Validation Protocol for Efficacy of Chemical Disinfectants

1.0 Objective

This document describes the testing procedure for the efficacy of different types of chemical disinfectants.

2.0 Scope

This document provides the procedure for validating the sanitizers and the sanitization procedure being followed in the manufacturing and the testing facilities in pharmaceuticals.

3.0 Reference Document

SOP for microbiological culture media preparation
Disinfectant Efficacy Test

4.0 Pre-Requisites

Prior to conducting/executing the General validation protocol following things  must be available

4.1  Microbial Standard Cultures

4.1.1  Bacillus subtilis

4.1.2  Escherichia coli

4.1.3  Staphylococcus aureus

4.1.4  Candida albicans

4.1.5  Aspergillus niger

4.1.6  Environment isolates

4.2  Disinfectants:  All disinfectants used in the facility       

4.3  Micropipettes

4.4  Sterilized tips and Petri plates

5.0 Responsibility

Responsibilities of Quality Assurance and Quality control Microbiology personnel involved in activities related to the General validation protocol are defined below

5.1  Quality Assurance (Validation)

5.1.1  Approval of protocol

5.2  Quality Control

5.2.1  Preparation of protocol

5.2.2  Review of protocol

5.2.3  Execution of protocol

6.0 Validation Method

Qualification Tests

A)  Suspension method (Validation of sanitizer)

B)  Surface spray/immersion or wipe method (Validation of Sanitization method)

6.1 Suspension Method

6.1.1 Objective:

To establish the test concentration and the contact time a suspension test is generally applied. The suspension test estimate the in vitro bactericidal activity of the disinfectant under precise experimental conditions including

•  Microbial strain

•  Preparation of inoculum

•  Volume of inoculum vs. Disinfectant

•  Temperature

•  Disinfectant concentration and contact period

•  Interfering substances (i.e. inorganic, organic matter)

6.1.2 Procedure:

6.1.2.1  Prepare the culture suspension from the original culture as per the SOP for preparation of Microbiological culture media.

6.1.2.2  Select the dilution which will yield 10⁵ to 10⁶ cells per ml.

6.1.2.3  Prepare 10 ml of test dilution to be tested with sterile distilled water.

6.1.2.4  Vortex the tube for 1 minute.

6.1.2.5  Add 0.1 ml of any one culture containing 10⁵ to 10⁶ cells per ml into the test disinfectant with the decided concentration.

6.1.2.6  The final concentration shall be 10⁴ to 10⁵ cells per the tube. Consider the preparation for 0 min.

6.1.2.7  Prepare like the same above for other 3 different sets for 3 different time intervals.

6.1.2.8  Give a contact time of 0 min, 5 min, 10 min and 15 min.

6.1.2.9  After the specified contact time, filter the samples through a 0.45 m membrane filter.

6.1.2.10  Give three washing of 100 ml each with 0.1 % sterile peptone water/ Sterile Water.

6.1.2.11  After filtration with the help of a sterile forceps take the membrane filter and place it on a Soybean casein digest agar.

6.1.2.12  Incubate the bacterial culture at 30-35 °C for 24 to 48 hours and fungal cultures at 20-25 °C for 72 to 120 hours.

6.1.2.13  After incubation count the number of colonies present on the membrane.

6.1.2.14  Note down the number of colonies.

6.1.2.15  This will be the Observed count after the exposure.

6.1.2.16  Select the plates, which have least to Nil counts.

6.1.2.17  Proceed in the same manner taking all the cultures to be tested.

6.1.2.18  Contact time for the usage of the disinfectant will be set on the basis of the results, which will have least counts.

6.2 Surface spray / Wipe method
6.2.1 Objective:

To establish the effectiveness of the test concentration and the contact time generally applied. The suspension test estimates the in vitro bactericidal activity of the disinfectant under precise experimental conditions including.

•  Microbial strain

•  Preparation of inoculum

•  Volume of inoculum vs. Disinfectant

•  Temperature

•  Disinfectant concentration and contact period

•  Interfering substances (i.e. inorganic, organic matter)

With the Spray/immersion or wipe method the following surfaces shall be taken for validation.

