A Jahurul Islam Company

VALIDATION PROTOCOL

TITLE: VALIDATION OF ANTIMICROBIAL EFFECTIVENESS TEST

Document No. AMVP/AET/01 Issue Date August 2, 2015 Revision No. 00
Copy No.
Supersedes New Document Effective Date
Page 1 of 7
Department Quality Control Review Date August 2, 2018

1.0 INTRODUCTION
Antimicrobial preservatives are substances added to aqueous pharmaceutical products. Nonsterile
dosage forms may have preservatives added to protect them from growth of microorganisms
introduced inadvertently during or subsequent to the manufacturing process. In the case of sterile
articles packaged in multi-dose containers, antimicrobial preservatives are added to inhibit the
growth of microorganisms that may be introduced during repeatedly withdrawing individual doses.
One or more antimicrobial preservatives ared expected in all sterile multidose units.
Antimicrobial preservatives should not be used as a substitute for good manufacturing practices,
solely to reduce the viable microbial population of a nonsterile product or control the
presterilization bioburden of a multidose formulation during manufacturing.
All useful antimicrobial agents are toxic substances. For maximum protection of patients, the
concentration of preservative effective in the final packaged product should be below a level that
may be toxic to human beings.
An antimicrobial preservative must be demonstrated for all injections packaged in multiple-dose
containers and for multiple-dose topical and oral dosage forms and for other dosage forms such as
ophthalmic, otic, nasal, irrigation, and dialysis fluids.

2.0 AIM
The aim of this validation protocol is:
 Growth promotion of the media used in this procedure.
 Suitability of the counting method in the presence of product.
 If the diluted product exhibits antimicrobial properties, using of specific neutralizer into
the diluents or the recovery media.

3.0 PRINCIPLE
To establish that this procedure is able to detect challenge microorganisms in the presence of a
product.

4.0 REFERENCE
4.1 USP 38, Chapter (51).
Prepared by: Checked by: Approved by:
Name Utpal Banik Md. Mostafizur Rahman Mohammad Saiful Islam
Designatio Sr. Executive, QC DM, QC AGM, QA
n
Sign. &
Date
Navana Health Care Ltd., Rupshi, Narayanganj, Bangladesh
A Jahurul Islam Company
VALIDATION PROTOCOL

TITLE: VALIDATION OF ANTIMICROBIAL EFFECTIVENESS TEST

Document No. AMVP/AET/01 Issue Date August 2, 2015 Revision No. 00
Copy No.
Supersedes New Document Effective Date
Page 2 of 7
Department Quality Control Review Date August 2, 2018

4.2 USP 38, Chapter (1227)

5.0 EQUIPMENT & APPARATUS

Laminar Air Flow, Sterile Petri dish, Micropipettes (20-200 µl and 100-1000µl), Incubator 30°C-35°C
and 20°C-25°C, Autoclave, Hot air oven, Media bottles, Test tubes Inoculating loops / spreader,
Burner, Balance, pH meter, UV reader.
8
6.0 PREPARATION OF INOCULUM (1.5X10 cfu/ml)
Inoculate the surface of a suitable volume of solid agar medium from a recently revived stock
culture of each of the specified microorganisms. The cultural conditions for the inoculums culture
are described in Table 3 in which the suitable media are Soyabean-Casein Digest or Sabouraud
Dextrose Agar medium. Harvest the bacterial and Candida albicans culture, use sterile saline, wash
the surface growth , collect into a suitable vessel, and add sufficient sterile saline to obtain a
microbial count of 1X108 colony-forming units(cfu)per ml. Harvest the cells of Aspergillus niger use
sterile saline containing 0.05% polysorbate 80, and add sufficient sterile saline to obtain a microbial
count of about 1X108 colony-forming units (cfu) per ml.
Standardize harvested inoculum with 0.5 Mc Farland standard for 1.5 x108 cfu per ml. with the help
of UV, where wavelength should be 600 nm. Match the absorbance of cell concentration with 0.5
Mc Farland standard. This value serves to calibrate the size of inoculum used in the test. Use the
bacterial and yeast suspension within 2hours of harvest or within 24 hours if stored between 2° and
8° C, but the fungal preparation and spore suspension may be stored between 2° and 8° C for up to 7
days. Confirm the challenge microorganisms by plate count method.

