Posted: January 15th, 2015

Sterility Testing of the sodium cromoglycate Eye Drop Formulation

Sterility Testing of the sodium cromoglycate Eye Drop Formulation

During this practical you will test the sterility of the sodium cromoglycate eye drop formulation (10ml, 2% w/v) that you manufactured during your recent  aseptic

class.  If you followed the correct procedure(s) throughout production of the sterile dosage form, the item should ‘in theory’ be free from microorganisms.  Within the

hospital and industrial settings it is important to conduct sterility testing, such as the one you are about to perform, to ensure product suitability for end use.

Should the product not pass the sterility test then aspects such as raw material sterility and operator technique would be investigated to minimise the risk to

patients.

4.3.1 Practical Aims

To carry out a test for sterility on cromoglycate eye drops produced.

To interpret the results of a sterility test.

4.3.2 Method

•    For eye drops containing a mercurial

The following steps are carried out in a laminar flow cabinet

1.    Transfer 1 ml of the eye drop formulation into each of:

2×50 ml Oxidised Brewer’s medium (Green) (aerobic bacteria)

2×50 ml Reduced Brewer’s medium (Yellow) (anaerobic bacteria)

2×50 ml Oxidised Brewer’s medium (Green) (fungi)

Here, ONE bottle of each pair is the test.

The following steps must not be carried out in a laminar flow cabinet

2.    To the second bottle of each pair add a few drops of S. aureus to Oxidised Brewer’s medium, C. sporogenes to Reduced Brewer’s medium and C. albicans to

Oxidised Brewer’s medium.

•    For eye drops containing a QAC with or without EDTA

The following steps are carried out in a laminar flow cabinet

1.    Transfer 1 ml of the eye drop formulation into each of:

2×50 ml Nutrient broth (NB) containing 0.1% (w/v) Lubrol W (aerobic bacteria)

2×50 ml Robertson’s cooked meat medium (RCMM) containing 0.1% (w/v) Lubrol W         (anaerobic bacteria)

2×50 ml Yeast dextrose broth (YDB) containing 0.1% (w/v) Lubrol W (fungi)

Here, ONE bottle of each pair is the test.

The following steps must not be carried out in a laminar flow cabinet

2.    To the second bottle of each pair add a few drops of S. aureus to NB, C. sporogenes to RCMM and C. albicans to YDB.

3.    All bottles should be incubated for 14 days, bacteria at 32°C and fungi at 25°C

For Information:

Standard methods of inactivation are shown in Table 1. As above, all tests are carried out in duplicate.

Antibacterial Agent    Method of Inactivation    Positive Aerobic Control
Quaternary ammonium compound (QAC)    Nonionic detergent    Staphylococcus aureus
Penicillin    ß-lactamase    Staphylococcus aureus
Sulphonamide    p-aminobenzoic acid (PABA)    Escherichia coli
Phenol    Dilution    Bacillus subtilis
Sodium Edetate (EDTA)    Dilution    Depends on the other ingredients
Mercurials    Thioglycollate    Depends on the other ingredients

Table 1. Means of Inactivation and the Positive Aerobic Control to be used

The positive control anaerobic bacterium is Clostridium sporogenes and the positive fungus control is Candida albicans.  The positive control for aerobic bacteria has

to be sensitive to the antibacterial agent.  Suitable controls are shown in Table 1.

Key Points

Sterility testing is a manual aseptic process for determining microbiological background contamination of pharmaceutical products using a system of sterile nutritional

media to test for aerobic bacterial, fungal and anaerobic bacterial growth.

A series of positive controls containing the inactivated ingredients of the product under study are provided, one for each of the representative groups of

microbiological contaminants stated previously.

The control organisms used are selected for their susceptibilities to the antimicrobial agent contained within the product(s) under test, these respective organisms

are inoculated into the controls at the end of the experiment.

The controls are mirrored by a series of test samples that have undergone similar inactivation without inoculation.  The active ingredients within the products are

inactivated to reveal any background microbial contamination, this is achieved by chemical processing or applying simple dilution of the active ingredient below its

prophylactic concentration.

Here, we will consider a number of examples.  Please refer to the schematic below. Water for injection is a straight forward test for contamination.  Benzylpenicillin

injection (i.e. an antibiotic) requires inactivation using benzylpenicillinase to break down the beta-lactam ring of the penicillin.  Contamination of this product may

arise from penicillin resistant organisms, bacterial genera, which are not affected by penicillin, organisms that may be in stasis at high concentrations of the

antibiotic of other effects associated with the minimum inhibitory concentration and sporing.  Cetrimide is both an antiseptic quaternary ammonium compound and

surfactant, it is neutralised using another non-ionic surfactant in the form of “Lubrol W” and applying a simple dilution.  The sulfonamide products are treated with

para-aminobenzoic acid (PABA), which acts as a competitor to the sulfonamide in bacterial enzyme processes for the synthesis of folic acid and simple dilution plays a

part in inactivation.  The oily phenol is a simple dilution of the active ingredient below its effective concentration.  The sulphacetamide eye drops are a mixture of

excipients: the sulfonamide active is dealt with using PABA, the phenyl mercuric nitrate element is inactivated with thioglycollate groups present in the nutritional

media whilst the sodium metabisulphite and sodium edetate are inactivated by simple dilution.

The nutrient media typically employed during such sterility testing are: nutrient broth, yeast dextrose broth, Brewer’s medium broth and Robertson’s cooked meat

medium.

4.3.3 Schematic

Sterility Testing of an Eye Drop Formulation
Practical Report

1.    Introduction

The rationale for using sterile products in pharmacy

The application of the allocated product

The rationale for the excipients used

2.    Methods

The method of sterilisation

Biological & physicochemical indicators used to validate the sterilisation process

The sterility testing method

3.    Results

The outcome(s) of the sterility indicators

The outcome(s) of sterility testing

4.    Discussion

The reliability of sterility testing outcomes

The importance of the sterilisation method

Parametric release

5.    References

References used

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