Disinfectant Validation in pharmaceuticals Microbiology lab
1. Objective:
The objective of this document is to provide guidelines for disinfectant Validation performed at QC microbiology laboratory. This test is applied to determine the disinfectants concentration and time required to inhibit or kill the microorganisms during cleaning.
2. Scope:
This will be applicable to all the disinfectant solutions used in Microbiology Lab.
This will be applicable to all the disinfectant solutions used in Microbiology Lab.
3. Responsibility:
Quality Control : a) To prepare and review Qualification Protocol and to monitor execution as per Qualification Protocol
b) To prepare and review Qualification Report as per Executed Qualification Protocol.
Quality Assurance:a) To approve Qualification Protocol and Report after review it as per Reference concept.
4. Procedure:
4.1 List of Culture to be used in this Validation:
Name of Culture
|
Strain No.
|
Classification
|
Escherichia coli
|
ATCC 8739
|
Gram negative
Vegetative Bacteria
|
Pseudomonas aeruginosa
|
ATCC 9027
|
Gram negative Vegetative Bacteria
|
Salmonella abony
|
NCTC 6017
|
Gram negative Vegetative Bacteria
|
Staphylococcus aureus
|
ATCC 6538
|
Gram positive Vegetative
Bacteria
|
Candida albicans
|
ATCC 10231
|
Vegetative yeast
|
Aspergillus niger
|
ATCC 16404
|
Fungal Spore
|
Bacillus subtilis
|
ATCC 6633
|
Gram
positive
Spore forming Bacteria
|
4.2 List of Disinfectants:
Sr. No.
|
Name of Disinfectant
|
Recommended Dilution
|
1
|
Germinil
|
1.0
% v/v
|
2
|
Iso Propyl Alcohol
|
70 % v/v
|
3
|
Dettol
|
2.5 % v/v
|
4
|
Savlon
|
2.5 % v/v
|
5
|
Sterilium
|
Ready to use
|
4.3 Prepare all the required media as Per SOP for Media Preparation.
4.4 Preparation and Standardization of Inoculum of Challenge organism as per SOP for culture dilution preparation
4.5 Disinfectant Challenge Testing :
4.5.1 To demonstrate the efficacy of a disinfectant within a pharmaceutical manufacturing environment, it is necessary to conduct the following tests:
4.5.2 Preparation of Disinfectants solution :
4.5.2.1 Measures require quantity of purified water with measuring cylinder and transfer half of the quantity in to a plastic container.
4.5.2.2 Take require quantity of active disinfectant in a measuring cylinder and transfer it to plastic container containing purified water. Mix it thoroughly by stirring.
4.5.2.3 Add remaining quantity of the purified water to make up the final volume.
4.5.2.4 Record the disinfectant prepared in Format.
4.5.2.5 Prepare disinfectant solution of 3 different concentrations for each disinfectant as per table below:
Sr. No.
|
Name of Disinfectant
|
1st Dilution
|
2nd Dilution
|
3rd Dilution
|
1
|
Germinil
|
0.5
% v/v
|
1.0
% v/v
|
1.5
% v/v
|
2
|
Isopropyl Alcohol
|
35 % v/v
|
70 % v/v
|
100 % v/v
|
3
|
Dettol
|
1.25 % v/v
|
2.5 % v/v
|
5.0 % v/v
|
4
|
Savlon
|
1.25 % v/v
|
2.5 % v/v
|
5.0 % v/v
|
5
|
Sterilium
|
As it is
| ||
4.5.3 Efficacy of working concentration and contact time (Dilution Test):
4.5.3.1 To screen the disinfectant for their efficacy at various concentrations and contact time using a wide range of standard test organisms.
4.5.3.2 Mark a set of tubes of each disinfectant concentration, with individual organism for 0, 5 & 10 minute.
4.5.3.3 Dispense 10 ml of different disinfectant concentration solution.
4.5.3.4 Add equal quantity of inoculum shows 10^3 cfu into each disinfectant concentration solution & mix them thoroughly.
4.5.3.5 Incubate the inoculated disinfectant concentration tubes at 20-25°C for 5 min. & 10 min. as mentioned on the tubes.
4.5.3.6 At the 0 min. interval from the time of inoculation of the culture take the sample and filter it through the filtration assembly aseptically through 0.45μm membrane filter. Wash the membrane filtration unit with 3 x 15 ml of Buffered peptone water.
