Protocol For Disinfectant Validation Disinfectant Validation Protocol

B.JAIN PHARMACEUTICALS PVT. LTD.
PROTOCOL FOR DISINFECTANT VALIDATION

Department Protocol No Revision No Page No.
QCM BJPL/DVP/GRL/001 01 1 of 13
TITLE DISINFECTANT VALIDATION PROTOCOL
INDEX
Sr. No. DESCRIPTION PAGE NO
1. Protocol approval 02
2. History Sheet 03
3. Objective 04
4. Scope 04
5. Site of study 04
6. Responsibilities 04
7. Standard Operating Procedure to be followed 04
8. Pre-Requisites 04-05
9. Validation Methodology And Plan 05-12
10. Acceptance criteria 12
11. Frequency 12
12. Result 12
13. Validation Report 12
14 Approval of validation report 12
15. Reference (If any) 12
16. Abbreviation 12-13

1.0 PROTOCOL APPROVAL
Name Designation Signature Date

Prepared By
Checked By
Name Designation Signature Date

Approved By
2.0 History Sheet
Nature of change/
Revision .No. Effective Date Reason for Sign-QA
Revision
01 28/11/2018 New Protocol

3.0 OBJECTIVE
The objective of this validation protocol is to evaluate the efficacy of a
disinfectant solution which are being used for the surface and area sanitization
of the controlled and critical clean rooms by using following test
 Use Dilution Test: Screening of the disinfectants for their efficacy at
various concentration and contact time against a wide range of standard
test organisms and environmental isolates.
 Surface Challenge Test: Using standard test microorganisms and
microorganisms that are typical environmental isolates, applying
disinfectants to surfaces at the selected use concentration with a specified
contact time, and determining the log reduction of the challenge
microorganisms.
4.0 SCOPE
 This protocol will define the procedure to be followed, for validating the
sanitizers and the sanitization procedure being followed in the
manufacturing and the testing facility B Jain Pharmaceuticals Pvt. Ltd.
 The same established data should be used in further routine usage.
5.0 SITE OF STUDY
Quality Control Department (Microbiology Section) of B Jain Pharmaceuticals
Pvt. Ltd.
6.0 RESPONSIBILITIES
6.1 Quality Control:
6.1.1 To prepare and check the protocol.
6.1.2 To provide all applicable documents for the generation of the protocol.
6.1.3 To provide personnel to assist in the execution of this protocol.
6.2 Quality Assurance:
6.2.1 To check and approve the protocol.
6.2.2 To approve deviation and to complete the final report.
6.2.3 Ensure the protocol completeness and technical accuracy.
7.0 STANDARD OPERATING PROCEDURE (SOP) TO BE FOLLOWED
 Procedure for maintenance and suspension preparation of microbial
cultures.
 Procedure for preparation of disinfectant and cleaning.
 Procedure for growth promotion test.
 Procedure for operation and calibration of BOD Incubators.
 Procedure for disposal of used or contaminated culture media
8.0 PRE-REQUISITES
8.1 Disinfectants: Disinfectant details such as name of the disinfectant, its
manufacturer, recommended concentration, action and its composition
will be mentioned in the validation report.
9.0 VALIDATION METHODOLOGY AND PLAN
9.1 Preparation of Challenge Inoculum
9.1.1 Challenge Inoculum should have a population of 105 - 106 CFU / ml.
9.1.2 Prepare the Soyabean casein digest agar and Sabouraud
Chloramphenicol agar media slant as per the SOP-
BJPL/SOP/QCM/004/D
9.1.3 Perform the growth promotion and pre-incubation test of the prepared
media slants.
9.1.4 Take out the working culture slant from the refrigerator 30 minutes
prior to the testing so as to acclimatize with the working environment
and place it under laminar airflow unit.
9.1.5 From the working culture slant streak a loopful of culture on the freshly
prepared slants.
9.1.6 Incubate the above inoculated slants at 32.5 ± 2.5° C for 24 - 48 hrs for
bacterial cultures and 22.5 ± 2.5° C for 3 - 7 days for fungal / yeast
cultures.
9.1.7 After completion of incubation add 5.0 ml of 0.9 % sterile normal saline
into the above slants aseptically and harvest the slant with the help of
sterile nichrome loop.
9.1.8 Transfer the whole content of the harvested slant in to a fresh sterile test
tube containing 45.0 ml of 0.9 % of sterile normal saline and vortex it for
2 - 3 minutes (Challenge Inoculum).
9.1.9 Perform the exercise from step 9.1.4 to 9.1.8 with all the culture
organisms, which are being used for validation.
9.1.10 Store the challenge inoculum of all the culture organisms in the
refrigerator at 2 - 8°C.
9.1.11 Perform 10 fold serial dilutions of challenge inoculum as mentioned
below to determine the initial microbial count of the challenge inoculum.
9.1.12 Take 1.0 ml from of the challenge inoculum and inoculate it into 9.0 ml
of 0.9 % sterile normal saline (1:10 dilution).
9.1.13 Perform the serial dilutions in the same manner ranging from 1:10 to1:
100000000.
9.1.14 Perform the exercise from step 9.1.1 to 9.1.13 with the challenge
inoculum of all the culture organisms, which are being used for
validation.
9.1.15 Enumerate the culture organisms by pour plate method and by
membrane filtration method.
9.1.15.1 Pour Plate Method
 Take 1.0 ml from each dilution and transfer in to sterile petriplates
in duplicate.
 Pour sterile molten Soyabean casein digest agar medium to all the
plates containing respective dilutions for bacterial cultures and
sterile molten sabouraud Chloramphenicol agar medium to all the
plates containing yeast and mold cultures.
 Incubate the plates at 32.5 ± 2.5°C for 48 - 72 hrs for bacterial
cultures and 22.5 ± 2.5C for 72 - 120 hrs for yeast and mold
cultures.
 After the specified incubation period count the number of colonies
and record as CFU / ml.
 After counting the colonies calculate the population of the
challenge inoculum as CFU / ml.
 Perform the whole exercise with the challenge inoculum of all the
culture organisms, which are being used for validation.
9.2 Preparation of the Disinfectant Solution
9.2.1 Prepare the disinfectant solution in Purified Water according to the
manufacturer recommended concentration.
9.2.2 Also prepare disinfectant solution + 50 % & - 50 % from the
manufacturer's recommended concentration to establish the efficacy of
the disinfectants.
9.2.3 Filter the prepared disinfectant solution using 0.2 µ-membrane filter.
9.2.4 Distribute 1.0 ml of the filtered disinfectant solution into sterile test
tubes.
9.2.5 Incase dilution is not recommended by the manufacturer then
disinfectant is to be validated only on manufacturer recommended
concentration.
9.3 Validation of the Neutralization Method:
The neutralization method use in the disinfectant validation study must
be initially validated using the following procedures as mentioned below.
9.3.1 Test Control Group

