Disinfectant Efficacy Validation Protocol

TABLE OF CONTENTS

APPROVAL SIGNATURE

OVERVIEW

• Objective
• Purpose & Scope
• Responsibility

EXECUTION TEAM

TRAINING RECORD

REQUIREMENTS FOR QUALIFICATION

SYSTEM/ EQUIPMENT DESCRIPTION

• Preparation of Bacillus subtilis spore culture
• Preparation of Challenge Inoculum
• Validation by Filtration Method
• Validation by Surface Method.

QUALIFICATION PROCEDURE OR METHODOLOGY

• General Recording Instructions
• General Safety Instruction for Execution
• Preparation of Bacillus subtilis spore culture

1. Take a preincubated sterile SCDA slant and a working culture of Bacillus subtilis
2. Pick up a loopful of Bacillus subtilis culture and streak on the surface of the sterile SCDA slant
3. Incubate the slant at 30 – 35 ° C for 7 days
4. Perform Spore staining for this incubated culture and observe for its purity and spore formation
5. If the culture shows spores (discard if contaminated), preserve this culture at 2-8 °C and use this spore for the preparation of Inoculum of Bacillus subtilis
6. Record the Result as per Annexure-02

• Preparation of Challenge Inoculum

1. Purpose
2. Requirement
3. Test Procedure

• Disinfectant Validation by Use Dilution Method
• Disinfectant Validation by Surface Method

ACCEPTANCE CRITERIA

• The reduction of Challenge Inoculum count should be more than 3 log for bacterial cultures

OBSERVED DEVIATION (IF ANY)

QUALIFICATION REPORT

ABBREVIATIONS

LIST OF ANNEXURES

REFERENCE DOCUMENT

APPROVAL SIGNATURE

• This is a specific protocol for Disinfectant Efficacy validation which is being used in the plant for Cleaning and Fumigation purpose. The Author's signature indicates that this document has been prepared following existing standards and adequately reflects the tasks and deliverables necessary for the disinfectant efficacy test.

• The reviewer’s signature indicates that this document has been reviewed and it accurately and completely reflects the tasks and deliverables necessary for the process.

ALSO READ: SOP for Validation of Laminar Air Flow

• The approver’s signature indicates that the documentation and information contained herein comply with applicable regulatory, corporate, divisional/departmental requirements, and current Good Manufacturing Practices.

OVERVIEW

Objective

To establish a methodology for Disinfectant efficacy Validation used in PharmaGuide Ltd.

Purpose & Scope

The purpose is to provide an outline for the efficacy of the Disinfectants used in the plant for the disinfection of surfaces, air and disinfection of hands etc.

Responsibility

To conduct the qualification study, a team shall be formed. The team shall contain members from the, Quality Control and Quality Assurance departments. The Validation team is described through the following responsibility.

Role	Responsibilities
Executive – QC (Microbiology)	Shall prepare the Qualification Protocol as per the guideline of Validation Master Plan
Section In-charge – Microbiology, Manager – QC, and Executive QA	Shall review the Qualification Protocol for correctness, completeness, and technical excellence.
QA Head	Qualification Protocol shall be approved by the QA Head prior to execution.
Executive – QC (Microbiology)	Shall conduct and record the qualification test as per the protocol.
Section In-charge – Microbiology, Manager – QC, and Executive QA	Shall review the executed protocol to check compliance and corrective actions for any discrepancies found. Also shall prepare the summary and conclusion of the qualification study.
QA Head	Shall review the qualification study and its summary report. Approval of the summary report and conclusion shall be done before the initiation of the installation qualification study.

EXECUTION TEAM

The following personnel shall be responsible for the execution of the qualification study;

1. Executive – QC (Microbiology): To conduct the qualification study as per protocol.
2. Executive - Formulations: To provide material for testing.
3. Executive Quality Assurance: To review & monitor the qualification study.

TRAINING RECORD

Purpose

The purpose of the training is to familiarize the trainees with the requirement of Disinfectant Efficacy Validation.

Scope

This Training is applicable to all concerned persons which are involved in this activity.

