QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number Disinfectant Efficacy Validation Effective Date

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VALIDATION PROTOCOL FOR DISINFECTANT EFFICACY VALIDATION

QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number TABLE OF CONTENTS Disinfectant Efficacy Validation Effective Date

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1.0APPROVAL SIGNATURE:.....................................................................................................................3 2.0OVERVIEW:.............................................................................................................................................5 2.1 Objective:.........................................................................................................................................5 2.2 Purpose & Scope:............................................................................................................................5 2.3 Responsibility:.................................................................................................................................5 3.0EXECUTION TEAM:..............................................................................................................................5 4.0TRAINING RECORD:.............................................................................................................................6 5.0REQUIREMENTS FOR QUALIFICATION:.......................................................................................6 6.0SYSTEM/ EQUIPMENT DESCRIPTION:............................................................................................7 6.1Preparation of Bacillus subtilis spore culture.........................................................................................7 6.2Preparation of Challenge Inoculum........................................................................................................7 6.3Validation by Filtration Method..............................................................................................................7 6.4Validation by Surface Method. ...............................................................................................................7 7.0QUALIFICATION PROCEDURE OR METHODOLOGY:...............................................................7 7.1 General Recording Instructions:...................................................................................................7 7.2 General Safety Instruction for Execution:....................................................................................8 7.3 Preparation of Bacillus subtilis spore culture:.............................................................................8 7.3.1 Take a preincubated sterile SCDA slant and a working culture of Bacillus subtilis.............8 7.3.2 Pick up a loopful of Bacillus subtilis culture and streak on surface of sterile SCDA slant...8 7.3.3 Incubate the slant at 30 35 C for 7 days...............................................................................8 7.3.4 Perform Spore staining for this incubated culture and observe for its purity and spore formation.......................................................................................................................................8 7.3.5 If the culture show spores (discard if contaminated), preserve this culture at 2-8 C and use this spore for the preparation of Inoculum of Bacillus subtilis.........................................8 7.3.6 Record the Result as per Annexure-02.......................................................................................8 7.4 Preparation of Challenge Inoculum:.............................................................................................8 7.4.1 Purpose: .......................................................................................................................................8 The purpose of the test is to prepare the challenge Inoculum of selected culture used for Disinfectant Efficacy Validation.................................................................................................8 7.4.2 Requirement: ..............................................................................................................................8 7.4.3 Test Procedure:............................................................................................................................8
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QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number Disinfectant Efficacy Validation Effective Date

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7.5 Disinfectant Validation by Use Dilution Method: .......................................................................9 7.6 Disinfectant Validation by Surface Method................................................................................10 8.0ACCEPTANCE CRITERIA:.................................................................................................................11 8.1 The reduction of Challenge Inoculum count should be more than 3 log for bacterial cultures........................................................................................................................................11 9.0OBSERVED DEVIATION (IF ANY):..................................................................................................12 10.0QUALIFICATION REPORT:.............................................................................................................12 This report shall also include the related documents and attachments / exhibit which were completed at the time of qualification activity.............................................................................................................12 11.0ABBREVIATIONS:..............................................................................................................................12 12.0LIST OF ANNEXURES:......................................................................................................................12 13.0REFERENCE DOCUMENT:..............................................................................................................12 Nil 12

1.0

APPROVAL SIGNATURE: This is specific protocol for Disinfectant Efficacy validation which is being used in the plant for Cleaning and Fumigation purpose. The Author signature indicates that this document has been prepared in accordance with existing standards and adequately reflects the tasks and deliverables necessary for disinfectant efficacy test. Prepared by / Functional area Designation Signature Date

Microbiology Section

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QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number Disinfectant Efficacy Validation Effective Date

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The reviewers signature indicates that, this document has been reviewed and it accurately and completely reflects the tasks and deliverables necessary for process. Checked by / Functional area Designation Signature Date

Quality Control-Microbiology Quality Control Validation QA

The approvers signature indicates that the documentation and information contained herein complies with applicable regulatory, corporate, divisional / departmental requirements, and current Good Manufacturing Practices. Approved by / Functional area Designation Signature Date

Quality Assurance

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QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number Disinfectant Efficacy Validation Effective Date

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2.0

OVERVIEW: 2.1 Objective: To establish methodology for Disinfectant efficacy Validation used in ABC Pharma Pvt. Ltd., Bhiwadi. 2.2 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. 2.3 Responsibility: To conduct the qualification study, a team shall be formed. The team shall contain the members from the, Quality Control and Quality Assurance departments. The Validation team is described through the following responsibility. Executive QC (Microbiology) Section In chargeMicrobiology, Manager QC, and Executive QA QA Head Executive QC (Microbiology) Section In chargeMicrobiology, Manager QC, and Executive QA QA Head Shall prepare the Qualification Protocol as per the guideline of Validation Master Plan and SOP QA004 for preparation of validation protocol and its control. Shall review the Qualification protocol completeness and technical excellence for correctness,

