Endotoxin limit tests

Where the test is conducted as a limit test, the specimen is determined to be positive or negative to the test judged against the endotoxin concentration specified in the individual monograph. Where the test is conducted as an assay of the concentration of endotoxin, with calculation of confidence limits of the result obtained, the specimen is judged to comply with the requirements if the result does not exceed (1) the concentration limit specified in the individual monograph and (2) the specified confidence limits for the assay. In either case the determination of the reaction endpoint is made with parallel dilutions of redefined endotoxin units. 

Bacterial endotoxin kinetic test or gel clot limit standard test method (provide reference number)... 

For limit test calculate the concentration of endotoxin in the test solution as follows ... 

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 ... 

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. ... 

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 PPC, required by the FDA Guideline, is a sample of the test material that contains a concentration of endotoxin that is double the labeled sensitivity of the LAL reagent.The PPC must be tested with each sample in a Limits test or kinetic LAL assay to ensure that a result is valid and free of interference. The most accurate and reliable technique is the hot spike method that requires adding 10 pi of endotoxin standard to the reaction vessel (tube or well) before addition of LAL. The gel clot method requires the addition of 10 pi of 20 A to the reaction mixture. Kinetic methods require the addition of 10 pi of a standard, 10 times greater than the spike concentration, to the wells or tubes designated as PPCs. Only inhibition is seen in gel-clot Limits tests, whereas both inhibition and enhancement are seen in kinetic LAL methods. 

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 ... 

Two pharmacopoeial limit tests exist. That for pyrogens uses rabbits to assess pharmacological activity and therefore the presence of pyrogens of all kinds. The test for bacterial endotoxins uses lysed amoebocytes (blood cells) of the horseshoe crab and is therefore termed the Limulus amoebocyte lysate (LAL) test. This may be extended to many drug and device products and clearly will be developed in the future to assess the presence of endotoxins in biotechnology products. 

The samples of products are incubated with Limulus amoebocyte lysate at 37°C. If endotoxins are present a solid gel forms, indicating the presence of endotoxins. The British Pharmacopoeia (2002) describes six separate methodologies for the test for endotoxin. These are (A) gel-clot limit test (B) gel-clot semi quantitative (C) turbidimetric kinetic method (D) chromogenic kinetic method (E) chro-mogenic end-point method and (F) turbidimetric end-point method. There are checks for interfering factors. Any validated method may be used, but the gel-clot method is the referee test in the case of dispute. Coloured products cannot be tested by... 

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). 

These tests would include compendial tests for Microbial Limits, Sterility, Bacterial Endotoxins, and Antimicrobial Effectiveness. 

Inhibition enhancement validation testing is performed on each raw material and finished product as part of the validation for bacterial endotoxin analysis in accordance with the current USP and the FDA Guidelines. Limits are determined from USP monographs or the FDA Guidelines and are used in calculating the maximum valid dilution (MVD). Serial dilutions are performed of spiked and unspiked product in order to determine the dilution required to overcome inhibition or enhancement (DROIE). If the DROIE does not exceed the MVD, the test is determined to be valid. Confirmation of the DROIE is performed on three finished product or raw material lots in order to complete the product validation. Products are tested at a dilution greater than the DROIE but less than the MVD. 

If the endotoxin concentration of one of the two solutions is lower and the other one is higher than this limit, repeat the test. 

Excipients used in injectable formulations have to meet several stringent requirements. A positive identification test uniquely applicable to the excipients is required (e.g., infrared spectrophotometry and chromatography). It is important that manufacturers identify and set appropriate limits for impurities. These limits should be based upon appropriate toxicological data, or the limits described in national compendial requirements. Manufacturing processes should be adequately controlled so that the impurities do not exceed such established specifications. Solvents or catalysts used in the excipient production process should be removed to appropriate levels. If naturally derived, excipients should meet endotoxin levels and may require further testing for bovine spongiform encephalopathy (BSE) /... 

These tests include compendial tests for microbial limits, sterility, bacterial endotoxins, and antimicrobial effectiveness. 

The observation of gel formation in a test tube as an endpoint for an endotoxin assay provides the means for a very simple test. The need for more objective quantitative methods has led scientists to develop a variety of automated methods for endotoxin measurement. This discussion is limited to the three methods that were accepted by the Food and Drug Administration (FDA). 

In vitro investigations have attempted to clarify the mechanism of immune activation but so far have provided limited data. Studies testing the hypothesis that implicated tryptophan or EBT can trigger PBMCs to release cytokines have been equivocal, although one study found that EBT activates eosinophils and induces IL-5 production from T cells. Another recent study found that certain lots of L-tryptophan could stimulate PBMCs to release granulocyte-macrophage colony-stimulating factor (GM-CSF) this response, however, was caused by endotoxin contamination and not associated with case lots of tryptophan. The mechanism of immune activation is clearly complex and may be difficult to reproduce with an in vitro assay. Similar difficulties have been encountered in the study of immune system activation in TOS. 

Finally, a number of Supplementary Chapters describe important topics such as endotoxin testing, statistical analysis of experimental results and guides for the nomenclature of complex natural or semi-synthetic drugs. Supplementary Chapter III F describes the validation of analytical procedures and contains a glossary of terms and their definitions, such as specificity, accuracy, precision, detection limit, etc. 

Even though the product has not yet been developed, a high-quality product specification can be proposed with tests and limits that the product should meet at the time of manufacture and at the end of shelf life. For the intravenous product example, tests might include appearance (clear, particle free), pH, osmolality, particulate levels, sterility and endotoxin levels. Appropriate standards or limits can also be proposed based on the knowledge of similar types of products that have already been developed and from standard pharmacopoeial monographs. 

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