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STERILITY TESTING FOR OILS AND OILY SOLUTIONS


Introduction
Sterilization can be defined as any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses and prions) from a surface, equipment, foods, medications, or biological culture medium. In practice sterility is achieved by exposure of the object to be sterilized to chemical or physical agent for a specified time. Various agents used as steriliants are: elevated temperature, ionizing radiation, chemical liquids or gases etc. The success of the process depends upon the choice of the method adopted for sterilization.
A sterility test is essentially a test which assesses whether a sterilized pharmaceutical or medical product is free from contaminating microorganisms by incubation of either the whole or a part of that product with a nutrient medium. It thus becomesa destructive test and is of questionable suitability for testing large, expensive or delicate products or equipment. Furthermore, by its very nature such a test is a statistical process in which part of a batch is randomly sampled and the chance of the batch being passed for use then depends on the sample passing the sterility test. The test is applied to substances or preparations which, according to the Pharmacopoeia, are required to be sterile. For example, products destined for parenteral administration, or for contact with broken skin, mucousal surfaces, or internal organs, where the threat of infection exists. However, a satisfactory result only indicates that no contaminating microorganism has been found in the sample examined in the conditions of the test.
The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any microorganisms which are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls.


Sterility Testing Methods
Tests for sterility are carried out majorly by two methods: 
(a) Membrane Filtration Method:
Membrane filtration is the technique recommended by most pharmacopoeias and, consequently, the method by which the great majority of products are examined. It involves filtration of fluids through a sterile membrane filter (pore size 0.45µm); any microorganism present being retained on the surface of the filter. After washing insitu, the filter is divided aseptically and portions are transferred to suitable culture media which are then incubated at the appropriate temperature for the required period of time. Water-soluble solids can be dissolved in a suitable diluent and processed in this way and oil-soluble products may be dissolved in a suitable solvent, e.g. isopropyl myristate.

(b) Direct Transfer / Inoculation Method:
 The direct inoculation procedure involves introducing test samples directly into nutrient media. The European Pharmacopoeia (2002) recommends two media: 
(i) fluid mercaptoacetate medium (also known as fluid thioglycollatemedium), which contains glucose and sodium mercaptoacetate (sodium thioglycollate) and is particularly suitable for the cultivation of anaerobic organisms (incubation temperature 30–35°C); and
(ii) Soyabean casein digest medium (also known as tryptone soya broth), which will support the growth of both aerobic bacteria (incubation temperature 30–35°C) and fungi (incubation temperature 20–25°C). Other media may be used provided that they can be shown to be suitable alternatives. Limits are placed upon the ratio of the weight or volume of added sample relative to the volume of culture medium so as to avoid reducing the nutrient properties of the medium or creating unfavourably high osmotic pressures within it.
In both cases use the same microorganisms as those described above under Growth promotion test of aerobes, anaerobes and fungi. Perform a growth promotion test as a positive control. Incubate all the containers containing medium for not more than 5 days. 
If clearly visible growth of microorganisms is obtained after the incubation, visually comparable to that in the control vessel without product, either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. The test for sterility may then be carried out without further modification. 
If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the control vessels without product, the product possesses antimicrobial activity that has not been satisfactorily eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity and repeat the method suitability test.


Methods Employed in Testing Sterility of Oils and Oily Solutions
The test may be carried out using the technique of membrane filtration or by direct inoculation of the culture media with the product to be examined. Appropriate negative controls are included. 
The technique of membrane filtration is used whenever the nature of the product permits, that is, for filterable aqueous preparations, for alcoholic or oily preparations and for preparations miscible with or soluble in aqueous or oily solvents provided these solvents do not have an antimicrobial effect in the conditions of the test.
Membrane Filtration
After transferring the content of the container or containers to be tested to the membrane add an inoculum of a small number of viable microorganisms (not more than 100 CFU) to the final portion of sterile diluent used to rinse the filter.
Procedure
Oils and oily solutions of sufficiently low viscosity may be filtered without dilution through a dry membrane. 
Viscous oils may be diluted as necessary with a suitable sterile diluent such as isopropyl myristate R shown not to have antimicrobial activity in the conditions of the test. 
Allow the oil to penetrate the membrane by its own weight then filter, applying the pressure or suction gradually. 
Wash the membrane at least three times by filtering through it each time about 100 ml of a suitable sterile solution such as peptone (1 g/l) TS1 containing a suitable emulsifying agent at a concentration shown to be appropriate in the method suitability test, for example polysorbate 80 at a concentration of 10 g/l. 
Transfer the membrane or membranes to the culture medium or media or vice versa as described above for aqueous solutions, and incubate at the same temperatures and for the same times.

Direct Inoculation Method (Rarely Used)
Oils cannot be directly inoculated into sterility test media as oil globules may enclose the contaminating cells and thus prevent their growth. If the oil is easily emulsified, it is then inoculated periodically during incubation. Otherwise, the oil is dissolved in a non germicidal solvent and either centrifuged under strict condition and the deposit is tested for sterility. After transferring the contents of the container or containers to be tested to the culture medium add an inoculum of a small number of viable microorganisms (not more than 100 CFU) to the medium. 


References 
Hugo and Russel’s Pharmaceutical Microbiology, Seventh Edition

WHO Monograph on Test For Sterility, Document QAS/11.413 FINAL March 2012, adopted at the Forty-sixth WHO Expert Committee on Specifications for Pharmaceutical Preparations in October 2011 for addition to the 4th Edition of the International Pharmacopoeia.

Sultana, Yashmin Jain, N. K, Sterilization Methods and Principles, issued 24th July 2008, obtained from: http//nsdl.niscair.res.in/jspui/handle/123456789/704

Prof. P. F. Olunrinola, Practical Schedules for PHCT 401: Aseptic Techniques, Aseptic Processing, Sterility Testing and Evaluation of Antimicrobial Agents, pg.26

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