Qualification Protocol shall be approved by the QA Head prior to execution Shall conduct and record the qualification test as per the protocol Shall review the executed protocol to check the compliance and corrective action for any discrepancies found. Also shall prepare the summary and conclusion of the qualification study. Shall review the qualification study and its summary report. Approval of the summary report and conclusion shall be done before initiation of installation qualification study

3.0

EXECUTION TEAM: Following personnel shall be responsible for the execution of qualification study; Executive QC (Microbiology) : To conduct the qualification study as per protocol.

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QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number Executive - Formulations Executive Quality Assurance 4.0 TRAINING RECORD: 4.1 Purpose: Disinfectant Efficacy Validation Effective Date : To provide material for testing. : To review & monitor the qualification study.

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The purpose of the training is to familiarize the trainees with the requirement of Disinfectant Efficacy Validation. 4.2 Scope: This Training is applicable to the all concerned persons which are involved in this activity. 4.3 Topics: The following topics shall be covered during training: 4.3.1 Purpose & procedure of Disinfectant Efficacy Validation. 4.3.2 Identifying the responsibility of involved person. 4.3.3 Documentation practices to be followed. 4.3.4 General precautions / guidelines to be followed during qualification. 4.3.5 Attach training record with the report as Annexure 01. 5.0 REQUIREMENTS FOR QUALIFICATION: 5.1 Following items shall be qualified before execution of Disinfectant Efficacy validation: 5.1.1 Microbial Culture 5.1.1.1 5.1.1.2 5.1.1.3 5.1.1.4 5.1.1.5 5.1.1.6 5.1.1.7 5.1.1.8 5.1.1.9 5.1.2 Media 5.1.2.1 5.1.2.2 5.1.2.3
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Staphylococcus aureus Bacillus subtilis Pseudomonas aeruginosa Clostridium sporogenes Candida albicans Aspergillus niger E. coli S. abony Environment isolate Sterile Molten Soybean Casein Digest Agar Sterile Molten Sabouraud Chloramphenicol Agar Reinforced Clostridial Agar

QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number 5.1.2.4 5.1.2.5 5.1.2.6 5.1.3 5.1.4 5.1.5 5.1.6 5.1.7 5.1.8 5.1.9 6.0 Sterile Peptone water Sterile Letheen broth Preincubated sterile SCDA, SCA plates Disinfectant Efficacy Validation Effective Date

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Sterile forceps and scissors Sterilized filtration unit and Membrane filters Manifold Vortex mixer Forceps Micropipette Biosafety Cabinet

5.1.10 Disinfectants to be validated SYSTEM/ EQUIPMENT DESCRIPTION: The validation will be performed under the following subheadings: 6.1 6.2 6.3 6.4 7.0 Preparation of Bacillus subtilis spore culture. Preparation of Challenge Inoculum. Validation by Filtration Method. Validation by Surface Method.

QUALIFICATION PROCEDURE OR METHODOLOGY: 7.1 General Recording Instructions: 7.1.1 Read the contents of the document thoroughly before proceeding for Execution of the activity (in case of doubts / contradictions / contact the approvers of the document for clarifications). 7.1.2 Recording of all the observations and data shall be as per good documentation practices SOP QA009. 7.1.3 Recording will be done on controlled copy of Disinfectant efficacy validation annexure. 7.1.4 In each of the tests/process outlined in the protocol the test parameter, specifications, acceptance criteria and any other requirement are pre-defined. Other columns viz. observations, verified by and other columns are to be filled in by the member of execution team manually. 7.1.5 Follow the specific instructions as mentioned in the particular test for verifying the parameters & recording the observations. 7.1.6 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. 7.1.7 All the deviations / discrepancies observed during the activity are to be explained & justified if acceptable.

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QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number 7.1.8 7.2 Disinfectant Efficacy Validation Effective Date

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Record all deviations / discrepancies observed during the execution in the Observed deviations format of 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. 7.2.1 All personnel involved with the execution shall identify hazards associated with performance of testing and precautions to be taken. 7.2.2 All personnel involved with the execution shall check that utilities are safely isolated when energizing or de-energizing. 7.2.3 All personnel involved in the execution shall inform the company management 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: 7.3.1 7.3.2 7.3.3 7.3.4 7.3.5 7.3.6 Take a preincubated sterile SCDA slant and a working culture of Bacillus subtilis. Pick up a loopful of Bacillus subtilis culture and streak on surface of 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 show 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.

