units are able to produce water of this quality. This topic and other requirements are under discussion within the Ph. Eur. committee on dialysis solutions. 

---