7.0 VALIDATION PROCEDURE

7.1 Growth promotion of media
7.1.1 Preparation of inoculum suspension(10-100cfu)
7.1.1.1Remove the desired lyophilized culture container from the refrigerator and take
the culture vial as well as desired quantity of hydration fluid vial.
7.1.1.2 Return the rest lyophilized culture container into the refrigerator.
7.1.1.3 Allow the taken lyophilized culture to equilibrate at room temperature.

Prepared by: Checked by: Approved by:

Name Utpal Banik Md. Mostafizur Rahman Mohammad Saiful Islam
Designatio Sr. Executive, QC DM, QC AGM, QA
n
Sign. &
Date
Navana Health Care Ltd., Rupshi, Narayanganj, Bangladesh
A Jahurul Islam Company
VALIDATION PROTOCOL

TITLE: VALIDATION OF ANTIMICROBIAL EFFECTIVENESS TEST

Document No. AMVP/AET/01 Issue Date August 2, 2015 Revision No. 00
Copy No.
Supersedes New Document Effective Date
Page 3 of 7
Department Quality Control Review Date August 2, 2018

7.1.1.4 Warm the hydration fluid at 34 o C- 38oC for few minutes.

7.1.1.5 Remove two pellets of culture and place into the 2ml hydration fluid vial with a
sterile forceps.
7.1.1.6Immediately recap the vial with the hydrated material and place into 34 o C- 38oC
incubator for 30 minutes to assure complete hydration.
7.1.1.7Replace the rubber stoppers, recap the vial and return the remaining lyophilized
culture in its original container into the refrigerator.
7.1.1.8Immediately following incubation, vortex the hydrated material to achieve equal
distribution of the strain throughout the hydrated suspension.
7.1.1.9 Prepare 9 ml phosphate buffer pH 7.2 according to manufacturer instruction and
autoclave at 121 o C for 15 minutes.
7.1.1.10 Remove 1 ml of the well mixed hydrated culture suspension and transfer to 9 ml
phosphate buffer solution.
7.1.1.11 This is the working culture suspension and this suspension contains 10-100
cfu/100µl.
7.1.1.12 Use this suspension within 30 minutes to ensure microorganisms viability.
7.1.2 Growth Promotion test of Agar or Solid Media by Pour plate method
7.1.2.1Label two sterile petridish by writing with culture name, Media B. No, Date of
incubation on the base.
7.1.2.2 Add each 100µl suspension of specific culture containing 10 to 100 cfu/ml into two
petridishes.
7.1.2.3 Aseptically pour the cooled media at 40 to 45 0C into the both labelled plates, mix
the plates by gently rotating clock wise and anti-clock wise direction.
7.1.2.4 Allow the plates to solidify at room temperature under Laminar Air Flow.
7.1.2.5 Simultaneously run negative control to verify testing conditions, using the same
procedure in place of the test organism use diluents i.e. 1.0 ml of sterile
phosphate buffer solution.
7.1.2.6 Incubate all the plates according the table:
Table-3
Organism Suitable Medium Incubation Inoculum Microbial recovery
Temperatur Incubation incubation time
Prepared by: Checked by: Approved by:
Name Utpal Banik Md. Mostafizur Rahman Mohammad Saiful Islam
Designatio Sr. Executive, QC DM, QC AGM, QA
n
Sign. &
Date
Navana Health Care Ltd., Rupshi, Narayanganj, Bangladesh
A Jahurul Islam Company
VALIDATION PROTOCOL