9.3.1.1 Filter 1.0 ml of the disinfectant solution (prepare as per section 9.2)
through a 0.45  membrane filter.
9.3.1.2 Give two washings of 100 ml each with 0.1 % sterile peptone water.
9.3.1.3 Give third washings of 100 ml with 0.1 % sterile peptone water which
is previously inoculated with 10 - 100 CFU / ml of the culture
organism.
9.3.1.4 After filtration / washing transfer the membrane on Soyabean casein
digest agar medium plate for bacterial cultures and Sabouraud
Chloramphenicol agar medium plate for yeast and mold cultures
respectively with the help of a sterile forcep.
9.3.1.5 Incubate the plates at 32.5 ± 2.5°C for 48 - 72 hrs for bacterial
cultures and 22.5 ± 2.5C for 72 - 120 hrs for yeast and mold cultures.
9.3.1.6 After the specified incubation period count the number of colonies and
record as CFU.
9.3.1.7 Perform the whole exercise with all the culture organisms, which are
being used for validation.
9.3.2 Positive Control Group
9.3.2.1 Filter 200 ml of 0.1 % sterile peptone water through a 0.45 
membrane filter.
9.3.2.2 After filtration of 200 ml of 0.1 % sterile peptone water again filter 100
ml of 0.1 % sterile peptone water, which is previously inoculated, with
10 - 100 CFU/ml of the culture organism.
9.3.2.3 After filtration transfer the membrane on Soyabean casein digest agar
medium plate for bacterial cultures and Sabouraud Chloramphenicol
agar medium plate for yeast and mold cultures respectively with the
help of a sterile forcep
cultures and 22.5 ± 2.5C for 72 - 120 hrs for yeast and mold cultures
record as CFU.
9.3.2.6 Perform the whole exercise with all the culture organisms, which are
being used for validation.
9.3.3 Negative Control Group
9.3.3.1 Filter 300 ml of 0.1 % sterile peptone water through a 0.45 
membrane filter in duplicate.
9.3.3.2 After filtration transfer the one membrane on Soyabean casein digest
agar medium plate and other on Sabouraud Chloramphenicol agar
medium plate respectively with the help of a sterile forcep.
9.3.3.3 Incubate the plates at 32.5 ± 2.5°C for 48 - 72 hrs for Soyabean casein
digest agar medium and 22.5 ± 2.5C for 72 - 120 hrs for Sabouraud
Chloramphenicol agar medium.
record as CFU.
9.3.4 Interpretation of the Result
9.3.4.1 Similar recovery should be observed in the test control group and the
positive control group for all the culture organisms.
9.3.4.2 In the Positive control group at least 70 % recovery should be achieve
for all the culture organisms.
9.3.4.3 In the Negative control group no colonies should be observe
9.4 Determination of the Efficacy of the Disinfectant by Use Dilution
Method: All the disinfectants, which are being used for the surface and
area sanitization of the controlled and the critical clean rooms, must be
initially validated using the following procedures as mentioned below.
9.4.1 Test Control
9.4.1.1 Transfer 1.0 ml of diluted disinfectant solution (prepare the
disinfection as per section 9.2) in each of four sterile test tubes and
add 1.0 ml of challenge inoculum having population between 105 - 106
CFU/ml in all the four test tubes separately.
9.4.1.2 Give a contact time for 1 minute to Ist test tube, 5 minute to 2nd test
tube, 10 minute to 3rd test tube.
9.4.1.3 After the specified contact time, take 1 ml of the sample from each of
the three test tubes in sterile Petri-plates separately.
9.4.1.4 After transferring the sample on Petri-dishes pour Soyabean casein
digest agar medium plate for bacterial cultures and Sabouraud
Chloramphenicol agar medium plate for yeast and mold cultures
respectively.
record as CFU.
9.4.1.7 Select the plates of particular contact time which have least to nil
colonies.
9.4.1.8 Perform the whole exercise with all the disinfectant concentration, at
all the contact period with all the challenge inoculum of all the culture
organisms, which are being used for validation.
9.4.2 Positive Control
9.4.2.1 Take 1.0 ml from of the challenge inoculum of the culture organism
and inoculate it into 9.0 ml of 0.9 % sterile normal saline (1:10
dilution).
9.4.2.2 Perform the serial dilutions in the same manner ranging from 1:10 to
1: 100000000.
9.4.2.3 Enumerate the culture organisms by Pour plate method.
9.4.2.4 Transfer 1.0 ml of each dilution in a sterile Petri- plate.
9.4.2.5 After transferring the sample pour Soyabean casein digest agar
medium for bacterial culture Sabouraud Chloramphenicol agar
medium for fungal growth.
record as CFU.
9.4.2.8 Perform the whole exercise with the challenge inoculum of all the
culture organisms, which are being used for validation.
9.4.3 Negative Control

