Disinfectant Validation Protocol

TABLE OF CONTENT

1. Protocol approval
2. Objective
3. Scope
4. Site of study
5. Responsibilities
6. Standard Operating Procedure to be followed
7. Pre-Requisites
8. Validation Methodology And Plan
9. Acceptance criteria
10. Frequency
11. Result
12. Validation Report
13. Approval of validation report
14. Reference (If any)
15. Abbreviation

PROTOCOL APPROVAL 

Prepared By	Name	Designation	Signature	Date
Checked By
Approved By	Name	Designation	Signature	Date

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 the following test

1. 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.
2. 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.

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 PharmaSky Pvt. Ltd.
• The same established data should be used in further routine usage.

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SITE OF STUDY

Quality Control Department (Microbiology Section) of PharmaSky Pvt. Ltd.

RESPONSIBILITIES

Quality Control:

• To prepare and check the protocol.
• To provide all applicable documents for the generation of the protocol.
• To provide personnel to assist in the execution of this protocol.

Quality Assurance:

• To check and approve the protocol.
• To approve deviation and to complete the final report.
• Ensure the protocol completeness and technical accuracy.

STANDARD OPERATING PROCEDURE (SOP) TO BE FOLLOWED

1. Procedure for maintenance and suspension preparation of microbial cultures.
2. Procedure for preparation of disinfectant and cleaning.
3. Procedure for growth promotion test.
4. Procedure for operation and calibration of BOD Incubators.
5. Procedure for disposal of used or contaminated culture media

PRE-REQUISITES

Disinfectants: Disinfectant details such as name of the disinfectant, its manufacturer, recommended concentration, action and its composition will be mentioned in the validation report.

VALIDATION METHODOLOGY AND PLAN

Preparation of Challenge Inoculum

• Challenge Inoculum should have a population of 105 - 106 CFU / ml.
• Prepare the Soyabean casein digest agar and Sabouraud Chloramphenicol agar media slant as per the SOP.
• Perform the growth promotion and pre-incubation test of the prepared media slants.
• 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.
• From the working culture slant streak a loopful of culture on the freshly prepared slants.
• 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.
• 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 a sterile nichrome loop.
• 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).
• Perform the exercise from the above step with all the culture organisms, which are being used for validation.
• Store the challenge inoculum of all the culture organisms in the refrigerator at 2 - 8°C.
• Perform 10-fold serial dilutions of the challenge inoculum as mentioned below to determine the initial microbial count of the challenge inoculum.
• 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).
• Perform the serial dilutions in the same manner ranging from 1:10 to1: 100000000.
• Perform the exercise from step 1 to 13 with the challenge inoculum of all the culture organisms, which are being used for validation.
• Enumerate the culture organisms by pour plate method and by membrane filtration method.

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• Pour Plate Method

1. Take 1.0 ml from each dilution and transfer in to sterile Petri plates in duplicate.
2. 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.
3. 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.
4. After the specified incubation period count the number of colonies and record as CFU / ml.
5. After counting the colonies calculate the population of the challenge inoculum as CFU / ml.
6. Perform the whole exercise with the challenge inoculum of all the culture organisms, which are being used for validation.

Preparation of the Disinfectant Solution

• Prepare the disinfectant solution in Purified Water according to the manufacturer recommended concentration.
• Also prepare disinfectant solution + 50 % & - 50 % from the manufacturer's recommended concentration to establish the efficacy of the disinfectants.
• Filter the prepared disinfectant solution using 0.2 µ-membrane filter.
• Distribute 1.0 ml of the filtered disinfectant solution into sterile test tubes.
• Incase dilution is not recommended by the manufacturer then disinfectant is to be validated only on manufacturer-recommended concentration.

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.

Test Control Group

• Filter 1.0 ml of the disinfectant solution (prepare as per above) through a 0.45 m membrane filter.
• Give two washings of 100 ml each with 0.1 % sterile peptone water.
• 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.
• 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.
• 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.
• Perform the whole exercise with all the culture organisms, which are being used for validation.

Positive Control Group

• Filter 200 ml of 0.1 % sterile peptone water through a 0.45 m membrane filter.
• 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.
• 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 sterile forceps.
• 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.
• Perform the whole exercise with all the culture organisms, which are being used for validation.

Negative Control Group

• Filter 300 ml of 0.1 % sterile peptone water through a 0.45 m membrane filter in duplicate.
• 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.
• 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.
• After the specified incubation period count the number of colonies and record as CFU.

Interpretation of the Result

• Similar recovery should be observed in the test control group and the positive control group for all the culture organisms.
• In the Positive control group at least 70 % recovery should be achieve for all the culture organisms.
• In the Negative control group no colonies should be observe

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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.

Test Control

• Transfer 1.0 ml of diluted disinfectant solution (prepare the disinfection as per above) in each of four sterile test tubes and add 1.0 ml of challenge inoculum having a population between 105 - 106 CFU/ml in all four test tubes separately.
• Give a contact time for 1 minute to Ist test tube, 5 minutes to 2nd test tube, 10 minutes to 3rd test tube.
• After the specified contact time, take 1 ml of the sample from each of the three test tubes in sterile Petri-plates separately.
• 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.
• 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.
• Select the plates of particular contact time which have least to nil colonies.
• Perform the whole exercise with all the disinfectant concentrations, at all the contact periods with all the challenge inoculum of all the culture organisms, which are being used for validation.