Stainless steel (SS)

•  Epoxy

•  Panel

•  PU Paint wall (Poly Urethane Paint)

6.2.2 Procedure:

6.2.2.1  Prepare the culture suspension from the original culture as per the SOP for preparation of Microbiological culture media.

6.2.2.2  Select the dilution which will yield 10⁴ to 10⁵ cells per ml.

6.2.2.3  Take plate of different surfaces such as SS, Epoxy, Panel, PU Paint wall present in the clean room having a surface area of 25 cm²

6.2.2.4  From the previously determined suspension having 10⁴ to 10⁵ cells per ml inoculate one culture on different surfaces mentioned above.

6.2.2.5  With the help of a sterile spatula spread the culture on the surface.

6.2.2.6  Keep it on the LAF bench for drying.

6.2.2.7  After the exposed duration for drying (a) spray the disinfectant (b) disinfect the surface by wipe method.

6.2.2.8  Give a contact time of 0 min, 5 min, 10 min and 15 min.

6.2.2.9  The exact procedure for sanitation followed in the clean room should be followed.

6.2.2.10  With the help of a sterile moistened swab, swab the surface gently covering all the area of the surface.

6.2.2.11  Place the swab sticks in a test tube having a sterile saline solution and do not hold the swab more than 24 hours.

6.2.2.12  Vortex the test tube gently for 5.0 minutes.

6.2.2.13  Aseptically filter the samples through a 0.45 m membrane.

6.2.2.14  Give three washing of 100 ml each with 0.1 % sterile peptone water / Sterile water.

6.2.2.15  After the filtration with the help of a sterile forceps take the membrane and place it on a Soybean casein digest agar.

6.2.2.16  Incubate the bacterial culture at 30-35 °C for 24 to 48 hours and fungal cultures at 20-25 °C for 72 to 120 hours.

6.2.2.17  After incubation count the number of colonies present on the membrane.

6.2.2.18  Proceed in the same manner taking all the cultures to be tested with the all mentioned disinfectants.

7.0 Acceptance criteria

7.1  The decrease in the bacterial load to the exposed disinfectant indicates that the disinfectant is capable of reducing the contaminant when used in the area. That shall be minimum of 4-log reduction for non-spore forming microorganisms, Yeast and minimum of 3-log reduction shall achieve for Spore-forming organisms, molds with the decided concentration.

7.2  Determine the contact period where the above said population log reduction of microorganisms achieved.

8.0 Observations

8.1  Record the observation of Suspension method in Annexure –I

8.2  Record the observation of Surface spray / Wipe method in Annexure –II

9.0 Test Process Flow Chart
9.1 Test for Suspension method

9.2 Test for Surface spray / Wipe method

10.0 Validation Matrix

Following test matrix is prepared for the initial analytical method validation and revalidation criteria for Efficacy of disinfectants.

Sr. No.	Test Description	Initial Validation	Revalidation
1	Suspension Method	All disinfectants used	Every new disinfectant
			Change in Disinfectant Concentration
			Change in Contact time
2	Surface Spray / Wipe Method	All disinfectants used	Every new disinfectant
			Change in Disinfectant Concentration
			Change in Contact time

11.0 Abbreviations

SOP: Standard Operating Procedure

° C: Degrees centigrade

% : Percentage

CFU: Colony forming units

SCA: Soybean casein digest agar

m: Micron

PU: Poly Urethane

Annexure –I

Results: Suspension Method

Name of the Disinfectant:
	Log reduction observed with Contact period (in Min.)
Name of the organism	0	5	10	15	Significant log reduction observed at
Bacillus subtilis
Escherichia coli
Staphylococcus aureus
Candida albicans
Aspergillus niger
Environmental isolate

Annexure –II

Results: Surface Spray / Wipe Method

Surface Selected:                                 Name of the Disinfectant:
Name of the organism	Log reduction observed with Contact period (in Min.)
	0	5	10	15	Significant log reduction observed at
Bacillus subtilis
Escherichia coli
Staphylococcus aureus
Candida albicans
Aspergillus niger
Environmental isolate

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