9.4.3.1 Transfer 1.0 ml of diluted disinfectant solution (prepare the
disinfection as per section 9.2) in each of two sterile test tubes and add
1.0 ml of 0.9 % sterile normal saline in each test tubes separately.
9.4.3.2 Transfer the content of both the test tubes in a sterile Petri-plate.
9.4.3.3 After transferring the sample on Petri-plate pour Soyabean casein
digest agar medium for bacterial culture Sabouraud Chloramphenicol
agar medium for fungal growth.
9.4.3.4 Incubate the plates at 32.5 ± 2.5°C for 48 - 72 hrs for Soyabean casein
digest agar medium and 22.5 ± 2.5C for 72 - 120 hrs for Sabouraud
Chloramphenicol agar medium.
record as CFU.
9.4.4 Interprettion of the Result
9.4.4.1 Calculate the log reduction for each culture organism at each contact
period, with each disinfectant concentration by using the following
formula –
Log Reduction = Log No - Log N
Where, No = The average count (Positive Control),
N = The average count (Test Control)
9.4.4.2 Determine the concentration of the disinfectant solution (use dilution)
and contact period at which a 5 log reduction or greater is achieved.
9.4.4.3 After determining the use dilution of the disinfectant solution,
determine the efficacy of the disinfectant by surface challenge method
using the same concentration of the disinfectant.
9.4.5 Acceptance Criteria
9.4.5.1 In the Test control five (3) Log reduction or greater should be achieve.
9.4.5.2 In the Positive control at least 70 % recovery should be achieve for all
the culture organisms.
9.4.5.3 In the Negative control no colonies should be observe.
9.5 Determination of the Efficacy of the Disinfectant by Surface
Challenge Method
9.5.1 Prepare the disinfectant solution as per recommended used dilution.
9.5.2 Select Nichomac surface, S.S surface and Epoxy coated surface area of
24 -30 cm2.
9.5.3 Marked three (3) Nichomac surfaces, S.S surface (3) and three (3)
Epoxy coated surfaces of 24 -30 cm2 size as Test control, Positive
control and Negative control separately.
9.5.4 Clean all the surfaces with sterile WFI and sterilized these surfaces in
the autoclave.
9.5.5 After sterilization spread 1.0 ml of challenge inoculum having
population between 105 - 106 CFU / ml on the Nichomac surface, S.S
surface and Epoxy coated surface marked as Test control and Positive
control separately with the help of sterile swab or spreader under LAF.
9.5.6 Allow the above spreaded surfaces along with unspreaded surface
(Negative control) for drying under LAF.
9.5.7 Take a lint free sterile duster and dipped / soaked in the disinfectant
solution of recommended used dilution.
9.5.8 Squeeze the above dipped / soaked duster for removal of excess
disinfectant solution.
9.5.9 Wipe the Nichomac surface, S.S Surface and Epoxy coated surface
marked as Test (Spreaded with challenge inoculum having population
between 105 - 106 CFU /ml) with the help of squeeze duster in one
direction for 3 - 4 time.
9.5.10 Allow the surfaces for drying for 10 minutes.
9.5.11 Test Control
9.5.11.1 With the help of a sterile moistened swab take the swab of the above
surface marked as Test control by moving the head of the swab slowly
over the area to be sampled in up and down followed by right and left
direction to cover the entire area.
9.5.11.2 Use different swabs for different surfaces and culture organisms.
9.5.11.3 After swabbing transfer the swab back into the tube aseptically.
9.5.11.4 Aseptically cut the swab stick and transfer the swab stick along with
the tube content in to another tube containing 10 ml 0.1% sterile
peptone water.
9.5.11.5 Gently vortex the tube containing swab stick and tube content.
9.5.11.6 After vortex transfer 1ml of the content of the tube in a Sterile Petri-
plate
9.5.11.7 After transferring the Sample on sterile Petri-plate pour Soyabean
casein digest agar medium plate for bacterial cultures and Sabouraud
Chloramphenicol agar medium plate for yeast and mold cultures.
cultures.
9.5.11.9 After the specified incubation period count the number of colonies
and record as CFU.
9.5.11.10 Perform the whole exercise at all the surfaces (Nichomac surface,
S.S. Surface and Epoxy coated surface) with the challenge inoculum
of all the culture organisms, which are being used for validation.
and record as CFU.
9.5.12 Positive Control