7.3

7.4

Preparation of Challenge Inoculum: 7.4.1 Purpose: The purpose of the test is to prepare the challenge Inoculum of selected culture used for Disinfectant Efficacy Validation. 7.4.2 Requirement: Sterile Peptone water, Sterilized tips, Micropipette, culture & Biosafety cabinet etc. 7.4.3 Test Procedure: 7.4.3.1 7.4.3.2 Take 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 in to 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.

7.4.3.3 7.4.3.4

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QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number 7.4.3.5 7.4.3.6 7.4.3.7 Disinfectant Efficacy Validation Effective Date

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7.4.3.8

7.4.3.9 7.4.3.10 7.5

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 to solidify 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 CFUs / 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: 7.5.1 Purpose: This test is performed so as to ensure the suitable concentration & contact time for the disinfectant to achieve 3 Log reduction by filtration technique. 7.5.2 Requirements: Filtration Funnels, flasks, vacuum pump, sterile membrane filters, sterile peptone water, medium etc. 7.5.3 Test procedure: 7.5.3.1 7.5.3.2 7.5.3.3 7.5.3.4 7.5.3.5 7.5.3.6 7.5.3.7 7.5.3.8 7.5.3.9 Dilute disinfectant solution as per the recommendation of manufacturer and also prepare 1/2 and 1/4 concentrations from the manufacturers 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 manufacturers recommended concentration of the disinfectant solution in 1st tube of each set. Add 10 ml of 50% strength of disinfectant solution as per manufacturer recommended concentration of the disinfectant in 2nd tube of each set. Add 10 ml of 25% strength of disinfectant solution as per manufacturer 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.

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QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number 7.5.3.10 7.5.3.11 7.5.3.12 7.5.3.13 7.5.3.14 7.5.3.15 7.5.3.16 7.5.3.17 7.5.3.18 7.5.3.19 7.5.3.20 7.5.3.21 7.5.3.22 7.5.3.23 7.5.3.24 7.6 Disinfectant Efficacy Validation Effective Date

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Arrange the sterile filter holders having 0.45 m membrane filters on the manifold and assemble the manifold to 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 individual filter holder for each tube. This is 0 min contact sample. Filter the solution meant for 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. After 5 minutes, mix and filter the solutions of 2nd set using 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 3 rd set using 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 point 5.1.1. Use same agar medium and incubation conditions as mentioned in point no. 7.4 - 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 7.5.4 Purpose To get a practical approach for the efficacy apply the disinfectants on to any of the surface which is present in that particular area which will be decontaminated by spraying the disinfectant. 7.5.5 7.5.6 Requirement: SS strip, Epoxy Coated strip, sterile swab. Procedure: 7.5.3.1 7.5.3.2 7.5.3.3 7.5.3.4 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 0 C for 1 hrs. Prepare different concentrations of the disinfectant solution as mentioned in the Use dilution method.

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QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number 7.5.3.5 7.5.3.6 7.5.3.7 7.5.3.8 Disinfectant Efficacy Validation Effective Date

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7.5.3.9 7.5.3.10 7.5.3.11 7.5.3.12 7.5.3.13 7.5.3.14 7.5.3.15 7.5.3.16 7.5.3.17 7.5.3.18 7.5.3.19 7.5.3.20 7.5.3.21 8.0

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 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 point 5.1.1. 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: 8.1 The reduction of Challenge Inoculum count should be more than 3 log for bacterial cultures.

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QUALITY CONTROL DISINFECTANT QUALIFICATION Protocol Name Protocol Number 9.0 OBSERVED DEVIATION (IF ANY): Disinfectant Efficacy Validation Effective Date

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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 QA006 Deviation Process. 10.0 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 / exhibit which were completed at the time of qualification activity. 11.0 ABBREVIATIONS: 11.1 11.2 11.3 11.4 11.5 SCDA RCA LAF SCA CFU : : : : : Soyabean Casein Digest Agar Reinforced Clostridial Agar Laminar Air Flow Sabouraud Chloramphenicol Agar Colony Forming Unit

12.0 LIST OF ANNEXURES: Annexure No. 01 02 03 04 05 Annexure Title Training Record Report for Bacillus subtilis Culture Report for Preparation of Challenge inoculum Report for Disinfectant Efficacy test by Use Dilution method. Report for Disinfectant Efficacy test by surface method.

13.0 REFERENCE DOCUMENT: Nil

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