Pharcal 10 12
Pharcal 10 12
Pharcal 10 12
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.
If broth was used to grow the microorganisms, the Bacterial cultures are incubated at a temperature of
microbes may be harvested through centrifugation, 30°C to 35°C for 18 to 24 hours, while C. albicans
washing, and resuspension in the appropriate cultures are incubated at a temperature of 25°C for
sterile suspending fluid. 44°C to 52 hours, and A. brasiliensis cultures are
incubated at a temperature of 20°C to 25°C for six
These microbial suspensions must contain a to ten days, or once good sporulation has been
microbial count of about 1x108 cfu/mL. The achieved.
bacterial and yeast suspensions must be used
within two hours if stored at room temperature, or For solid cultures, bacterial mediums and the
within 24 hours if stored between 2°C to 8°C. The medium containing C. albicans are collected
A. brasiliensis spore suspension may be aseptically. Adjust the viable cell count to around
maintained for up to seven days at 2°C to 8°C. 10* CFU/mL and suspend the collected cells in
physiological saline. For A. brasiliensis,
European Pharmacopoeia physiological saline containing 0.05 w/v%
A Casein Soybean Digest agar for bacteria and polysorbate 80 should be used to suspend the
Sabouraud Dextrose agar for fungi is used. An cultivated cells of A. brasiliensis, and the spore
inoculum from a fresh stock of the challenge count should be set at around 10* CFU/mL. If
microorganisms will be inoculated on the surface of necessary, filter the spore suspension to get rid of
the agar. Analogous to JP and USP, the cultures hyphae using sterile gauze or glass wool. All of the
containing bacteria will be incubated at 30°C to cells that have been produced in this manner must
35°C for 18-24 hours. Those that contain be centrifuged to eliminate any remaining medium
C.albicans will be incubated at 20°C to 25°C for 48 components. These suspensions are then utilized
hours. On the other hand, a culture of A.brasiliensis as inocula.
will be incubated at 20°C to 25°C for a week.
For liquid cultures, with the exception of A.
A sterile suspending fluid, containing 9 g/L of brasiliensis, remove the medium from after each of
sodium chloride R, is utilized in the harvest of C. the four strains has been grown in fluid Sabouraud
albicans. The microbial count should be around 108 Glucose Media or Soybean Casein Digest medium
microorganisms per mL. In order to achieve this in a centrifuge. After being washed in physiological
count, a suspending fluid is added. saline, the cells are re-suspended in the same
solution with the inoculum's viable cell count
The same concentration of sterile suspending fluid adjusted to approximately 10° CFU/mL.When
is used in the culture of A. brasiliensis but with the cultivating strains other than the five test cultures,
addition of 0.5g/L polysorbate 80. The spore count choose a culture medium that will promote the
should also be around 108 microorganisms per mL. growth of the strain in question. An appropriate
method for that strain should also be used to
A suitable sample is then collected from the prepare the cell suspension. If it is not possible to
suspension and plate count or membrane filtration inoculate the microbial suspensions into the test
method is utilized to determine the number of specimens within 2 hours of them being prepared
colony-forming units per mL. from the cultivations on agar media or in liquid
media, keep them at 2 - 8°C and use them within
Japanese Pharmacopeia 24 hours. Usually, the spore of A. brasiliensis can
Inoculation of the five test microorganisms is be kept at 2 to 8°C for up to 7 days. Calculate the
performed on the surface of agar plates and/or agar theoretical viable cell count per mL or per gram of
slants. Soybean Casein Digest Agar is utilized for the product present just after inoculation, then
the growth of E. coli, P. aeruginosa, and S. aureus. compare it to the actual viable cell count of the
Whereas Sabouraud Glucose Agar is utilized for inocula just before use.
the growth of C. albicans and A. brasiliensis.
Overall comparisons
4
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 are 5 days and 3 days, respectively. At least 50% of
conditions for bacterial cultures. However, each the standardized cell counts should be acquired
pharmacopeia has different procedures and from the colony counts on the agar media.
guidelines in the incubation of fungi.
Overall comparisons
The USP states that C. albicans should be The USP and JP are essentially the same in terms
incubated for 44 to 52 hours, and A. brasiliensis of their requirements for the growth promotion test,
should be incubated for 6 to 10 days at a except the USP does not provide room for
temperature of at 22.5 ± 2.5°C. Whereas the JP substitution like the JP does. They, however, both
instructs that cultures of C. albicans are to be require the agar colony counts to reach at least
incubated at a temperature of 25°C for 44°C to 52 50% of the standardized inoculum.
hours, and A. brasiliensis cultures are to be
incubated at a temperature of 20°C to 25°C for six The Ph. Eur. did not provide any specifications.
to ten days, or once good sporulation has been
achieved. Lastly, the Ph. Eur. states that cultures of Counting method suitability
C.albicans are incubated at 20°C to 25°C for 48 United States Pharmacopoeia
hours, and cultures of A.brasiliensis are incubated Preparation of a 10-1 dilution is done by adding 1
at 20°C to 25°C for a week. mL of the specific product with 9 mL of saline or
other neutralizing agent. Continue the dilution
Growth Promotion tests scheme to 10-2 and 10-3 dilution levels. An
United States Pharmacopoeia appropriate number of challenge organisms is
The test requires media that can support microbial added to each tube of diluted product and mixed. A
growth. Hence, each batch of the ready-prepared suitable volume from each dilution is then plated to
medium and each batch of medium prepared from yield less than 250 cfu/plate for bacteria and yeast
dehydrated medium or additional ingredients are (ideally between 25 to 250 cfu) or less than 80
tested. For solid media, the counts must reach at cfu/plate for A. brasiliensis (ideally between 8 to 80
least 50% of the calculated value for a standardized cfu) with the plating being performed minimally in
inoculum. On the other hand, freshly prepared duplicate.
inoculum must have comparable growth to those of
previously tested and approved media. The positive control for the procedure is prepared
by introducing the same inocula into saline, and
European Pharmacopoeia transferring saline in similar volumes to the agar
The Ph. Eur. did not specify a method to test plates. The suitable recovery scheme provides 50%
growth promotion of the media to be used. of the saline control count, which is averaged.
