UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY – DEPT. OF PHARMACY

PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023

COMPARATIVE ANALYSIS OF COMPENDIAL ANTIMICROBIAL EFFECTIVENESS TESTS

Arreola, R., Barroga, P., Beltran, C., Bocalan, M.

ABSTRACT
Since antimicrobial preservatives are utilized in the pharmaceutical industry to inhibit microorganism growth
and maintain product stability, antimicrobial effectiveness tests have been established by the different
pharmaceutical compendiums. The main compendia that the paper focused on are the Japanese
Pharmacopoeia (JP), United States Pharmacopeia (USP), and European Pharmacopoeia (Ph. Eur.). The
paper analyzed the three compendiums for their similarities and differences in terms of product categories,
challenge microorganisms, procedures, and criteria.

Keywords: Antimicrobials, Compendia, Pharmaceuticals, Preservatives, Testing

INTRODUCTION must be used at minute concentrations, ranging

Preservatives are natural or synthetic chemicals
added to food, pharmaceutical, and cosmetic
products to counteract microbial contamination and
decomposition, and chemical change (Shaikh et al.,
2016). There are many ways to classify
preservatives. According to Shaikh et al. (2016),
based on source, there are natural and artificial
preservatives, and based on the mechanism of
action, there are antioxidants, chelating agents, and
antimicrobials.
Antimicrobials, in particular, are included in
pharmaceutical preparations to either inhibit growth
or kill microorganisms that were accidentally
introduced during product manufacture or through
its use (Moser & Meyer, 2011). These are of great
importance in multiple-dose products, since
withdrawing product multiple times comes with the
possibility of contamination, and thus, product
instability (Fahelelbom & El-Shabrawy, 2007).
Products with high water content are also prone to
microbial proliferation, and require preservatives to
maintain protection from potentially pathogenic
microorganisms (Fahelelbom & El-Shabrawy,
2007).
Such antimicrobial preservatives are further
classified as parabens, acids and their salts,
quaternary ammonium compounds, mercurials,
alcohols, phenols, and biguanides (Fahelelbom &
El-Shabrawy, 2007). Typically, sodium benzoate,
potassium sorbate, and methyl paraben are
employed in liquid pharmaceuticals (Boukarim et
al., 2009). Since these are potentially toxic, these
from 0.1% to 0.2%, 0.1% to 0.2%, and 0.1% to
0.25% (w/w), respectively (Boukarim et al., 2009).
There are many different ways to characterize
antimicrobial preservatives, such as electrokinetic
capillary electrophoresis (CE), high performance
liquid chromatography (HPLC), gas
chromatography (GC), thin layer chromatography
(TLC), and UV-visible spectrophotometry
(Fahelelbom & El-Shabrawy, 2007). To test the
antimicrobial properties and effectiveness of the
preservatives, antimicrobial effectiveness tests are
used in the pharmaceutical industry.
Antimicrobial effectiveness tests are indispensable
during the manufacture of pharmaceutical products,
as these determine the safety of a broad range of
products and dosage forms (Sutton & Porter, 2002).
It assures that the effectiveness of the
preservatives or the antimicrobial activity of the
product is sufficient to make it safe to use or intake
in a given period (Sutton & Porter, 2002).
There are three major pharmacopeia used as
reference in the pharmaceutical industry. Each of
these compendiums present minor differences in
their antimicrobial effectiveness tests. The
procedures are the following: the United States
Pharmacopoeia (USP) <51> Antimicrobial
Effectiveness Testing, the European
Pharmacopoeia (Ph. Eur.) 5.1.3 Efficacy of
Antimicrobial Preservation, and the Japanese
Pharmacopoeia (JP) 19, Preservative Effectiveness
Tests.
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 UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023
The purpose of this comparative analysis is to
review each of the three different antimicrobial
effectiveness tests under the USP, Ph. Eur., and JP.
ANALYSIS
Overview of the procedures
The USP clearly states that the antimicrobial
effectiveness test must be conducted on
aqueous-based, multiple-dose topical and oral
dosage forms and for other dosage forms such as
ophthalmic, otic, nasal, irrigation, and dialysis
fluids.
The Ph. Eur. notes the use of the efficacy of
antimicrobial preservation test for aqueous
products and products in multidose containers. It
also states that it is only used to demonstrate
efficacy of antimicrobial activity, but was not