Topics

1. The following topics shall be covered during the training:
2. Purpose & procedure of Disinfectant Efficacy Validation.
3. Identifying the responsibility of an involved person.
4. Documentation practices to be followed.
5. General precautions/guidelines to be followed during qualification.
6. Attach the training record with the report as Annexure – 01.

REQUIREMENTS FOR QUALIFICATION

The following items shall be qualified before the execution of Disinfectant Efficacy validation:

• Microbial Culture

1. Staphylococcus aureus
2. Bacillus subtilis
3. Pseudomonas aeruginosa
4. Clostridium sporogenes
5. Candida albicans
6. Aspergillus niger
7. E. coli
8. S. abony
9. Environment isolate

• Media

1. Sterile Molten Soybean Casein Digest Agar
2. Sterile Molten Sabouraud Chloramphenicol Agar
3. Reinforced Clostridial Agar
4. Sterile Peptone water
5. Sterile Letheen broth
6. Preincubated sterile SCDA, SCA plates

• Sterile forceps and scissors
• Sterilized filtration unit and Membrane filters
• Manifold
• Vortex mixer
• Forceps
• Micropipette
• Biosafety Cabinet
• Disinfectants to be validated

SYSTEM/ EQUIPMENT DESCRIPTION

The validation will be performed under the following subheadings:

• Preparation of Bacillus subtilis spore culture.
• Preparation of Challenge Inoculum.
• Validation by Filtration Method.
• Validation by Surface Method.

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QUALIFICATION PROCEDURE OR METHODOLOGY

General Recording Instructions

• Read the contents of the document thoroughly before proceeding for the Execution of the activity (in case of doubts/contradictions / contact the approvers of the document for clarifications).
• Recording of all the observations and data shall be as per good documentation practices SOP.
• The recording will be done on a controlled copy of the Disinfectant efficacy validation annexure.
• In each of the tests/processes outlined in the protocol the test parameter, specifications, acceptance criteria and any other requirements are pre-defined. Other columns viz. observations, verified by and other columns are to be filled in by the member of the execution team manually.
• Follow the specific instructions as mentioned in the particular test for verifying the parameters & recording the observations.
• Recording the requested information in a particular test indicates that it has been inspected/verified according to specifications by visual examination, product literature, using a measuring device etc.
• All the deviations/discrepancies observed during the activity are to be explained & justified if acceptable.
• Record all deviations/discrepancies observed during the execution in the Observed deviations format of the protocol.

General Safety Instruction for Execution

Safety will be one of the key considerations during the execution of this document. The following guidelines must be observed during the execution stage.

• All personnel involved with the execution shall identify hazards associated with the performance of testing and precautions to be taken.
• All personnel involved with the execution shall check that utilities are safely isolated when energizing or de-energizing.
• All personnel involved in the execution shall inform the company management of any hazard, to themselves or others, associated with the materials, equipment, method of working and the precautions to be taken.

Preparation of Bacillus subtilis spore culture

• Take a preincubated sterile SCDA slant and a working culture of Bacillus subtilis.
• Pick up a loopful of Bacillus subtilis culture and streak on the surface of the sterile SCDA slant.
• Incubate the slant at 30 – 35°C for 7 days.
• Perform Spore staining for this incubated culture and observe for its purity and spore formation.
• If the culture shows spores (discard if contaminated), preserve this culture at 2-8°C and use this spore for the preparation of Inoculum of Bacillus subtilis.
• Record the Result as per Annexure-02.

Preparation of Challenge Inoculum

Purpose

The purpose of the test is to prepare the challenge Inoculum of the selected culture used for Disinfectant Efficacy Validation.

Requirement

Sterile Peptone water, Sterilized tips, Micropipette, culture & Biosafety cabinet etc.