9.5.12.1 With the help of a sterile moistened swab take the swab of the surface
marked as Positive control by moving the head of the swab slowly over
the area to be sampled in up and down followed by right and left
direction to cover the entire area.
9.5.12.2 Use different swabs for different surfaces and culture organisms.
the tube content in to another tube containing 10 ml 0.1% Sterile
peptone water.
9.5.12.5 Gently vortex the tube containing swab stick and tube content and
make serial dilution by taking 1 ml of the content of the tube into
9.0 ml of 0.9 % sterile normal saline (1: 10).
9.5.12.6 Perform the serial dilutions in the same manner ranging from 1:10 to
1: 100000000.
9.5.12.7 Enumerate the culture organisms by Pour Plate Method.
9.5.12.8 Transfer 1.0 ml of each dilution in a sterile Petri-Plate.
9.5.12.9 After transfer, pour Soyabean casein digest agar medium plate for
bacterial cultures and Sabouraud Chloramphenicol agar medium
plate for yeast and mold cultures.
cultures.
and record as CFU.
9.5.13 Negative Control
9.5.13.1 With the help of a Sterile moistened swab take the swab of the surface
marked as Negative control by moving the head of the swab slowly
over the area to be sampled in up and down followed by right and left
direction to cover the entire area in duplicate.
9.5.13.2 Use different swabs for different surfaces.
the tube content in to another tube containing 10 ml 0.1% Sterile
peptone water.
9.5.13.5 Gently vortex the tube containing swab stick and tube content.
9.5.13.6 After vortex transfer 1.0ml of the content of the tube in two sterile
Petri-Plates.
9.5.13.7 After transfer, pour Soyabean casein digest agar medium for bacterial
growth Sabouraud Chloramphenicol agar medium plate for fungal
count respectively in each plate.
9.5.13.8 Incubate the plates at 32.5 ± 2.5°C for 48 - 72 hrs for Soyabean
casein digest agar medium and 22.5 ± 2.5C for 72 - 120 hrs for
Sabouraud Chloramphenicol agar medium.
and record as CFU.
9.5.14 Interpretation of the Result
9.5.14.1 Calculate the log reduction for each culture organism at each surface
(Nichomac surface, S.S. Surface and Epoxy coated surface), using
recommended disinfectant concentration (use dilution) at each time
exposure period by using the following formula
Log Reduction = Log No - Log N
Where, No = The average count (Positive Control)
N = The average count (Test Control)
9.5.14.2 The decrease in the microbial load (log reduction) to the exposed
disinfectant concentration indicates that the disinfectant
concentration is capable of reducing the contaminant when used in
the area.
9.5.15 Acceptance Criteria
9.5.15.1 In the Test control five (5) Log reduction or greater should be achieve
using recommended disinfectant concentration (use dilution) at 10
minute exposure period.
9.5.15.2 In the Positive control at least 70 % recovery should be achieve for all
the culture organisms.
9.5.15.3 In the Negative control no colonies should be observed.
10.0 ACCEPTANCE CRITERIA
 The decrease in the bacterial count to the exposed disinfectant indicates
that the disinfectant is capable of reducing the contaminants when used
in the area. That shall be minimum of 4 log reduction for non-spore
forming microorganisms, yeast and minimum 3 log reduction shall
achieve for spore forming organism, mold with the decided concentration.
 Determine the contact period where the above said populations log
reduction of microorganisms achieved.
 Microbial recovery must not be less than 70% of the spiked amount.
11.0 FREQUENCY
 Whenever a new disinfectant is received.
 If the manufacturer revises the concentration of the ingredients.
 Any change or modification in the validation test procedure
12.0 RESULT
Record all the observations during the validation study and results in the
validation report.
13.0 VAIDATION REPORT
The validation report shall consist of summery document in narrative
form which shall describe the activity performed along with observation. The
report shall also include Attachment / Annexure which shall completed at
the time of validation.
14.0 APPROVAL OF VALIDATION REPORT