developed to be used in routine control tests.
The JP likewise highlights the microbial
contamination risks associated with products in
multidose containers and aqueous products.
The three tests were compared in terms of their (1)
products and categories, (2) challenge
microorganisms, (3) test strain preparation, (4)
growth promotion tests, (5) counting method
suitability, (6) test procedure, and (7) interpretation
of results and acceptance criteria.
Products and their Categories
United States Pharmacopoeia
Since the criteria of antimicrobial effectiveness are
a function of the route of administration of the
products, the USP developed four categories based
on this.
Category 1 includes injections; other parenterals
including emulsions, otic products, sterile nasal
products, and ophthalmic products made with
aqueous bases or vehicles.
Category 2 is composed of topical products made
with aqueous bases or vehicles and nonsterile
nasal products and emulsions, including those
applied to mucous membranes.
Category 3 comprises oral products, not including
antacids, made with aqueous bases or vehicles.
Category 4 only has antacids made with an
aqueous base.
European Pharmacopoeia
The Ph. Eur. loosely grouped products using their
routes of administration, where each classification
has a different acceptance criteria. The first
category comprises parenterals, eye, intrauterine,
and intramammary preparations. The next category
includes ear, nasal, and cutaneous preparations, as
well as preparations for inhalation. The last
category is composed of oral, oromucosal, and
rectal preparations.
Japanese Pharmacopeia
The products here have been divided into Category
I and Category II. Category I refers to products with
aqueous bases or vehicles. On the other hand,
Category II comprises all dosage forms from
Category I that are produced with non-aqueous
bases or vehicles. Moreover, it is important to note
that oil-in-water emulsions are under Category I,
while water-in-oil emulsions are classified under
Category II.
Category I is divided into four subcategories: IA, IB,
IC, and ID.
Category IA includes injections and sterile products
produced through dissolution or suspension in
aqueous vehicles, such as ophthalmic, otic, and
nasal preparations.
Category IB includes non-sterile topical products
that are produced through dissolution or
suspension in aqueous vehicles, such as
ophthalmic, otic, and nasal preparations, and
inhalations. Furthermore, products under category
IB may also be topically applied to mucous
membranes.
Category IC includes orally administered
preparations, other than antacids, that are
produced through dissolution or suspension in
aqueous vehicles. Lastly, Category ID includes
antacids with aqueous bases or vehicles.
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 UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023
Overall comparisons
While the USP and Ph. Eur. both categorize
products based off of the route of administration,
the JP first categorizes pharmaceuticals based on
the vehicle used, wherein those with aqueous
bases are placed in subcategories based on the
route of administration. Here, the USP and JP have
similarities.
The USP and JP both have four categories based
on the route of administration, from injections,
topicals, non-antacid oral products, and antacids.
The Ph. Eur., however, only has three categories,
and includes products that were not mentioned in
the other two pharmacopoeia, such as intrauterine,
intramammary, oromucosal, and rectal
preparations.
Challenge microorganisms
United States Pharmacopoeia
The organisms chosen by the USP are Escherichia
coli (ATCC 8739), Pseudomonas aeruginosa
(ATCC 9027), Staphylococcus aureus (ATCC
6538), Candida albicans (ATCC 10231), and
Aspergillus brasiliensis (ATCC 16404). Seed-lot
culture maintenance techniques are used.
European Pharmacopoeia
The test microorganisms used are P. aeruginosa,
S. aureus, C. albicans, and A. brasiliensis.
However, E. coli is mainly recommended for oral
preparations only. E. coli could still be used for
sterile multi-dose parenterals but it is not
mandatory. The use of Zygosaccharomyces rouxii
is in oral preparations that have a high
concentration of sugar.
Japanese Pharmacopeia
Seed-lot culture maintenance techniques are
utilized to ensure that the viable microorganisms for
inoculation are not more than five passages
removed from the initial master seed-lot. The test
microorganisms are E. coli, P. aeruginosa, S.
aureus, C. albicans, and A. brasiliensis.