Test Procedure

• Take the required types of working cultures (Spore culture in the case of Bacillus subtilis).
• Inoculate a small loop of each working culture (Spore culture in the case of Bacillus subtilis) and inoculate into different bottles containing 50 ml sterile saline and dilute the suspension serially using 10 fold dilution method from 10-1 to 10-10.
• Pipette out 1.0 ml of 10-4 diluted culture onto each of two sterile Petri dishes
• Repeat this step for 10-5, 10-6, 10-7, 10-8, 10-9& 10-10. Repeat the above 4 steps for all other cultures (as per point no. 5.1.1) also.
• Add 20-25 ml of Agar Medium (SCDA for Aerobic bacteria, RCA for Anaerobic bacteria, and SCA for Fungi) that is melted and cooled to approximately 45°C.
• Cover the Petri dishes and mix the sample with agar by tilting or rotating the Petri dishes.
• Allow solidifying at room temperature. Invert the Petri plates and incubate. (Aerobic bacteria at 30° to 35° for 48 hrs. Anaerobic bacteria at 30° to 35° for 48 hrs in an anaerobic jar, Candida albicans at 20° to 25° for 72 hrs., and Aspergillus niger at 20° to 25° for 5 days.)
• Preserve all the dilutions at 2-8 ° C. Examine the plates, count the number of colonies and express the average of 2 plates in terms cfu/plate. Select the dilutions containing 100000 to 1000000 CFU’s / ml, preserve at 2-8°C and discard all other dilutions
• Use the preserved dilute culture suspensions within 7 days.
• Record the data as per Annexure-03.

Disinfectant Validation by Use Dilution Method

Purpose

This test is performed to ensure the suitable concentration & contact time for the disinfectant to achieve ≥ 3 Log reduction by filtration technique.

Requirements

Filtration Funnels, flasks, vacuum pump, sterile membrane filters, sterile peptone water, medium etc.

Test Procedure

• The dilute disinfectant solution as per the recommendation of the manufacturer and also prepare 1/2 and 1/4 concentrations from the manufacturer's recommendation.
• Use sterile purified water or sterile water for injection as diluent.
• Arrange 3 sets (3 bottles in each set) of sterile empty 25 ml tubes (for one culture).
• Add 10 ml of the manufacturer’s recommended concentration of the disinfectant solution in 1st tube of each set.
• Add 10 ml of 50% strength of the disinfectant solution as per the manufacturer's recommended concentration of the disinfectant in the 2nd tube of each set.
• Add 10 ml of 25% strength of the disinfectant solution as per the manufacturer's recommended concentration of the disinfectant in 3rd tube of each set.
• Arrange one sterile bottle containing 10 ml of sterile peptone water (for initial count of the challenge Inoculum).
• Add 1 ml of prepared challenge inoculum of E.coli to this 100 ml of diluent.
• Dilute this solution using 10-fold dilution methods from 10-1 to 10-4.
• Arrange the sterile filter holders having 0.45 μm membrane filters on the manifold and assemble the manifold to a vacuum source.
• Add 1 ml of the Challenge Inoculum of E.coli to all the tubes of 3 sets.
• Mix and immediately filter the solutions of 1st set using an individual filter holder for each tube. This is ‘0 min’ contact sample.
• Filter the solution meant for the initial count using another filter holder. Rinse each membrane filter with 3X100 ml of sterile 0.1% peptone water.
• After rinsing, place each membrane filter on the surfaces of individual agar medium plates.

ALSO READ: SOP for Preparation and Sterilization of Microbial Media

• After 5 minutes, mix and filter the solutions of 2nd set using an individual filter holder for each tube. This is ‘5 min’ contact sample. Rinse each membrane filter with 3X100 ml sterile 0.1% peptone water.
• After rinsing, place each membrane filter on the surfaces of individual agar medium plates.
• After another 5 minutes, mix and filter the solutions of 3rd set using an individual filter holder for each tube. This is ‘10 min’ contact sample.
• Rinse each membrane filter with 3X100 ml sterile 0.1% peptone water.
• After rinsing, place each membrane filter on the surfaces of individual agar medium plates.
• Repeat the whole procedure for each organism as per above point.
• Use same agar medium and incubation conditions as mentioned in point no. ‘Preparation of Challenge Inoculum’.
• Examine the plates, count the number of colonies on each plate.
• Compare the ‘0 min’ ‘5 min’ ’10 min’ contact culture plates with ‘Initial count’ plates.
• Record the results as per Annexure-04.

