Report for Disinfectant Efficacy Validation

TABLE OF CONTENTS

1. Summary and Results
2. Objective
3. Scope
4. Validation Team Responsibility
5. Procedure
6. Validation Procedure
7. Acceptance Criteria
8. Training
9. Summary and Report
10. Conclusion and Remarks
11. Post-Approval

1. SUMMARY AND RESULTS

Sr. No.	Validation Parameter	Acceptance Criteria	Results
1.	Disinfectant efficacy validation of XYZ using dilution method - 0.5%, 2.5%, 5%	There should be at least a 90% population reduction between the initial challenge dilution and the recovered CFU	Overall, there was a population reduction of between 78.3% and 97.6% for concentrations 0.5% and 5% at contact times 5 and 10 minutes respectively.
2.	Disinfectant efficacy validation of XYZ using contact method – 1.5%, 2.5%, 5%	There should be at least a 90% population reduction between the initial challenge dilution and the recovered CFU	Overall, there was a population reduction of between 74.5 % and 95.1% for concentrations 0.5% and 5% at contact times 5 and 10 minutes respectively.
XYZ are effective disinfectant agents at 0.5% for 5 minutes, and 5% for 5 minutes respectively.
XYZ of make PharmaGuide Ltd stand validated as disinfectants and can be used for effective and routine disinfection purposes.

1. OBJECTIVE

To prepare the report for the disinfectant efficacy validation of XYZ that was done using different concentrations against the acceptance criteria as per Protocol No: PG/DV/VP/001-01.

2. SCOPE

This is a report of the efficacy of the disinfectants to be used for the cleaning of the Production Area and the Microbiology Laboratory at PharmaGuide Limited. It covers the methodology that was employed to determine the efficacy, the data that was generated and an evaluation and summary of the said analytical data to conclude that the disinfectants, which will be used in rotation are effective.

3. VALIDATION TEAM RESPONSIBILITY

Functional Department	Name	Designation	Responsibility
Quality Control			Reporting of the activity Evaluation and submission of the results.
Quality Assurance			Review and approval of report
Production			Checking / Evaluation of documents and co-ordination.

4. PROCEDURE

4.1 Description of the Study

This validation was done using two methods, that is, the dilution method and contact or template method. It involved the validation of XYZ at different concentrations and contact times to determine their efficacy as disinfectants. This validation was done as per protocol PG/DV/VP/001-01 and the methodology and results are described in this report.

4.2 Details of the Requirements

Name of Disinfectant	Batch No.	Active Ingredient	Expiry Date	Manufacturer
XXX
YYY

ALSO READ: SOP for Efficacy Test Procedure of Antimicrobial Preservatives

Challenge Organisms Used	ATCC / NTCC No.
Escherichia Coli	ATCC 8739
Staphylococcus Aureus	ATCC 6538
Salmonella Abony	NCTC 6017
Pseudomonas Aeruginosa	ATCC 9027

Equipment	Identification No.
Praano Autoclave
Sledoc Autoclave
pH Meter
Analytical Balance
Colony Counter
Biosafety Cabinet
Vacuum Pump
Incubators

Culture Media Used	Lot No.	Validity
Soyabean Casein Digest Agar
Peptone Water
Normal Saline
Sabouraud Dextrose Agar

All the templates and glassware were sterilized before use.

ALSO READ: Sterility Test Validation Protocol

4.3 Preparation of Disinfectant concentrations

XXX was prepared in different concentrations using pre-sterilized purified water. The details of preparation, the concentration and amounts used are recorded in Annexure 01.

4.4 Preparation of Challenge Organisms

The challenge organisms were prepared and quantified according to SOP and the preparation details recorded in Annexure 02. Stable suspensions containing between 10-100 CFU/ml of each organism were selected as dilutions to use. From them, back calculation was done to estimate the culture population in the initial stock and then one that yielded 1 x 108 was selected.

4.5 Preparation of Test Kits and Glassware

All the test kits and glassware were sterilized prior to the validation activity.

5. VALIDATION PROCEDURE

All validation activities were performed under the Biosafety Cabinet while following aseptic techniques.

5.1 Dilution Method

Positive Product Control

• 10 ml of disinfectant solution of each concentration was dispensed into sterile test tubes in duplicate. 1 ml of the chosen inoculum of challenge organism was inoculated into each test tube separately. One of the duplicate concentrations was held for a contact time of 5 minutes while the other was held for 10 minutes.
• After the specified contact time, a sterile 0.45 µm membrane was taken and pre-wetted with about 10 ml of pre-sterilized 0.1% peptone water. The prepared disinfectant solution was filtered through the sterilized membrane which was then rinsed with 3 x 100ml of pre-sterilized 0.1% peptone water to remove traces of the disinfectant solution on the membrane.
• The filtered membrane was then placed on the surface of an agar plate. This was repeated for all the concentrations and all challenge organisms. The agar plates with the membrane were incubated as per the type of challenge microorganism. Bacterial challenged filter membranes were incubated at 30-35ºC for 48-72 hours and Yeast challenged filter membranes at incubated at 20-25ºC for 5-7 days.