Furthermore, it is preferable to use strains that,
depending on the product's characteristics, could
contaminate the product and grow on or in it. For
instance, it is preferable to use Z. rouxii (NCYC
381; IP 2021.92; NBC 1960) for products like
syrups that contain a lot of sugar. The test strains
can be obtained by growing them on either liquid or
solid agar.
Overall comparisons
All three pharmacopeias utilize a standard of test
microorganisms: E. coli (ATCC 8739), P.
aeruginosa (ATCC 9027), S. aureus (ATCC 6538),
C. albicans (ATCC 10231), and A. brasiliensis
(ATCC 16404). However, the Ph. Eur. states that
the use of E. coli as a test microorganism is mainly
for oral preparations and is optional for testing in
multidose parenterals. In addition, the JP and Ph.
Eur. have an additional guideline wherein Z. rouxii
may be used as a test microorganism in oral
preparations that have a high concentration of
sugar, such as syrups.
Test Strain Preparation
United States Pharmacopoeia
The bacteria are to be grown in Soybean-Casein
Digest broth or agar at a temperature of 32.5 ±
2.5°C for 18 to 24 hours. The microbial recovery
incubation time for these bacteria is from three to
five days. Both of the fungi are to be grown in
Sabouraud Dextrose agar or broth at 22.5 ± 2.5°C.
C. albicans should be incubated for 44 to 52 hours,
while A. brasiliensis should be incubated for 6 to 10
days. The microbial recovery incubation time for C.
albicans is three to five days, while it is three to
seven days for A. brasiliensis.
If the microorganisms are grown in agar, sterile
saline test solution is used to wash the surface
growth of bacterial and C. albicans cultures. These
are then collected in a suitable vessel.
As for the A. brasiliensis spores, a sterile saline test
solution mixed with 0.05% of polysorbate 80 is
used to harvest. The spore suspension is then
aseptically treated to remove any hyphae.
It must be certified that no residual growth medium
from the inoculum is mixed into the suspension.
This may be done through centrifugation and
resuspension in its corresponding sterile
suspending fluid.
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 UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023
If broth was used to grow the microorganisms, the
microbes may be harvested through centrifugation,
washing, and resuspension in the appropriate
sterile suspending fluid.
These microbial suspensions must contain a
microbial count of about 1x108 cfu/mL. The
bacterial and yeast suspensions must be used
within two hours if stored at room temperature, or
within 24 hours if stored between 2°C to 8°C. The
A. brasiliensis spore suspension may be
maintained for up to seven days at 2°C to 8°C.
European Pharmacopoeia
A Casein Soybean Digest agar for bacteria and
Sabouraud Dextrose agar for fungi is used. An
inoculum from a fresh stock of the challenge
microorganisms will be inoculated on the surface of
the agar. Analogous to JP and USP, the cultures
containing bacteria will be incubated at 30°C to
35°C for 18-24 hours. Those that contain
C.albicans will be incubated at 20°C to 25°C for 48
hours. On the other hand, a culture of A.brasiliensis
will be incubated at 20°C to 25°C for a week.
A sterile suspending fluid, containing 9 g/L of
sodium chloride R, is utilized in the harvest of C.
albicans. The microbial count should be around 108
microorganisms per mL. In order to achieve this
count, a suspending fluid is added.
The same concentration of sterile suspending fluid
is used in the culture of A. brasiliensis but with the
addition of 0.5g/L polysorbate 80. The spore count
should also be around 108 microorganisms per mL.
A suitable sample is then collected from the
suspension and plate count or membrane filtration
method is utilized to determine the number of
colony-forming units per mL.
Japanese Pharmacopeia
Inoculation of the five test microorganisms is
performed on the surface of agar plates and/or agar
slants. Soybean Casein Digest Agar is utilized for
the growth of E. coli, P. aeruginosa, and S. aureus.
Whereas Sabouraud Glucose Agar is utilized for
the growth of C. albicans and A. brasiliensis.
Bacterial cultures are incubated at a temperature of
30°C to 35°C for 18 to 24 hours, while C. albicans
cultures are incubated at a temperature of 25°C for
44°C to 52 hours, and A. brasiliensis cultures are
incubated at a temperature of 20°C to 25°C for six
to ten days, or once good sporulation has been
achieved.
For solid cultures, bacterial mediums and the
medium containing C. albicans are collected
aseptically. Adjust the viable cell count to around