TITLE: VALIDATION OF ANTIMICROBIAL EFFECTIVENESS TEST

Document No. AMVP/AET/01 Issue Date August 2, 2015 Revision No. 00
Copy No.
Supersedes New Document Effective Date
Page 4 of 7
Department Quality Control Review Date August 2, 2018

e time
Escherichia coli Soyabean-Casein 32.5±2.5°C 18 to 24 3 to 5 days
(ATCC No. 8739) Digest agar hours
Pseudomonas Soyabean-Casein 32.5±2.5°C 18 to 24 3 to 5 days
aeruginosa Digest agar hours
(ATCC No. 9027)
Staphylococus Soyabean-Casein 32.5±2.5°C 18 to 24 3 to 5 days
aureus Digest agar hours
(ATCC No. 6538)
Candida albicans Sabouraud 22.5±2.5°C 44 to 52 3 to 5 days
(ATCC No. 10231) Dextrose Agar hours

Aspergillus niger Sabouraud 22.5±2.5°C 6 to 10 days 3 to 7 days

(ATCC No. 16404) Dextrose Agar.

7.1.2.7 Counts obtained must be at least 50 % of the calculated value for standardized
inoculums.

7.2 Suitability of counting method in the presence of product:

7.2.1 Preparation of inoculums ( less than 250 cfu and 80 cfu per ml)
8
7.2.1.1 Serially dilute the inoculum (1.5X10 cfu/ml) prepared in 6.0 and find a dilution
from which a inoculum give less than 250 cfu per one ml of bacteria and yeast or
less than 80 cfu per one ml of Aspergillus niger when mixed with diluted product
and finally plated for counting.
8
7.2.1.2 For example, add 0.1 ml of inoculum from (1.5X10 cfu/ml) into 9.9 ml saline so
6
this dilution will contain 1.5x10 cfu/ml. From this dilution take 0.1 ml and
transfer it into a tube containing 9.9 ml saline so this dilution will
4
contain1.5x10 cfu/ml in this way dilute the inoculums to find a dilution form
which 1 ml inoculum when mixed with 10 ml diluted product will give less than
250 cfu per one ml of bacteria and yeast or less than 80 cfu per one ml of
Aspergillus niger.

Prepared by: Checked by: Approved by:

Name Utpal Banik Md. Mostafizur Rahman Mohammad Saiful Islam
Designatio Sr. Executive, QC DM, QC AGM, QA
n
Sign. &
Date
Navana Health Care Ltd., Rupshi, Narayanganj, Bangladesh
A Jahurul Islam Company
VALIDATION PROTOCOL

TITLE: VALIDATION OF ANTIMICROBIAL EFFECTIVENESS TEST

Document No. AMVP/AET/01 Issue Date August 2, 2015 Revision No. 00
Copy No.
Supersedes New Document Effective Date
Page 5 of 7
Department Quality Control Review Date August 2, 2018

7.2.2 Preparation of Product

-1
7.2.2.1 Prepare a 10 dilution by adding 1 ml of product to 9 ml saline and mark the tube
-1
with 10 .
-2 -1
7.2.2.2 Prepare 10 dilution by adding 1 ml of 10 dilution to 9 ml saline and mark the
-2
tube with 10 .
-3 -2
7.2.2.3 Prepare 10 dilution by adding 1 ml of 10 dilution to 9 ml saline and mark the
-3
tube with 10 .
7.2.3 Working procedure
-1
7.2.3.1 Add 1 ml of each challenged organism to each tube of diluted product (10 ,
-2 -3
10 , 10 ) mix and then plate 1ml from each dilution to yield less than 250cfu
bacteria and yeast per plate or less than 80 cfu per one ml of Aspergillus niger.
7.2.3.2 Perform this plating in duplicate for all test organisms.
7.2.3.3 Introduce the same inocula into same amount of saline and transfer similar
volumes of saline to agar plates for a positive control.
7.2.4 Acceptance criteria: a suitable recovery scheme is the one that provides at least 50% of
the saline control count. If the diluted product exhibit antimicrobial properties incorporate
specific neutralizer into the diluents.

