In-Vitro Evaluation of Preservatives and Antiseptics

Introduction

In-Vitro evaluation refers to studies performed on antimicrobials outside the normal biological context. These antimicrobials are either biostatic or biocidal; this determines their suitability.
Preservatives are antimicrobials utilized for their ability to inhibit the micro organisms that usually affect food, pharmaceutical and cosmetic products.
Antiseptics are products that are suitable for application on living and animal
tissues for the purposes of eliminating bacterial or fungal infections.
Antibiotics is a general term used to refer to compounds utilized in management
of infections that affect humans. These could be antibacterial, antiviral or antifungal.
Disinfectants are usually applied to the environment especially on surfaces or inanimate objects where they eliminate micro-organisms

Preservatives

• Food and pharmaceutical products provide favourabe environment for
  growth of microbes.
• This results in changes that can affect product stability, odour, odour, acidity, among other things; therapeutic efficacy may also be lost.
• Ideal preservatives have to be biostatic (at least), chemically stable, non irritant and compatible with both active pharmaceutical ingredient (s) and excipients in the formulation.
• Inclusion of Preservatives may be:
  • Obligatory: for injections and multidose eye drops
  • Recommended: for aseptically prepared aqueous products
  • Permitted: for oil-in-water creams, liniments and hydrophilic ointments
  • Not permitted: for injection more than 15ml in volume used as single used and intraocular administration.

Presevatives used in the Pharmaceutical Industry

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Microbial Challenge Test

• The product in its final container is used for the test
• The product is inoculated with 1X106 cells per gram or ml.
• The inoculated product is properly mixed.
• The product is then incubated at room temperature (depending on the product).
• 250ul will be collected at 6h, 24h, 48h, 7 days, 14 days and 28 days.
• Viable cell counts using pour plate or spread plate technique will be done using the collected samples.
• Each sample should replicated at least 5 times.
• A test which shows a sterile product after 24 hours indicates an agent that will give produce satisfactory in the field (EP specification)

Factors to Consider

• Selection of Organism for the test
• Single or mixed Challenge
• State of the organism used in the challenge
• Selection of the growth medium
• Number of organisms used
• Number of challenges
• Challenge within the container
• Storage of product
• Inclusion of organic matter
• Frequency of sampling

Antiseptics

Antiseptics are used for:

1. Cleaning skin, open wounds and body cavities (e.g. nostrils, ears, etc.)
2. Cleaning the skin prior to surgery

• They should be able to prevent infection in the parts of the body
  where they are applied.
• Properties of an ideal antiseptic include:
  • Broad spectrum
  • Biocidal
  • Retains activity in the presence of organic matter
  • Rapid action especially for oral antiseptics
  • Must not suppress immunity
  • High activity at small concentration
  • Cheap and available

Evaluation of Antiseptics

 In-vivo

1. Skin infection tests
2. Skin antisepsis test

In-Vitro

1. Skin penetration test
2. Biostatic
3. Minimum inhibitory concentration
4. Biocidal
5. MBC
6. Extinction time test
7. Other tests
   1. Agar diffusion in determining Spectrum of activity
   2. Phagocyte toxicity test

Minimum Inhibitory Concentration

The MIC is the minimum concentration of a compound which does not support the growth of micro-organism. This is done using Mueller Hinton Broth
Method

• A serial dilution is done using the stock solution (usually 2-fold).
• The 0.1ml of the inoculum is a added to each tube in the series.
• The tubes are incubated for 24hr.
• Microbial growth is examined.
• If turbidity is in doubt, an indicator can be used to confirm acidity of the medium

Minimum Bacteriocidal Concentration

This is the minimum concentration which kills off all the cells in a microbial population.
Method

• Complete the steps for MIC above
• A loopful of the reaction mixture will be transferred from each tube to a corresponding tube, containing fresh broth which will act as a recovery medium.
• The newly inoculated broth medium will be incubated at 32ºC and 35ºC for 48hr.
• Absence of growth in recovery medium shows death.

INACTIVATING AGENTS AND METHODS

• Alcohols: Dilution to less than 1%.
• Antibiotics: Addition of penicillinase
• Benzoic acid: Polysorbates
• Phenols: Dilution to less than 0.01%

Extinction time

• This is the time it would take a specified concentration of an antimicrobial
  agent to kill all the cells in a given population.
• A shorter extinction time is associated with a more biocidal agent.

Method

• A solution of antimicrobial agent is prepared with sterile distilled water
• A measured volume of the solution is mixed with specified volume of microbial culture.
• At intervals, a loopful is transferred to suitable recovery medium.
• All recovery medium are incubated for 72 hours
• The shortest time corresponding to absence of cell growth is the extinction time.

EQUATIONS
C^t=K
Where:

C = Concentration of antimicrobial

T= time of collection

^ = Dilution Coefficient

Graphicallly; Log t = logk-nlogC
Mathematically;

n= (logt2-logt1)/(logC1-logC2)