10* CFU/mL and suspend the collected cells in
physiological saline. For A. brasiliensis,
physiological saline containing 0.05 w/v%
polysorbate 80 should be used to suspend the
cultivated cells of A. brasiliensis, and the spore
count should be set at around 10* CFU/mL. If
necessary, filter the spore suspension to get rid of
hyphae using sterile gauze or glass wool. All of the
cells that have been produced in this manner must
be centrifuged to eliminate any remaining medium
components. These suspensions are then utilized
as inocula.
For liquid cultures, with the exception of A.
brasiliensis, remove the medium from after each of
the four strains has been grown in fluid Sabouraud
Glucose Media or Soybean Casein Digest medium
in a centrifuge. After being washed in physiological
saline, the cells are re-suspended in the same
solution with the inoculum's viable cell count
adjusted to approximately 10° CFU/mL.When
cultivating strains other than the five test cultures,
choose a culture medium that will promote the
growth of the strain in question. An appropriate
method for that strain should also be used to
prepare the cell suspension. If it is not possible to
inoculate the microbial suspensions into the test
specimens within 2 hours of them being prepared
from the cultivations on agar media or in liquid
media, keep them at 2 - 8°C and use them within
24 hours. Usually, the spore of A. brasiliensis can
be kept at 2 to 8°C for up to 7 days. Calculate the
theoretical viable cell count per mL or per gram of
the product present just after inoculation, then
compare it to the actual viable cell count of the
inocula just before use.
Overall comparisons
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 UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023
All three pharmacopeias have similar incubation
conditions for bacterial cultures. However, each
pharmacopeia has different procedures and
guidelines in the incubation of fungi.
The USP states that C. albicans should be
incubated for 44 to 52 hours, and A. brasiliensis
should be incubated for 6 to 10 days at a
temperature of at 22.5 ± 2.5°C. Whereas the JP
instructs that cultures of C. albicans are to be
incubated at a temperature of 25°C for 44°C to 52
hours, and A. brasiliensis cultures are to be
incubated at a temperature of 20°C to 25°C for six
to ten days, or once good sporulation has been
achieved. Lastly, the Ph. Eur. states that cultures of
C.albicans are incubated at 20°C to 25°C for 48
hours, and cultures of A.brasiliensis are incubated
at 20°C to 25°C for a week.
Growth Promotion tests
United States Pharmacopoeia
The test requires media that can support microbial
growth. Hence, each batch of the ready-prepared
medium and each batch of medium prepared from
dehydrated medium or additional ingredients are
tested. For solid media, the counts must reach at
least 50% of the calculated value for a standardized
inoculum. On the other hand, freshly prepared
inoculum must have comparable growth to those of
previously tested and approved media.
European Pharmacopoeia
The Ph. Eur. did not specify a method to test
growth promotion of the media to be used.
Japanese Pharmacopoeia
The test uses Sabouraud Glucose Agar Medium for
fungi, and Soybean-Casein Digest Agar Medium for
bacteria. If other media offer comparable nutritional
components and growth-promoting qualities for the
microorganisms under test, those may also be
utilized.
The material to be used in actual product testing
should be utilized for the growth promotion test
using challenge strains or those deemed
comparable. The maximum incubation periods for
Sabouraud Glucose Agar Medium and
Soybean-Casein Digest Agar Medium, respectively,
are 5 days and 3 days, respectively. At least 50% of
the standardized cell counts should be acquired
from the colony counts on the agar media.
Overall comparisons
The USP and JP are essentially the same in terms
of their requirements for the growth promotion test,
except the USP does not provide room for
substitution like the JP does. They, however, both
require the agar colony counts to reach at least
50% of the standardized inoculum.
The Ph. Eur. did not provide any specifications.
Counting method suitability
United States Pharmacopoeia
Preparation of a 10-1 dilution is done by adding 1
mL of the specific product with 9 mL of saline or
other neutralizing agent. Continue the dilution
scheme to 10-2 and 10-3 dilution levels. An
appropriate number of challenge organisms is
added to each tube of diluted product and mixed. A