The validation report consist of Summary/Conclusion shall be approved by
Head –QA/Nominee.
15.0 REFERENCE (IF ANY)
Chapter 1072 - Disinfectants & Antiseptics (United State Pharmacopoeia - 38).
16.0 ABBREVIATION
VP : Validation Protocol
QC : Quality Control
ATCC : American Type Culture Collection
SOP : Standard Operating Procedure
ml : Milliliter
No. : Number
% : Percentage
oC : Degree centigrade
mm : Millimeter
hrs : Hours
CFU : Colony Forming Unit
µ : Micron
cm : Centimeter
LAF : Laminar Air Flow
IPA : Isopropyl Alcohol
WFI : Water for Injection
S.S : Stainless Steel

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B.JAIN PHARMACEUTICALS PVT. LTD.

PROTOCOL FOR DISINFECTANT VALIDATION

Sr. No. DESCRIPTION PAGE NO

7. Standard Operating Procedure to be followed 04

9. Validation Methodology And Plan 05-12

10. Acceptance criteria 12

13. Validation Report 12

14 Approval of validation report 12

15. Reference (If any) 12

16. Abbreviation 12-13

1.0 PROTOCOL APPROVAL

Name Designation Signature Date

Name Designation Signature Date

2.0 History Sheet

01 28/11/2018 New Protocol

9.3.1 Test Control Group

9.4.3 Negative Control

9.5.12 Positive Control

14.0 APPROVAL OF VALIDATION REPORT

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