Positive Control

• 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).
• Perform the serial dilutions in the same manner ranging from 1:10 to 1: 100000000.
• Enumerate the culture organisms by Pour plate method.
• Transfer 1.0 ml of each dilution in a sterile Petri- plate.
• After transferring the sample pour Soyabean casein digest agar medium for bacterial culture Sabouraud Chloramphenicol agar medium for fungal growth.
• 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.
• Perform the whole exercise with the challenge inoculum of all the culture organisms, which are being used for validation.

Negative Control

• Transfer 1.0 ml of diluted disinfectant solution (prepare the disinfection as per section above) in each of two sterile test tubes and add 1.0 ml of 0.9 % sterile normal saline in each test tubes separately.
• Transfer the content of both the test tubes in a sterile Petri-plate.
• After transferring the sample on Petri-plate pour Soyabean casein digest agar medium for bacterial culture Sabouraud Chloramphenicol agar medium for fungal growth.
• 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.
• After the specified incubation period count the number of colonies and record as CFU.

Interpretation of the Result

• 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)

• Determine the concentration of the disinfectant solution (use dilution) and contact period at which a 5 log reduction or greater is achieved.
• 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.

Acceptance Criteria

• In the Test control five (3) Log reductions or greater should be achieved.
• In the Positive control at least 70% recovery should be achieved for all the culture organisms.
• In the Negative control, no colonies should be observed.

Determination of the Efficacy of the Disinfectant by Surface Challenge Method

• Prepare the disinfectant solution as per recommended used dilution.
• Select Nichomac surface, S.S surface and Epoxy coated surface area of 24 -30 cm2.
• 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.
• Clean all the surfaces with sterile WFI and sterilized these surfaces in the autoclave.
• 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.
• Allow the above spreaded surfaces along with unspreaded surface (Negative control) for drying under LAF.
• Take a lint free sterile duster and dipped / soaked in the disinfectant solution of recommended used dilution.
• Squeeze the above dipped / soaked duster for removal of excess disinfectant solution.
• 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.
• Allow the surfaces for drying for 10 minutes.

Test Control

• 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.
• Use different swabs for different surfaces and culture organisms.
• After swabbing transfer the swab back into the tube aseptically.
• 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.
• Gently vortex the tube containing the swab stick and tube content.
• After vortex transfer 1ml of the content of the tube in a Sterile Petri-plate
• After transferring the Sample on a sterile Petri plate pour Soyabean casein digest agar medium plate for bacterial cultures and Sabouraud Chloramphenicol agar medium plate for 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.
• 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.
• After the specified incubation period count the number of colonies and record as CFU.
• 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.

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Positive Control

• 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.
• Use different swabs for different surfaces and culture organisms.
• After swabbing transfer the swab back into the tube aseptically.
• 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.
• 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).
• Perform the serial dilutions in the same manner ranging from 1:10 to 1: 100000000.
• Enumerate the culture organisms by Pour Plate Method.
• Transfer 1.0 ml of each dilution in a sterile Petri-Plate.
• After transfer, pour Soyabean casein digest agar medium plate for bacterial cultures and Sabouraud Chloramphenicol agar medium plate for 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.
• 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.

Negative Control

• 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.
• Use different swabs for different surfaces.
• After swabbing transfer the swab back into the tube aseptically.
• 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.
• Gently vortex the tube containing swab stick and tube content.
• After vortex transfer 1.0ml of the content of the tube in two sterile Petri-Plates.
• After transfer, pour Soyabean casein digest agar medium for bacterial growth Sabouraud Chloramphenicol agar medium plate for fungal count respectively in each plate.
• 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.
• After the specified incubation period count the number of colonies and record as CFU.

Interpretation of the Result

• 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)

• 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.

Acceptance Criteria

• In the Test control five (5) Log reduction or greater should be achieve using recommended disinfectant concentration (use dilution) at 10 minute exposure period.
• In the Positive control at least 70 % recovery should be achieve for all the culture organisms.
• In the Negative control no colonies should be observed.

ACCEPTANCE CRITERIA

1. 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 a minimum of 4 log reduction for non-spore forming microorganisms, and yeast and a minimum 3 log reduction shall achieve for spore-forming organisms, mold with the decided concentration.
2. Determine the contact period where the above said populations log reduction of microorganisms achieved.
3. Microbial recovery must not be less than 70% of the spiked amount.

FREQUENCY

1. Whenever a new disinfectant is received.
2. If the manufacturer revises the concentration of the ingredients.
3. Any change or modification in the validation test procedure

RESULT

Record all the observations during the validation study and results in the validation report.

VALIDATION REPORT

The validation report shall consist of summary document in narrative form which shall describe the activity performed along with observation. The report shall also include an Attachment / Annexure which shall be completed at the time of validation.

APPROVAL OF VALIDATION REPORT

The validation report consisting of the Summary/Conclusion shall be approved by the Head –QA/Nominee.

REFERENCE (IF ANY)

Chapter 1072 - Disinfectants & Antiseptics (United State Pharmacopoeia - 38).

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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