suitable volume from each dilution is then plated to
yield less than 250 cfu/plate for bacteria and yeast
(ideally between 25 to 250 cfu) or less than 80
cfu/plate for A. brasiliensis (ideally between 8 to 80
cfu) with the plating being performed minimally in
duplicate.
The positive control for the procedure is prepared
by introducing the same inocula into saline, and
transferring saline in similar volumes to the agar
plates. The suitable recovery scheme provides 50%
of the saline control count, which is averaged.
In case the diluted product exhibits antimicrobial
properties, specific neutralizers may need to be
incorporated into the diluents or the recovery
media. If there are no suitable neutralizing agents,
the level of inoculum may be increased to measure
a 3 log unit reduction.
The ability of the procedure to measure
preservative efficacy may be compromised if the
method requires a greater dilution (10-2 and 10-3),
as it will affect the measured recovery. It may be
difficult to measure a 3 log unit reduction for highly
diluted inoculum.
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 UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023
The reported recovery of the method cannot be
less than 1 cfu/plate on average. Membrane
filtration may be used to filter larger volumes of
dilutions to avoid a decrease in the reported
recovery, and/or to assist in neutralizing the
antimicrobial properties of the product.
European Pharmacopoeia
The Ph. Eur. did not specify a method to determine
the suitability of counting method in the presence of
product.
Japanese Pharmacopoeia
In preparation of the product, 1 g or 1 mL must be
diluted to nine times its mass of physiological
saline, or other appropriate neutral diluting
solutions. Prepare two more dilutions for the
solutions by serial ten-fold dilution, and then add
the suitable count of the test cells in each specific
test tube. Mix the tubes then inoculate so they
would yield less than 250 CFU/plate (bacteria and
C. albicans) or less than 80 CFU/plate (A.
brasiliensis).
The same inocula is introduced into saline. Similar
volumes of saline are transferred to the agar plates.
Suitable recovery scheme is one that provides at
least 50% of the average saline control count. The
method is valid to verify the suitability in the
specimen.
If growth in the cells were inhibited, an inactivator
may be added to a buffer solution for the use of
dilution. Confirmation that the inactivator has no
effect on the growth of the microorganism is
important.
If the preservative or product itself affects the
determination of the viable cell count, and there is
no suitable inactivator, the viable cell counts must
be calculated using the membrane filtration
method.
Validation study states that if the cell recovery
count is not less than 50% of the inoculated cell
counts the ones set on zero days may be used as
theoretical inoculate cell count.
Overall comparisons
Both the USP and JP introduce the inocula to
saline, which is transferred to the agar plates. The
suitable recovery scheme for both is set at at least
50% of the average saline control count.
Antimicrobial properties are to be neutralized for
USP and JP by the use of neutralizers or
inactivators. Both pharmacopeias also state that
membrane filtration is also to be used for larger
dilutions and for cases in which antimicrobial
properties need to be neutralized without an
inactivator.
The Ph. Eur. did not provide any specifications.
Product Testing Procedure
United States Pharmacopoeia
The test may be conducted in five of the original
containers if these containers can be entered
aseptically and have a sufficient volume of product.
Otherwise, the test must be conducted in five
sterile, capped bacteriological containers of
sufficient size wherein the appropriate volume has
been transferred.
Each container is then inoculated and mixed with
one of each standardized suspensions. The volume
of the inocula should be between 0.5% to 1% of the
volume of the product.
For products belonging to the first three categories,
the concentration of test microorganisms should
bring the final concentration of the test preparation
to between 10 x 105 to 10 x 106 cfu/mL of the
product. For Category 4, the final concentration
after inoculation should be between 10 x 103 to 10 x
104 cfu/mL of the product.
The initial concentration of viable microorganisms
in each test preparation is estimated based on the
concentration of microorganisms in the
standardized inocula, as determined by the
plate-count method.
The inoculated containers must then be incubated
