Antimicrobial Effectiveness test http://www.gibraltarlabsinc.com/services/microbiology.html

USP <51>

Gina Marino

Proprietary and Confidential © Gibraltar Laboratories, Inc.

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Summary

• What is AET ?

• Why add a preservative

• AET Procedure

• Neutralization Validation

• Interpretation of Results

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What is Antimicrobial Effectiveness Test?

• AET demonstrates effectiveness of preservative in a product

• Compendia Test

• Not yet harmonized around the world

• Testing to confirm that preservatives added in a formulation will work as expected

over time

• Mostly used during formulation development and in stability programs

• organisms-bacteria, fungus, mold Product requirements typically 20-100mL of

product

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Compendial names

• Antimicrobial Effectiveness Test (USP)

• Preservative Challenge Test-CTFA(Cosmetic Toiletry Fragrance Association)

• Efficacy of Antimicrobial Preservation (EP)

•

• Preservation Effectiveness Test (JP) Test

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Name Change of USP Chapter

• Preservative Challenge Test-Old USP

• Antimicrobial Effectiveness Testing(AET)-New USP

• Do you agree in the new name?????

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Why Add a Preservative to a Product?

• To prevent contamination of organisms and make the product safe to use.

• How do we contaminate these products with various microorganisms?

• Please take a Guess ??????

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Did you Guess correctly?

1. Contamination During Manufacturing-non sterile dosage forms

2. End user contamination—after the product container is opened/closed

repeatedly and is in contact with hands, skin ,mucus membrane.

3. Repeated withdrawal of individual doses-Oral products

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How Much Preservative to Add?

• Since these preservatives are toxic substances!

• Formulators can add antimicrobial preservatives below a level that can be toxic to human

beings but can still protect the products from microbial growth

• Therefore-----

• Goal is to add lowest concentration possible that will still inhibit growth of test organisms

allowing the product still to pass the AET test

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Background History

• When in time line did preservative become important

• After-WWII

• Vasaline was replaced with PEG

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AET Test

BASIC PROCEDURE

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Product categories Category

Product Description

1 Injections, other parenteral including emulsions, otic products ,sterile nasal

products and ophthalmic products made with aqueous bases

2 Topically used products made with aqueous bases or vehicles, non-sterile nasal

products and emulsions, including those applied to mucus membrane

3 Oral products, other than antacids made with aqueous bases

4 Antacids made with aqueous base

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Test Organisms/Basic Procedure

• Candida Albicans ATCC No 10231

• Aspergillus Brasiliensis ATCC No 16404

• Escharichia coli ATCC 8739

• Pseudomonas aeruginosa ATCC No 9027

• Staphylococcus aureus ATCC No 6538

• Environmental Isolates

• For parenteral, one might want to consider challenging with organisms associated with

nosocomial infections

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Basic Procedure

Separate containers for each organism to be tested, including appropriate

controls

– 6 Aliquots of products need to be measured out (20 g each)

– Prepare the cultures to be used

– Inoculate needs to be at the right levels of microorganisms

– The cultures must be freshly prepared(24 hours)

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Basic Procedure

• Inoculate the products individually with specific organism, 1 organism per

aliquot

• The concentration of organisms should achieve between 10^5 to 10^6 cfu/ml

• Incubate Microbial Suspension

• Sample at Day 7, 14, and 28

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Basic Procedure

• At Day 0 -Perform inoculum recovery to make sure that the original inoculation levels are at

their specified targets and to also estimate the concentration of organisms in the challenged

products.

• Store products, protected from light at 22.5 +/- 2.5 Deg. Celsius for the time specified in the

tables

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Basic Procedure

• At the test time, remove aliquots and perform plate counts.

• Perform 10-fold serial Dilutions Plate dilutions to determine number of survivors Calculate the

log reduction

• Determine the log-10 of the concentration of the organisms remaining in the samples and

compare the results to the required results from the tables in the individual chapters.

• The requirements are different based on what type of category they are.

• Also- no increase in a microorganism count is defined as not more than 0.5 log-10 increase in

counts

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Interpretation of results Category 1-Inject

Bacteria

Not less than 1.0 log reduction from initial calculated count at 7 Days. Not less than 3.0 log reduction from initial count at 14 days.

No increase from count at 14 days to the count at 28 days

Yeast/Mold

No increase from the initial count calculated at 7,14, and 28 days

Category 2- Creams

Bacteria

Not less than 2.0 log reduction from the initial calculated count at 14 Days. No increase from the count at 14 days to the count at 28 days,

Yeast & Mold No increase from initial count @14 and 28 Days

Category 3-Orals

Bacteria

Not less than 1.0 log reduction from the initial calculated count at 14 days and no increase from the count at 14 days to the count at 28 days.

Yeast & Mold---Same as above

Category 4 Bacteria and Yeast and Mold-No increase from initial calculated count at 14 and 28 days

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USP<1227>

(Neutralization Validation)USP

• Validation for a neutralizing substance

• The neutralizer(inactive agent) must have the following properties:

• It must not have inhibitory effects on the microorganisms

• Should completely overcome the activity of the preservative

• If it inactivates the preservative by combining with it, the resultant product must not be toxic to

the microorganisms

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Three ways to neutralize

• Chemical inhibition

• Dilution

• Filtration and washing

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Neutralization/Basic Method

Divided into three groups

Test group Product Initial 1:10 Dilution Neutralized product with

inoculum

Toxicity group Peptone Initial 1:10 Dilution Challenge inoculum in

buffered solution

Viability Group

Control Group

Buffer No dilution No exposure to

neutralization—Basing

our recovery on this

group

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Neutralization Validation

• Neutralizer Efficacy-We need to demonstrate that we are getting at least 70 % recovery of the

challenge microorganisms at various dilutions of the product compared to the control group.

• Viable count= the control count

• Neutralizer Toxicity-The neutralizer is not, itself, toxic to the microorganisms.

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GIBRALTAR

LABORATORIES 122 Fairfield Road

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Fairfield, New Jersey 07004

(973) 227-6882

kkohan@gibraltarlabsinc.com

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Proprietary and Confidential © Gibraltar Laboratories, Inc.

www.gibraltarlabsinc.com / (973) 227-6882