7.3 Validation of neutralization method

7.3.1 Add suitable amount of neutralizer according to the following table into diluents and
record the procedure into the annexure (F/NHCL/MB/21).
Neutralizer Biocide class Potential action of
biocides
Bisulfate Gluteraldehyde, Mercurials Non sporing bacteria
Dilution Phenolics, alcohol, aldehydes, sorbate -
Glycine Aldehydes Growing cell
Lecithin Quarternary Ammonium Compounds (QACs) Bacteria
Parahydroxybenzoates (parabens),bis-biguanides

Prepared by: Checked by: Approved by:

Name Utpal Banik Md. Mostafizur Rahman Mohammad Saiful Islam
Designatio Sr. Executive, QC DM, QC AGM, QA
n
Sign. &
Date
Navana Health Care Ltd., Rupshi, Narayanganj, Bangladesh
A Jahurul Islam Company
VALIDATION PROTOCOL

TITLE: VALIDATION OF ANTIMICROBIAL EFFECTIVENESS TEST

Document No. AMVP/AET/01 Issue Date August 2, 2015 Revision No. 00
Copy No.
Supersedes New Document Effective Date
Page 6 of 7
Department Quality Control Review Date August 2, 2018

Thioglycollate Mercurals Staphylococci and

Spores
Thiosuphate Mercurals,halogens,aldehydes Staphylococci
2+ 2+
Mg or Ca ions EDTA -

7.3.2 A validated method of neutralizing the antimicrobial properties of a product must meet
two criteria: Neutralizer efficacy and Neutralizer toxicity.
7.3.3 Neutralization procedure must meet these two criteria by comparing recovery results for
treatment groups. The first is the test group in which the product is subject to
neutralization method then a low level of challenge microorganism is inoculated for
recovery. The Second is the peptone control group in which the neutralization method is
used with peptone as the test solution. The Third is the viability group in which the actual
inoculum is used without exposure to the neutralization scheme.
7.3.4 Acceptance criteria: similar recovery between the test group and the peptone group
demonstrate adequate neutralizer efficacy and similar recovery between the peptone
group and viability group demonstrate adequate neutralizer toxicity.
If no suitable neutralizing agent or method is found and method suitability requires
significant dilution, use higher level of inoculums 10 7-108cfu/ ml so that a 3 log reduction
can be measured.

8.0 RELATED DOCUMENTS

8.1 SOP of Microbiological Media Preparation- SOP/NHCL/MB/002.
8.2 Operation of oven (fn-120) SOP/CEPHA/MB/004.
8.3 Operation of laminar air flow unit (hs 1601 k, class 100) SOP/CEPHA/MB/006.

9.0 CHANGE CONTROL

Head of the Department is only authorized to make any changes in this SOP through the change
control SOP.

10.0 LIST OF DISTRIBUTION

Prepared by: Checked by: Approved by:

Name Utpal Banik Md. Mostafizur Rahman Mohammad Saiful Islam
Designatio Sr. Executive, QC DM, QC AGM, QA
n
Sign. &
Date
Navana Health Care Ltd., Rupshi, Narayanganj, Bangladesh
A Jahurul Islam Company
VALIDATION PROTOCOL

TITLE: VALIDATION OF ANTIMICROBIAL EFFECTIVENESS TEST

Document No. AMVP/AET/01 Issue Date August 2, 2015 Revision No. 00
Copy No.
Supersedes New Document Effective Date
Page 7 of 7
Department Quality Control Review Date August 2, 2018

Sl. Division/Department/Section Copy Sl. No. Division/Department/Section Copy

No. No. No.
01 QUALITY ASSURANCE N/A
(Master Copy)
02 QUALITY CONTROL 01

11.0 CHANGE HISTORY

Sl. No. Name of initiator Revision No. Effective date Reason for change
01 Utpal Banik 00 New

12.0 ANNEXURE
12..1Annexure I: Validation Report of Antimicrobial Effective Test (F/NHCL/MB/21).

Prepared by: Checked by: Approved by:

Name Utpal Banik Md. Mostafizur Rahman Mohammad Saiful Islam
Designatio Sr. Executive, QC DM, QC AGM, QA
n
Sign. &
Date
Navana Health Care Ltd., Rupshi, Narayanganj, Bangladesh