at 22.5 ± 2.5°C. These are then sampled at
appropriate intervals, at 7, 14, and 28 days.
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 UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023
For each interval, changes in appearance, and the
number of cfu present in each test are determined
by plate-count procedure with a minimum of
duplicate plates. The cfu is averaged before
determination of deduced cfu/mL. If membrane
filtration is used, duplicate membrane filters should
be used for each estimate.
The changes in log10 of the concentration of cfu/mL
of each microorganism at each interval are then
calculated using the calculated cfu/mL
concentrations from the beginning of the test.
These changes are expressed in terms of log
reductions, which are the difference between the
log10 values of the starting concentration of cfu/mL
in the suspension and the log10 unit value of cfu/mL
of the survivors at that time point.
European Pharmacopoeia
The method used the agar medium from the initial
cultivation of the microorganisms. Inoculate a
series of containers of the product to be examined,
each with a suspension of one of the test
organisms to give an inoculum of 105 to 106
microorganisms per mL or per g of the preparation.
The volume of the suspension of inoculum should
not exceed 1% of the volume of the product. Mix to
ensure homogeneity, and maintain the inoculated
product at a temperature of 20°C to 25°C while
being protected from light.
A sample, typically 1 mL or 1 g, is removed from
the containers at zero hour and at appropriate
intervals for the type of product being tested.
Determine the number of viable microorganisms by
plate count or membrane filtration.
Residual antimicrobial activity is eliminated by
dilution, filtration, or using a specific inactivator.
When diluting, a due allowance is made to maintain
a reduced sensitivity in recovery for small numbers
in microorganisms. When a specific activator is
used, the system’s ability to support growth must
be confirmed with the use of controls.
The procedure is validated in terms of its ability to
demonstrate the needed reduction for the count of
viable microorganisms.
Japanese Pharmacopoeia
For products under Category I, the five containers
containing the product will be injected with each of
the cell suspensions.
If there are difficulties in injecting the cell
suspension into the container or the volume of the
product in each container is too small to be tested,
transfer a sufficient volume of the product into each
of the alternative sterile containers aseptically. After
that, the inoculum is mixed. Calculate the viable cell
count of the test preparations at 0, 7 (only for
Category IA), 14, and 28 days after the inoculation
containers have been incubated at 20°C to 25°C
with protection from light. When noticeable changes
in the test preparations have occurred (such as
color changes, the emergence of a foul odor, or the
formation of a fungus), note them. It is important to
note them to determine the preservative’s efficacy.
This is expressed as changes in terms of log
reduction against the count of inoculated cells. In
general, the Plate-count method (Pour-plate
methods; Surface-spread method) or the
Membrane Filtration method in "Microbiological
Examination of Non-sterile Products (4.05)" are
used to calculate the viable cell counts. For items in
Categories I and II, alternative microbiological
techniques, including automated approaches, may
be used as long as they produce results that are at
least as good as those obtained using
pharmacopeial techniques.
Processes for Category II products are the same as
those outlined for Category I products, but extra
considerations and procedures are needed for both
the uniform dispersion of the test microorganism in
the product and the calculation of viable cell counts
in the test preparations. For semisolid ointment
bases, heat the test preparation to 45°C to 50°C
until it becomes oily. The cell suspension is added
then evenly distributed in the inoculum with a sterile
glass rod or spatula. To promote uniform
dispersion, surfactants can also be used, but it is
important to make sure they do not interfere with
the test microorganisms' ability to survive or
weaken the product's ability to preserve itself. To
evenly distribute the test preparations in the buffer
solution or liquid medium in order to determine the
viable cell count, a surfactant or emulsifier may be
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 UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023