4.5.3.7 Aseptically transfer the membrane filter to the 30 ml of sterile Soyabean Casein Digest Medium and incubate at 30-35°C for 5 days. For fungal check use 30 ml of sterile Sabouraud Dextrose Broth and incubate at 20-25°C for 5 days.
4.5.3.8 Repeat the above procedures for rest of the organism and different disinfectant concentration and contact times for 5 min. and 10 min.
4.5.3.9 After completion of incubation record the observation of growth in Record.
4.5.3.10 Observe the growth in the form of turbidity.
4.5.3.11 In case of failed above all concentration increase the concentration and repeat the above test.
4.5.3.12 Acceptance Criteria: At least one of the concentrations of each disinfectant solution should be able to give 3-log reduction for each organism (No growth should be observed).
4.5.4 Efficacy of working concentration and contact time at working area (Surface Challenge Test):
4.5.4.1 Use the selected dilution of each disinfectant, studied in the earlier method for the surface challenge study. Use the surfaces such as Vinyl flooring, SS sheet, Tiles.
4.5.4.2 Apply equal quantity of inoculum of the challenge organism shows 10^3 cfu to 5 X 5 cm2 area of the selected surface at three locations to cover 0 min., 5 min. and 10 min. contact time.
4.5.4.3 Clean the surface with selected dilution of disinfectant using mop. After mopping take the swab for each 0 min., 5 min. and 10 min. contact time for each organism and put it in 10 ml of sterile buffered peptone water and filter it using the filtration assembly aseptically through 0.45μm membrane filter. Wash the membrane filtration unit with 3 x 15 ml of Buffered peptone water.
4.5.4.4 Aseptically transfer the membrane filter to the 30 ml of sterile Soyabean Casein Digest Medium and incubate at 30-35°C for 5 days. For fungal check use 30 ml of sterile Sabouraud Dextrose Broth and incubate at 20-25°C for 5 days.
4.5.4.5 Repeat the above procedures for rest of different surfaces.
4.5.4.6 Acceptance Criteria: The Efficacy of the Working Concentration should be able to give 3-log reduction for each organism at particular surface (No growth should be observed).
4.5.5 Efficiency check for disinfectant solution during Hold time (Hold time study):
4.5.5.1 Freshly prepare the effective concentration of each disinfectant.
4.5.5.2 Use the resistant bacteria/ fungi found in the part 4.5.3 and Part 4.5.4 for the hold time efficiency of the disinfectant study.
4.5.5.3 Study it for time intervals at the 1 hrs, 6 hrs, 9 hrs, 24 hrs, 28 hrs intervals from the time of the preparation of disinfectant.
4.5.5.4 Take 10 ml of he selected dilution of the disinfectant in the sterile test tube.
4.5.5.5 Add equal quantity of inoculum shows 10^3 cfu into each disinfectant concentration solution & mix them thoroughly.
4.5.5.6 At the 1 hour interval from the time of preparation of the disinfectant filter the sample through the filtration assembly aseptically through 0.45μm membrane filter. Wash it with 3x 15 ml of Buffered peptone water.
4.5.5.7 Aseptically transfer the membrane filter to the 30 ml of sterile Soyabean Casein Digest Medium and incubate at 30-35°C for 5 days. For fungal check use 30 ml of sterile Sabouraud Dextrose Broth and incubate at 20-25°C for 5 days.
4.5.5.8 Similarly follow the same procedure for different time intervals stated above.
4.5.5.9 In case of failed in above all surfaces increase the concentration and repeat the above test.
4.5.6 Acceptance Criteria: The effective Concentration of disinfectant should be able to give 3-log reduction at each time interval (No growth should be observed).
Negative control: Filter 10 ml sterilized Buffered peptone water through 0.45 μm membrane filter for negative control. Wash the filter with 30 ml buffered peptone water. Aseptically transfer the membrane filter to the 30 ml of SCDM and incubate at 30-35° for 5 days. For fungal check use 30 ml of sterile SDB and incubate at 20-25°C for 5 days.
5. Revalidation Criteria
Requalification shall be carried out through change control procedure in following cases if:
· Introduction of new disinfectant.
· Change in Concentration of existing disinfectant
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