USP <51> Antimicrobial

Effectiveness Testing

Product Categories

What sorts of pharmacopeial articles might benefit from the inclusion of an

antimicrobial preservative?

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Product Categories

Category 1:

“Injections, other parenterals including emulsions, otic products, sterile nasal
products, and ophthalmic products made with aqueous bases or vehicles.”

Category 2:

“Topically used products made with aqueous bases or vehicles, nonsterile nasal
products, and emulsions, including those applied to mucous membranes.”

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Product Categories

Category 3:

“Oral products, other than antacids, made with aqueous bases or vehicles.”

Category 4:

“Antacids made with an aqueous base.”

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What is Aqueous according to <51>?

For the purpose of the test, aqueous is defined as a water activity of more
than 0.6

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Inappropriate Reasons for Including Antimicrobial Preservatives

What are some inappropriate reasons for including antimicrobial preservatives in

a product?

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Product Categories

Antimicrobial preservatives should not be used as a substitute for good

manufacturing practices, solely to reduce the viable microbial population of a
nonsterile/ contaminated sterile product, or to control the pre-sterilization
bioburden of a multidose formulation during preparation.

In the case of sterile aqueous based articles packaged in multiple-dose
containers, suitable antimicrobial preservatives are added to inhibit the growth of
microorganisms (bacteria, yeast) that may be inadvertently introduced during or
after the manufacturing process (from repeatedly withdrawing individual doses).

One or more antimicrobial preservative(s) are expected in all sterile multidose
units.

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Antimicrobial Effectiveness

Antimicrobial effectiveness, whether inherent in the product or produced

because of the addition of an antimicrobial preservative, must be demonstrated
for all aqueous based injections packaged in multiple-dose containers and for
other products containing antimicrobial preservatives.

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Why Include the "Minimum Amount"?

Antimicrobial preservatives are inherently toxic. If not so, they would not kill
bacteria. Sometimes, they can be diluted fast enough to be acceptable even if
the added concentration is toxic.

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Self-Preserved Products?

Can a product possibly be self-preserving?

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Unopened Containers

The procedures and acceptance criteria for effectiveness apply to a product in the
original, sealed container in which it was distributed by the manufacturer.

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Secondary Containers

The test need not be conducted in these containers, but care should be taken to
avoid using materials that can interact with the preservative in the containers
that are used for antimicrobial effectiveness testing.

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General Considerations

The ability of the procedure to detect challenge microorganisms in the presence

of a suitably neutralized product to be tested must be established. The suitability
of the procedure must be reconfirmed if a change is made in materials or
methods or if a change is made in the product or direct product contact
materials that may affect the outcome of the test.

The growth-promoting capabilities of media used in this procedure must be
established.

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Growth Promotion and B/F Testing

Every batch of ready prepared media or each batch of medium prepared either
from dehydrated medium or from the ingredients described.

For solid media, counts obtained must be at least 50% of the calculated value
for a standardized inoculum. For a freshly prepared inoculum, growth of the
microorganisms occurs comparable to that previously obtained with a previously
tested and approved batch of medium

Method Suitability has to be demonstrated. Either dilution, inclusion of

neutralizers, membrane filtration.

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Microorganisms

Candida albicans (yeast)

Aspergillus brasiliensis (mold)

Escherichia coli (Gram-negative rod)

Pseudomonas aeruginosa (Gram-negative rod)

Staphylococcus aureus (Gram-positive coccus)

What is significant about the list of species employed?

Why is it important to include such a broad spectrum of microorganism types?

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Suitability of the Counting Method in the Presence of Product

Prepare a 10−1 dilution by adding 1 mL of product (by volume) to 9 mL of saline

or other neutralizing diluent. Continue this dilution scheme to 10−2 and 10−3
dilution levels. Add an appropriate number of challenge organisms to each tube
of diluted product, mix, and then plate a suitable volume from each dilution to
yield less than 250 cfu/plate for bacteria and yeast (ideally between 25 and 250
cfu) or less than 80 cfu/plate for A. brasiliensis (ideally between 8 and 80 cfu).