added. Lecithin, polysorbate 80, or sorbitan

days’ count at 28 days
monooleate may be added to enhance miscibility
between the buffer solution or liquid medium and Yeast and molds No increase from the initial
the semi-solid ointments or oils into which the test calculated count at 14 and
microorganisms were inoculated. These 28 days
substances work to neutralize or inactivate many of
the most popular preservatives. For Category 3 Products

Overall comparisons Bacteria NLT 1.0 log reduction from

USP and Ph. Eur. showed similarities in the the initial count at 14 days,
volumes to be utilized from the inocula. The and no increase from the 14
incubation period is the same for the three days’ count at 28 days
compendiums with a range from 20°C to 25°C and
should be protected from light. All of the three use Yeast and molds No increase from the initial
the plate-count method and membrane filtration. JP calculated count at 14 and
contains more detail to the procedures. 28 days

Interpretation of Results and Acceptance For Category 4 Products

Criteria
United States Pharmacopoeia Bacteria, yeast, No increase from the initial
The conditions for antimicrobial effectiveness are and molds calculated count at 14 and
met if the criteria in Table 1 are met. There are 28 days
different criteria for each category. The criteria for Note. “No increase” in counts is defined as NMT
bacteria and yeast and molds must all be passed 0.5 log10 unit more than the value to which it is
for a preservative to pass the antimicrobial compared.
effectiveness test.
European Pharmacopoeia
Table 1. The A category is the optimal efficacy, while B is
Criteria for Tested Microorganisms the criteria to be satisfied when A could not be
For Category 1 Products achieved. NI means there should be no increase in
the number of viable microorganisms while Nr
Bacteria NLT 1.0 log reduction from means there should be no recovery. The different
the initial calculated count at criteria for the three different categories may be
7 days, NLT 3.0 log viewed in Tables 2, 3, and 4.
reduction from the initial
count at 14 days, and no Table 2.
increase from the 14 days’ Parenteral preparations, eye preparations,
count at 28 days intrauterine preparations and intramammary
preparations
Yeast and molds No increase from the initial
Log reduction
calculated count at 7, 14,
and 28 days 6 hr 24 hr 7 d 14 d 28d
For Category 2 Products Bacteria A 2 3 - - NR
Bacteria NLT 2.0 log reduction from B - 1 3 - NI
the initial count at 14 days,
and no increase from the 14 Fungi A - - 2 - NI

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 UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023

B - - - 1 NI IA Bacteria At 7 days: Not less

than 1.0 log reduction
Table 3. from the initial count
Ear preparations, nasal preparations, preparations
for cutaneous application and preparations for At 14 days: Not less
inhalation than 3.0 log reduction
Log reduction from the initial count.

24 hr 7d 14 d 28d At 28 days: No
Bacteria A 2 3 - NI increase from the 14
day's count.
B - - 3 NI
Fungi At 7, 14 and 28 days:
Fungi A - - 2 NI No increase from the
initial count.
B - - 1 NI
IB Bacteria At 14 days: Not less
Table 4. than 2.0 log reduction
Oral preparations, oromucosal preparations and from the initial count.
rectal preparations
At 28 days: No
Log reduction increase from the 14
day's count.
14 d 28d
Fungi At 14 and 28 days: No
Bacteria 3 NI
increase from the
Fungi 1 NI initial count.

IC Bacteria At 14 days: Not less

Japanese Pharmacopoeia
The preservative efficacy is interpreted based on than 1.0 log reduction
the criteria set in Table 5. In case microorganisms from the initial count.
that are not being tested are found in the product, At 28 days: No
there is a strong chance of contamination, and/or increase from the 14
the contamination level in a nonsterile product day's count.
exceeds the microbial enumeration limit, these
require caution in handling the product and Fungi At 14 and 28 days: No
changes in the manufacturing process.
increase from the
Table 5. initial count.
Interpretation criteria by product category
ID Bacteria At 14 and 28 days: No
Category Microorganism Interpretation increase from the
Criteria
initial count.

