SOP for Disinfectant Validation

Objective

The objective of this validation 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 concentrations and contact times 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 SOP defines the procedure to be followed, for validating the sanitizers and the sanitization procedure being followed in the manufacturing and the testing facility.
• The same established data should be used in further routine usage.

Responsibilities

1. Quality control:

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

2. Quality Assurance:

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

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Documents Required

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

Validation Policy and Plan

1. 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 a 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 into 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, that 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 the above step 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.

2. Pour Plate Method

• Take 1.0 ml from each dilution and transfer in to sterile Petri plates 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.

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3. Preparation of the Disinfectant Solution

• Prepare the disinfectant solution in Purified Water according to the manufacturer's 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 a 0.2 μ-membrane filter.
• Distribute 1.0 ml of the filtered disinfectant solution into sterile test tubes.
• In case dilution is not recommended by the manufacturer then disinfectant is to be validated only on manufacturer-recommended concentration.

4. Validation of the Neutralization Method

The neutralization method used in the disinfectant validation study must be initially validated using the following procedures as mentioned below:

A.Test Control Group:

• Filter 1.0 ml of the disinfectant solution (prepared as per section 3) 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 culture 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.

B. 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 the 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 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.

C. Negative Control Group

• Filter 300 ml of 0.1 % sterile peptone water through a 0.45 m membrane filter in duplicate.
• After filtration transfer one membrane on the Soyabean casein digest agar medium plate and the other on the 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.

6. 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 achieved for all the culture organisms.
• In the Negative control group, no colonies should be observed

7. Determination of the Efficacy of the Disinfectant by Use Dilution Method

Determination of the Efficacy of the Disinfectant by Use Dilution Method

A. Test Control

• Transfer 1.0 ml of diluted disinfectant solution (prepare the disinfection as per above) in each of the 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 of 1 minute to 1st 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 to 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 that have the 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.

B. 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 cultureSabouraud 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.

C. Negative Control

• Transfer 1.0 ml of diluted disinfectant solution (prepare the disinfection as per the section above) in each of two sterile test tubes and add 1.0 ml of 0.9 % sterile normal saline in each test tube separately.
• Transfer the content of both test tubes in a sterile Petri plate.
• After transferring the sample on a 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.

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Interpretation of Results

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 the 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

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

8. Determination of the Efficacy of the Disinfectant by Surface Challenge Method

• Prepare the disinfectant solution as per the recommended 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 -30cm2 size as Test control, Positive control, and Negative control separately.
• Clean all the surfaces with sterile WFI and sterilize these surfaces in the autoclave.
• After sterilization spread 1.0 ml of challenge inoculum having a 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-spread surfaces along with unsprayed surfaces (Negative control) to dry under LAF.
• Take a lint-free sterile duster and dip/soak in the disinfectant solution of the recommended dilution.
• Squeeze the above dipped/soaked duster for the removal of excess disinfectant solution.
• Wipe the Nichomac surface, S.S Surface, and Epoxy coated surface marked as Test (Spread with challenge inoculum having a population between 105 - 106 CFU /ml) with the help of a squeeze duster in one direction for 3 - 4 times.
• Allow the surfaces to dry for 10 minutes.

A. 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 into 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 it 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.

B. 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 up and down followed by right and left directions 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 into another tube containing 10 ml 0.1% Sterile peptone water.
• Gently vortex the tube containing the 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 SabouraudChloramphenicol 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 it 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.

C. 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 up and down followed by right and left directions 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 into a tube containing 10 ml 0.1% Sterile peptone water.
• Gently vortex the tube containing the 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 SabouraudChloramphenicol 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 and22.5 ± 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

1. In the Test control, five (5) Log reductions or greater should be achieved using the recommended disinfectant concentration (use dilution) at 10 10-minute exposure period.
2. In the Positive control, at least 70 % recovery should be achieved for all the culture organisms.
3. In the Negative control, no colonies should be observed.

9. Frequency of validation \ Revalidation

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

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