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Laxmi Yadav, Mumbai August 10 , 2022
In order to control the microbial quality of the entire medicinal range, the Indian Pharmacopoeia Commission (IPC) has come out with revised draft general chapters on microbiology in Indian Pharmacopoeia (IP).

The revised draft general chapters on microbiology include revised bacterial endotoxin, revised effectiveness of antimicrobial preservatives, and revised microbial contamination in non-sterile products. The IPC has invited stakeholders’ comment on it by September 20, 2022.

The Bacterial Endotoxins Test (BET) is an in vitro assay for detection and quantification of bacterial endotoxins, a component of the cell wall of gram-negative bacteria.

The test for bacterial endotoxins measures the activity of bacterial endotoxins that may be present in the sample or on the article to which the test is applied using a lysate derived from the hemolymph cells or amoebocytes of the horseshoe crab, Limulus polyphemus. Other species of horseshoe crab namely Tachypleus gigas, Tachypleus tridentatus and Carcinoscorpius rotundicauda also yield amoebocyte lysate having similar activity.

The addition of a solution containing endotoxins to a solution of the lysate produces turbidity or gelation of the mixture. However, addition of a chromogenic substrate to a solution of the lysate results in development of colour due to release of chromophore from the substrate upon activation by the endotoxin present in the solution. The rate of reaction depends on the activity of endotoxin, the pH and the temperature. The reaction requires the presence of certain bivalent cations, a clotting cascade enzyme system and clottable protein, all of which are provided by the lysate.
The following methods can be used to monitor the endotoxin activity in a product official in the pharmacopoeia and to determine whether the product complies with the limit specified in the monograph. Gel-Clot Limit Test Method, Semi-quantitative Gel-Clot Method, Kinetic Turbidimetric Method, Kinetic Chromogenic Method, End-Point Chromogenic Method.
In the event of doubt or dispute, the final decision is made based upon the gel-clot limit test unless otherwise specified in the monograph for the product being tested. The endotoxin limit for a given test preparation is calculated from the expression K/M, where M is the maximum recommended bolus dose administered to an adult (taken as 70 kg for this purpose) per kg of body weight. When the product is to be injected at frequent intervals or infused continuously, M is the maximum total dose administered in a single hour period.
The test for effectiveness of antimicrobial preservatives shall be demonstrated during development of pharmaceutical preparation and during commercial manufacturing. The test is not intended to be used for routine control purposes. The efficacy of antimicrobial preservation of a pharmaceutical preparation on its own or, if necessary, with the addition of a suitable preservative has to be ascertained during the development of the product. The primary purpose of adding antimicrobial preservatives to dosage forms is to prevent adverse effects arising from contamination by microorganisms that may be introduced inadvertently during or subsequent to the manufacturing process. However, antimicrobial agents should not be used solely to reduce the viable microbial count as a substitute for good manufacturing procedures. There may be situations where a preservative system may have to be used to minimize proliferation of microorganisms in preparations that are not required to be sterile. It should be recognized that the presence of dead microorganisms or their metabolic by-products may cause adverse reactions in sensitized persons. Any antimicrobial agent may show the protective properties of a preservative.

However, for the protection of the consumer the concentration of the preservative shown to be effective in the final packaged product should be considerably below the concentrations of the preservative that may be toxic to human beings.

Antimicrobial efficacy test must be demonstrated for multiple dose parenteral, otic, nasal, ophthalmic, oral and topical products made with aqueous bases or vehicles, the effectiveness of any added preservatives, during the shelf-lives of the preparations to ensure that the antimicrobial activity has not been impaired by storage.
The tests apply only to the product in the original, unopened container in which it was supplied by the manufacturer. The test consists of challenging the preparation in its final container with a prescribed inoculum of suitable microorganisms, storing the inoculated product at a prescribed temperature, withdrawing samples from the container at specified intervals of time and counting the organisms in the samples removed. The preservative properties of the product are considered adequate if, in the conditions of the test, there is a significant fall or no increase in the number of microorganisms in the inoculated preparation after storage for the times and at the temperatures prescribed. Single strain challenges (rather than mixed cultures) should be used throughout.

The following test organisms are used in the test-- Candida albicans ATCC 10231, Aspergillus brasiliensis ATCC 16404, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538.

Under the revised microbial contamination in non-sterile products, a slew of tests is designed for the estimation of number of viable aerobic microorganisms present, for detecting the presence of designated microbial species and acceptance criteria in pharmaceutical substances, non sterile dosage forms, herbs, processed herbs and herbal products.
The Total Aerobic Viable Count test is one of them. The test will allow enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions. Membrane Filtration, Plate count methods, Surface-spread method can be used for it.

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