The reported recovery of the method cannot be Both the USP and JP introduce the inocula to
less than 1 cfu/plate on average. Membrane saline, which is transferred to the agar plates. The
filtration may be used to filter larger volumes of suitable recovery scheme for both is set at at least
dilutions to avoid a decrease in the reported 50% of the average saline control count.
recovery, and/or to assist in neutralizing the
antimicrobial properties of the product. Antimicrobial properties are to be neutralized for
USP and JP by the use of neutralizers or
European Pharmacopoeia inactivators. Both pharmacopeias also state that
The Ph. Eur. did not specify a method to determine membrane filtration is also to be used for larger
the suitability of counting method in the presence of dilutions and for cases in which antimicrobial
product. properties need to be neutralized without an
inactivator.
Japanese Pharmacopoeia
In preparation of the product, 1 g or 1 mL must be The Ph. Eur. did not provide any specifications.
diluted to nine times its mass of physiological
saline, or other appropriate neutral diluting Product Testing Procedure
solutions. Prepare two more dilutions for the United States Pharmacopoeia
solutions by serial ten-fold dilution, and then add The test may be conducted in five of the original
the suitable count of the test cells in each specific containers if these containers can be entered
test tube. Mix the tubes then inoculate so they aseptically and have a sufficient volume of product.
would yield less than 250 CFU/plate (bacteria and Otherwise, the test must be conducted in five
C. albicans) or less than 80 CFU/plate (A. sterile, capped bacteriological containers of
brasiliensis). sufficient size wherein the appropriate volume has
been transferred.
The same inocula is introduced into saline. Similar
volumes of saline are transferred to the agar plates. Each container is then inoculated and mixed with
Suitable recovery scheme is one that provides at one of each standardized suspensions. The volume
least 50% of the average saline control count. The of the inocula should be between 0.5% to 1% of the
method is valid to verify the suitability in the volume of the product.
specimen.
For products belonging to the first three categories,
If growth in the cells were inhibited, an inactivator the concentration of test microorganisms should
may be added to a buffer solution for the use of bring the final concentration of the test preparation
dilution. Confirmation that the inactivator has no to between 10 x 105 to 10 x 106 cfu/mL of the
effect on the growth of the microorganism is product. For Category 4, the final concentration
important. after inoculation should be between 10 x 103 to 10 x
104 cfu/mL of the product.
If the preservative or product itself affects the
determination of the viable cell count, and there is The initial concentration of viable microorganisms
no suitable inactivator, the viable cell counts must in each test preparation is estimated based on the
be calculated using the membrane filtration concentration of microorganisms in the
method. standardized inocula, as determined by the
plate-count method.
Validation study states that if the cell recovery
count is not less than 50% of the inoculated cell The inoculated containers must then be incubated
counts the ones set on zero days may be used as at 22.5 ± 2.5°C. These are then sampled at
theoretical inoculate cell count. appropriate intervals, at 7, 14, and 28 days.
Overall comparisons
6
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023
8
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023
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.
9
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY – DEPT. OF PHARMACY
PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY (PHA 6114)
S.Y. 2022-2023
Ph. Eur., on the other hand, has two different The USP and JP have similar requirements for the
criteria: one that expresses the recommended growth promotion test, where they make use of
efficacy, and one for justified cases when the first Soybean Casein Digest medium and Sabouraud
one cannot be attained. The log reductions are also Glucose Agar medium. The JP allows for
different compared to the USP and JP. substitutions, unlike the USP. They, however, both
require the agar colony counts to reach at least
CONCLUSION 50% of the standardized inoculum.
Preservatives are essential in pharmaceutical
products packaged in multidose containers and In terms of the counting method suitability, both the
those containing a large amount of water USP and JP mix the inoculum and saline, which is
(Fahelelbom & El-Shabrawy, 2007; Moser & Meyer, to be transferred to the agar plates. The suitable
2011). recovery scheme for both is set at at least 50% of
the average saline control count.
One type of preservative is antimicrobial
preservatives, of which their activity may be Antimicrobial properties are to be neutralized for
assessed through antimicrobial effectiveness tests 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 formulations, and thus, the safety and stability of
membrane filtration is also to be used for larger the resulting product.
dilutions and for cases in which antimicrobial
properties need to be neutralized without an REFERENCES:
inactivator. European Pharmacopoeia. 8 (2014), 5.1.3.
Effectiveness of Antimicrobial Preservation.
The Ph. Eur. did not specify the use or methods for
a growth promotion test and counting method Fahelelbom, K. M., & El-Shabrawy, Y. (2007).
suitability. Analysis of preservatives in pharmaceutical
products. Pharm Rev, 5(1).
For the product testing procedures, USP and Ph.
Eur. use the same volumes for the inocula. The Japanese Pharmacopoeia. JP <19> Preservative
incubation period is the same for the three effectiveness tests.
compendiums, ranging from 20°C to 25°C, making
sure that these are protected from light. All three Moser, C. L., & Meyer, B. K. (2011). Comparison of
use the plate-count method and membrane compendial antimicrobial effectiveness tests: a
filtration. JP contains more detail to its procedures. 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.