This plating should be performed minimally in duplicate (although a greater
number of replicates can be useful to minimize variability in the plate count
estimate). A positive control for this procedure is to introduce the same inocula
into saline, and transfer similar volumes of saline to agar plates. A suitable
recovery scheme is the one that provides at least 50% of this saline control
count (averaged).
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Suitability of the Counting Method in the Presence of Product

If the diluted product exhibits antimicrobial properties, specific neutralizers may need to be
incorporated into the diluents or the recovery media.

The ability of the procedure to measure preservative efficacy may be compromised if the method
suitability requires significant dilution (10−2 or 10−3) as this will affect the measured recovery
(e.g., it may be difficult to measure a 3 log10 unit reduction for a 105–106 inoculum). If no suitable
neutralizing agent or method is found and method suitability requires significant dilution, a higher
level of inoculum (e.g., 107–108) may be used so that a 3 log 10 unit reduction can be measured.

Reported recovery cannot be less than 1 cfu/plate on average (or 100 cfu/mL if 1 mL is plated in
duplicate at the 10−2 dilution).

Membrane filtration may be used to filter larger volumes of dilutions to overcome this difficulty or
to assist in the neutralization of antimicrobial properties.

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Microorganisms in Separate Containers or Together?

To save time, is it OK to add all five test species into the same container?

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Amount of Inoculum to Add

The chapter states that the volume of inoculum should not exceed 0.5 – 1.0% of
the total volume of the product.

Why?

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Number of Microorganisms to Add

105 – 106 cfu/mL of product are added for categories 1-3. Where a large dilution
(greater than 10-2 -10-3 is required) a higher inoculum 107 to 108 cfu/ mL may be used.

Only 103-104 cfu/mL are added for category 4.

What does this suggest about preservation of category 4 products?

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Incubation Temperatures

Inoculated products are supposed to be incubated at 22.5 ± 2.5o. In other USP

microbiology chapters, incubation was at 32.5 ± 2.5o for bacteria, and 22.5 ±
2.5o for fungi.

Why is only one, lower, temperature used?

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Criteria for Passage

Table 3 (first section):

For Category 1 Products

▸ Bacteria: Not less than 1.0 log reduction from the initial calculated count at day 7,
not less than 3.0 log reduction from the initial count at day 14, and no
increase from day 14 to day 28 [initial count?]

▸ Yeasts No increase from the initial calculated

and Molds: count at day 7, 14, or 28

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Criteria for Passage

Table 3 (second section):

For Category 2 Products

▸ Bacteria: Not less than 2.0 log reduction from the initial count at day 14, and
no increase from day 14 to day 28

▸ Yeasts No increase from the initial calculated

and Molds: count at day 14 or 28

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Criteria for Passage

Table 3 (third section):

For Category 3 Products

▸ Bacteria: Not less than 1.0 log reduction from the initial count at day 14, and no
increase from day 14 to day 28

▸ Yeasts No increase from the initial calculated

and Molds: count at day 14 or 28

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Criteria for Passage

Table 3 (fourth section):

For Category 4 Products

▸ Bacteria, Yeasts No increase from the initial

and Molds: calculated count at day 14 or 28

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Criteria Difference Questions

Note that requirements decrease in stringency as you go from Category 1 to 4.

What does this suggest about relative importance of preservation?

What about category 4?

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Fungal Preservative Efficacy

The requirements for fungal control never exceed that of fungistasis.

Why might this be so?

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Initial Calculated Count

All 4 categories contain statements about no increase from the initial calculated
counts at particular time points.

How does one determine the initial time counts?

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About That 0.5 Log Difference

“‘No increase’ is defined as not more than 0.5 log10 unit higher than the previous
value measured.”

Why should this be so defined?

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About That 0.5 Log Difference

Log10 of 1,000 cfu/mL = 3.0 (at day 14)

“‘No increase’ is defined as not more than 0.5 log10 unit higher than the
previous value measured.”

Does 1,500 cfu/mL (at day 28) meet the requirement?

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When It’s All Said and Done…

Nobody actually bothers with this microbiological determination of antimicrobial

effectiveness once it is established.

Once established, all that is necessary to do is determine the concentration of
antimicrobial agent chemically from then on Right?

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