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 UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023
Fungi At 14 and 28 days: No
increase from the
initial count.
II Bacteria At 14 and 28 days: No
increase from the
initial count.
Fungi At 14 and 28 days: No
increase from the
initial count.
Note. “No increase from the initial count” means not
more than 0.5 log10 increase from the initial
calculated count.
Overall comparisons
The intervals for testing the samples are the same
for USP and JP, which are 7, 14, and 28 days. Ph.
Eur. also makes use of these days, but has an
additional interval for 6 and 24 hours.
All three pharmacopeias use log reduction as the
measurement for the acceptance criteria.
The USP and JP share the same acceptance
criteria for the products under USP Categories 1 to
4 and JP Category IA to ID. However, JP has an
additional classification for nonaqueous products,
which has the same acceptance criteria for
Category IA.
Ph. Eur., on the other hand, has two different
criteria: one that expresses the recommended
efficacy, and one for justified cases when the first
one cannot be attained. The log reductions are also
different compared to the USP and JP.
CONCLUSION
Preservatives are essential in pharmaceutical
products packaged in multidose containers and
those containing a large amount of water
(Fahelelbom & El-Shabrawy, 2007; Moser & Meyer,
2011).
One type of preservative is antimicrobial
preservatives, of which their activity may be
assessed through antimicrobial effectiveness tests
(Sutton & Porter, 2002), contained in the three
major pharmacopeias, the USP, Ph. Eur., and JP.
There are similarities and differences in the test
products categories, challenge microorganisms,
procedures, and criteria.
In terms of the products and their categories, all the
compendia essentially divide the criteria based on
the route of administration. The JP, however, first
divides products based on whether or not they
make use of an aqueous vehicle. Then, the
aqueous products are subdivided into four based
on the routes of administration similar to those of
the USP. The Ph. Eur. only has three categories,
and includes products administered through routes
that were not mentioned in the other two.
As for the challenge microorganisms, all three use
the same ones: E. coli, P. aeruginosa, S. aureus, C.
albicans, and A. brasiliensis. In Ph. Eur., E. coli is
an optional test microorganism for multidose
parenterals, as it is mainly for oral preparations and
is optional for testing in multidose parenterals. Both
JP and Ph. Eur. have an additional guideline
wherein Z. rouxii may be used in testing oral
preparations with high sugar concentrations.
In preparing these test strains, all three
pharmacopeias have similar incubation conditions
for bacterial cultures. However, each pharmacopeia
has different procedures and guidelines in the
incubation of fungi.
The USP and JP have similar requirements for the
growth promotion test, where they make use of
Soybean Casein Digest medium and Sabouraud
Glucose Agar medium. The JP allows for
substitutions, unlike the USP. They, however, both
require the agar colony counts to reach at least
50% of the standardized inoculum.
In terms of the counting method suitability, both the
USP and JP mix the inoculum and saline, which is
to be transferred to the agar plates. The suitable
recovery scheme for both is set at at least 50% of
the average saline control count.
Antimicrobial properties are to be neutralized for
USP and JP by the use of neutralizers or
10
 UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023

inactivators. Both pharmacopeias also state that
membrane filtration is also to be used for larger
dilutions and for cases in which antimicrobial
properties need to be neutralized without an
inactivator.
The Ph. Eur. did not specify the use or methods for
a growth promotion test and counting method
suitability.
For the product testing procedures, USP and Ph.
Eur. use the same volumes for the inocula. The
incubation period is the same for the three
compendiums, ranging from 20°C to 25°C, making
sure that these are protected from light. All three
use the plate-count method and membrane
filtration. JP contains more detail to its procedures.
formulations, and thus, the safety and stability of
the resulting product.
REFERENCES:
European Pharmacopoeia. 8 (2014), 5.1.3.
Effectiveness of Antimicrobial Preservation.
Fahelelbom, K. M., & El-Shabrawy, Y. (2007).
Analysis of preservatives in pharmaceutical
products. Pharm Rev, 5(1).
Japanese Pharmacopoeia. JP <19> Preservative
effectiveness tests.
Moser, C. L., & Meyer, B. K. (2011). Comparison of
compendial antimicrobial effectiveness tests: a
review. Aaps Pharmscitech, 12(1), 222-226.

As for the interpretation of results and acceptance Shaikh, S. M., Doijad, R. C., Shete, A. S., &
criteria, the intervals for testing the samples are the Sankpal, P. S. (2016). A Review on: Preservatives
same for USP and JP, which are 7, 14, and 28 used in Pharmaceuticals and impacts on Health.
days. The Ph. Eur., in addition to these intervals, PharmaTutor, 4(5), 25-34.
also uses 6 and 24 hours.
Sutton, S. V., & Porter, D. (2002). Development of
The effectiveness of the preservatives, and thus the the antimicrobial effectiveness test as USP
acceptance criteria, are expressed through the chapter< 51>. PDA Journal of Pharmaceutical
changes of log reduction of the initial and final Science and Technology, 56(6), 300-311
concentration for all the three.
U.S. Pharmacopeia. 38 (2015), <51> Antimicrobial
The USP and JP share the same log reduction Effectiveness Testing.
values for the products under USP Categories 1 to
4 and JP Category IA to ID, which also contain the
same products for each category. In JP, Category II
(nonaqueous products) shares the same criteria as
Category IA.

Ph. Eur., on the other hand, has two different

criteria: one that expresses the recommended
efficacy, and one for justified cases when the first
one cannot be attained. The accepted log reduction
values are also different compared to the USP and
JP.

Despite the differences in the antimicrobial

effectiveness tests of the three different
pharmacopeias, these are harmonized in certain
aspects. These ultimately provide guidelines that
uphold the effectiveness of antimicrobial
preservatives to be used in various pharmaceutical
11