Disinfectant Validation by Surface Method

Purpose

To get a practical approach for efficacy apply the disinfectants to any of the surfaces which is present in that particular area that will be decontaminated by spraying the disinfectant.

Requirement

SS strip, Epoxy Coated strip, sterile swab.

Procedure

• Take 3 sets (3 strips in each set) each of S.S and Epoxy coated strips having a surface area of 25 cm2.
• Take one more strip each of SS and Epoxy coated.
• Wrap all the strips with aluminum foil and Depyrogenate it in hot air oven at 250°C for 1 hrs.
• Prepare different concentrations of the disinfectant solution as mentioned in the ‘Use dilution method’.
• Arrange the strips in the LAF.
• Add 1 ml of Challenge Inoculum to each Epoxy coated and SS strips.
• Allow the culture to dry on the surface of strips for 30 minutes or until they dry.
• Spray the manufacturer-recommended concentration of disinfectant on the surfaces of 1st strip of each set, 50% of manufacturer recommended concentration of disinfectant on surfaces of 2nd strip of each set and 25% dilution of manufacturer recommended concentration of disinfectant on surfaces of 3rd strip of each set.
• Do not spray the disinfectant on 10th strip, which will be used to determine the ‘Initial count’.
• With the help of different, individual sterile swabs, swab the disinfectant exposed culture surfaces of strips of 1st set. Add each swab to individual test tube containing 10 ml of sterile Letheen broth.
• Mix, filter and follow further process as mentioned for ‘0 min’ contact sample of ‘Use dilution Method’.
• Swab the culture surfaces (Not exposed to disinfectant) of SS and Epoxy strip surfaces and add each swab to individual test tube containing 10 ml of sterile Letheen broth. Dilute this solution up to 10-4
• Mix, filter and follow further process as mentioned for ‘Initial count sample’ of ‘Use dilution Method’.
• After 5 minutes, swab the disinfectant exposed culture surfaces of all the strips of 2nd set with the help of different, individual sterile swabs and add each swab to individual test tube containing 10 ml of sterile Letheen broth.
• Follow the filtration, plating and incubation procedure as mentioned in ‘5 min’ contact sample of ‘Use dilution Method'.
• After further 5 minutes, swab the disinfectant exposed culture surfaces of the strips of 3rd set with the help of different, individual sterile swabs and add each swab to individual test tube containing 10 ml of sterile Letheen broth.
• Follow the filtration, plating and incubation procedure as mentioned in ‘10 min’ contact sample of ‘Use dilution Method'.
• Repeat the whole procedure for each Challenge inoculum as mentioned in above point.

• Examine the plates; count the number of colonies on each plate.
• Compare the ‘0 min’ ‘5 min’ ’10 min’ contact culture plates with ‘Initial count’ plates.
• Record the results as per Annexure-05.

ACCEPTANCE CRITERIA

The reduction of Challenge Inoculum count should be more than 3 log for bacterial cultures.

OBSERVED DEVIATION (IF ANY)

• Allot a sequential number starting from 01 for each deviation/discrepancy observed during the execution of the activity. Record the details of the test/parameter during which the deviation/discrepancy was observed, along with a brief description of the deviation/discrepancy.
• The deviation/discrepancy shall be addressed as per the SOP “Deviation Process”.

QUALIFICATION REPORT

• The qualification report shall consist of a summary document, in narrative form, which shall briefly describe the activity performed along with the observations recorded in relevant exhibits.
• This report shall also include the related documents and attachments / exhibits which were completed at the time of qualification activity.

ABBREVIATIONS

SCDA : Soyabean Casein Digest Agar

RCA : Reinforced Clostridial Agar

LAF : Laminar Air Flow

SCA : Sabouraud Chloramphenicol Agar

CFU : Colony Forming Unit

ANNEXURES

01 - Training Record

02 - Report for Bacillus subtilis Culture

03 - Report for Preparation of Challenge inoculum

04 - Report for Disinfectant Efficacy test by Use Dilution method.

05 - Report for Disinfectant Efficacy test by surface method.

REFERENCE DOCUMENT

Nil

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