Positive Control

• For positive control, 1 ml of each chosen dilution of challenge organism was inoculated into 100 ml of 0.1% peptone water and the entire solution was held for maximum contact time. After the specified contact time, a sterile membrane was taken and pre-wetted with at least 10 ml of pre-sterilized 0.1% peptone water before the inoculated solution was mixed and filtered through.
• The membrane filter was then be rinsed with 3 x 100ml of 0.1% peptone water, placed on agar plate as per the organism and incubated. The Bacterial challenged membranes were put on SCDA and incubated at 30-35℃ for 48 to 72 hours while Yeast challenged membranes were put on SDA plates and incubated at 20-25℃ for 5 to 7 days.

Negative Control

• 10ml of pre-sterilized 0.1% peptone water was held for maximum contact time after which a sterile membrane was taken and pre-wetted with at least 10ml of pre-sterilized 0.1% peptone water. The 0.1% peptone water was then filtered through the sterilized membrane.
• The membrane was rinsed with 3 x 100 ml of 0.1% peptone water and placed on the surface of an SCDA plate which was incubated at 30-35℃ for 48-72 hours. Another negative was prepared in a similar way and the membrane was placed on an SDA plate then incubated at 20-25℃ for 5-7 days.
• After completion of the incubation periods, the positive product control, positive control and negative control plates were observed. The population reduction was calculated and recorded in Annexure 03.
• The population reduction was calculated as per the following formula;

ALSO READ: Microbial Limit Test Validation Protocol

5.2 Contact Method (Template Method)

Positive Product Control

• The effective concentrations of each disinfectant from the dilution method were used for the contact method.
• Templates of 10 x 10cm were taken and inoculated in with 1 ml of the challenge organism, which was then spread over with an L-spreader and allowed to dry in the Biosafety Cabinet. The disinfectant was then sprayed uniformly onto the template and was allowed to stand for contact time as determined in dilution method.
• After that, a sterilized swab was used to swab the template in overlapping vertical and horizontal strokes and then collected into a test tube containing 0.9 % sterilized saline solution (10 ml). The swab sample was agitated slowly to dislodge and disperse the cells, then filtered through a pre-wetted sterile 0.45µm filter membrane and rinsed with 3 x 100 ml of 0.1% peptone water to remove traces of the disinfectant solution on the membrane.
• The filtered membrane was then placed on the surface of an SCDA plate. This was repeated for all the disinfectants and challenge organisms. Bacterial challenged filter membranes were incubated at 30-35℃ for 48-72 hrs and Yeasts challenged filter membranes on SDA plates were incubated at 20-25℃ for 5-7 days.

Positive control

• Templates of 10 x 10cm were taken and inoculated with 1 ml of the challenge organism which was then spread with an L-spreader and allowed to air dry under the Biosafety cabinet.
• After the maximum contact time, a sterilized swab was used to swab the template in overlapping horizontal and vertical strokes and collected test tubes containing sterilized saline solution.
• The swab sample was agitated slowly to dislodge and disperse the cells and was filtered through sterile 0.45µm membrane and then finally rinsed with 3 x 100 ml of pre-sterilized 0.1% peptone water and placed on agar plates as per the organism and incubated. The filtered Bacterial challenged filter membranes were put on SCDA plates and incubated at 30-35℃ for 48-72 hours while the Yeast challenged filter membranes were put on SDA plates and incubated at 20-25℃for 5-7 days.

Negative Control

• One template 10 x 10cm was selected and held for specific contact time. The Template was swabbed to ensure the Bioburden of the template, the swab sample was filtered and rinsed with 3 x 100 ml of 0.1% peptone water and placed on an SCDA plate which was incubated at 30-35℃ for 48 hrs.
• Another negative control was prepared in a similar way and the membrane was placed on an SDA plate then incubated at 20-25℃ for 5-7 days. After completion of the incubation periods, the positive product control, positive control and negative control plates were observed. The population reduction was calculated and recorded in Annexure 04.
• The population reduction was calculated as per the following formula;

6. ACCEPTANCE CRITERIA

• The minimal concentration of the disinfectant with the lowest contact time that was able to demonstrate a minimum 90% population reduction of the challenged organisms was accepted.
• The negative controls of all the tests and media were sterile and showed no evidence of microbial growth.

7. TRAINING

The personnel involved in the validation activity were trained as per the protocol PG/DV/VP/001-01.

8. SUMMARY

The activities were performed as per the protocol PG/DV/VP/001-01 and observations, their evaluation and summary recorded in this report. The validation was done using three different disinfectants and the minimal concentrations of the said disinfectants that yielded at least a 90% population reduction at the lowest contact were accepted for use.

9. DEVIATIONS / FAILURES

No deviations / failures were observed during this disinfectant efficacy validation.

10. CONCLUSION AND REMARKS

XYZ are effective as disinfectants at 0.5 % and 5 % respectively for contact times of 5 minutes.

11. POST-APPROVAL

The post-approval of this Protocol for Validation was the joint responsibility of the following functional areas.

ALSO READ: Disinfectant Efficacy Validation Protocol