Isopropyl alcohol 70% is one of the most commonly used disinfectants and the pharmaceutical industry. It is used for surface disinfection, equipment sanitization, hand decontamination in cleanrooms and aseptic processing areas.
It is necessary to validate the shelf life of 70% isopropyl alcohol to ensure effectiveness. It is done to know how long a stored IPA solution remains effective when stored. This post will explain the principal, requirements and microbiological methods used for shelf life validation of 70% IPA solution.
IPA solution disinfects the surface by denaturing proteins and dissolving lipids present in microbial cell membranes. It had its optimum microbial activity when diluted with purified in 70:30 ratio. After some time of storage alcohol concentration may drop in solution due to evaporation of alcohol reducing its disinfection efficiency. Validation of IPA solution helps to determine the duration for which the prepared 70% IPA solution remains effective.
Mix the solution well and label the container with:
At each time interval, evaluate the disinfectant efficacy using both methods described below.
1. Surface Swab Method
2. Settle Plate Method
Remove following culture slant from the refrigerator and allow it to attain room temperature.
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
Salmonella, and
Wild culture
2. Add 1 ml of the broth culture into 9 ml of sterile saline solution (0.9% sodium chloride solution) to obtain a test dilution of 10-1.
3. Transfer 1 ml of the 10-1 dilution into 9 ml of sterile saline solution to give 10-2 dilution.
4. Similarly serially dilute the culture suspension to obtain dilutions of 10-3, 10-4, 10-5 and 10-6
5. Take 1 ml of the culture suspension from dilutions 10-3, 10-4, 10-5 and 10-6 in duplicate into sterile petridishes.
6. Pour approximately 15 to 20 ml of sterile Soyabean Casein Digest Agar cooled to about 45°C in each plate. Incubate petri plates at 32.5 ± 2.5°C for 24 to 48 hours.
7. Count the number of colonies on each plate and select the dilution, which gives a count of not less than 105.
8. Apply 1 ml of each culture suspension containing cell concentration not less than 105 cfu/ml, separately on the floor approx 25 cm2 area, and allow to air dry.
9. After drying, take a surface swab as per latest SOP for Swab Testing, and carry out the determination of total aerobic count per cm2 within 4 hours of sampling.
10. Immediately clean the floor with IPA 70% v/v solution and allow to stand for 30 minutes to facilitate the action of the disinfectant solution on the challenge test organisms.
11. After 30 minutes, take a swab and detect the bacterial count as per SOP for swab testing.
12. Repeat the same procedure, steps no. 9 to 12 to determine the efficacy of 70% IPA solution after 24 hrs, 48 hrs, 72 hrs and 96 hrs at room temperature.
Log Reduction = log10(N0) − log10(N)
Where:
N0 = number of organisms before disinfection
N = number of organisms after disinfection
2. After plate exposure time, immediately clean the floor with IPA 70% v/v solution.
3. After proper cleaning, spray the area with IPA 70 % v/v solution and immediately close the room for 30 minutes to facilitate the action of disinfectant solution.
4. After 30 minutes of contact time, immediately expose the plate as per latest SOP for plate exposure and detect the bacterial count per location per 2 hours.
5. Repeat the same procedure, steps no. 1 to 4 to determine the efficacy of IPA 70% v/v solution after 24 hrs, 48 hrs, 72 hrs and 96 hrs at room temperature
Validation of shelf life of 70% IPA is a critical GMP requirement in pharmaceutical manufacturing facilities. By performing validation using both the surface web method and settle plate method, manufacturers can confirm that the 70% IPA maintains its antimicrobial property throughout its use.
Generally, a freshly prepared 70% IPA solution remains effective for 96 hours if it is stored in a clean and closed container at controlled temperature. Some pharmaceutical facilities assign longer or shorter shelf lives based on their own validation data.
It is necessary to validate the shelf life of 70% isopropyl alcohol to ensure effectiveness. It is done to know how long a stored IPA solution remains effective when stored. This post will explain the principal, requirements and microbiological methods used for shelf life validation of 70% IPA solution.
Principle of 70% IPA Disinfection
IPA solution disinfects the surface by denaturing proteins and dissolving lipids present in microbial cell membranes. It had its optimum microbial activity when diluted with purified in 70:30 ratio. After some time of storage alcohol concentration may drop in solution due to evaporation of alcohol reducing its disinfection efficiency. Validation of IPA solution helps to determine the duration for which the prepared 70% IPA solution remains effective. Materials and Equipment
In order to efficiently conduct validation of diluted disinfectant storage conditions and period, ensure that the following requirements are fulfilled.- 99% Isopropyl Alcohol (IP/BP/USP grade)
- Purified Water or WFI
- Clean stainless-steel or glass mixing vessel
- Sterile containers or spray bottles for storage
- Soybean Casein Digest Agar (SCDA)
- Sterile swabs
- Validated Laminar air flow
- Calibrated Incubators
- Approved Test Methods for Swab testing and Plate Exposure
- Test Organisms – E. coli, Salmonella abony, Staph. aureus, C. albicans, and Environmental isolate (wild culture)
Preparation of 70% v/v IPA Solution
Add 700 ml isopropyl alcohol and 300 ml purified water using a clean and calibrated measuring cylinder into a clean container.Mix the solution well and label the container with:
- Name: 70% v/v isopropyl alcohol
- Date of preparation:
- Batch number:
- Use before date:
- Prepared by / Signature:
- Verified by / Signature:
Validation Duration
Perform validation tests at interval of 0 hr (just after solution preparation), 24 hrs, 48 hrs, 72 hrs and 96 hrs.At each time interval, evaluate the disinfectant efficacy using both methods described below.
1. Surface Swab Method
2. Settle Plate Method
Method 1: Surface Swab Method
A defined surface area, usually 25 cm2 is knowingly contaminated with known population organisms. After treatment 70% IPA solution, samples are collected using sterile swabs and count is determined to know the reduction percentage of microbial load.Remove following culture slant from the refrigerator and allow it to attain room temperature.
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
Salmonella, and
Wild culture
Procedure
1. Using a sterile loop, inoculate bacterial culture from each slant separately into 50 ml of sterile Soyabean casein digest medium and incubate at 32.5 ± 2.5°C for 24 to 48 hrs.2. Add 1 ml of the broth culture into 9 ml of sterile saline solution (0.9% sodium chloride solution) to obtain a test dilution of 10-1.
3. Transfer 1 ml of the 10-1 dilution into 9 ml of sterile saline solution to give 10-2 dilution.
4. Similarly serially dilute the culture suspension to obtain dilutions of 10-3, 10-4, 10-5 and 10-6
5. Take 1 ml of the culture suspension from dilutions 10-3, 10-4, 10-5 and 10-6 in duplicate into sterile petridishes.
6. Pour approximately 15 to 20 ml of sterile Soyabean Casein Digest Agar cooled to about 45°C in each plate. Incubate petri plates at 32.5 ± 2.5°C for 24 to 48 hours.
7. Count the number of colonies on each plate and select the dilution, which gives a count of not less than 105.
8. Apply 1 ml of each culture suspension containing cell concentration not less than 105 cfu/ml, separately on the floor approx 25 cm2 area, and allow to air dry.
9. After drying, take a surface swab as per latest SOP for Swab Testing, and carry out the determination of total aerobic count per cm2 within 4 hours of sampling.
10. Immediately clean the floor with IPA 70% v/v solution and allow to stand for 30 minutes to facilitate the action of the disinfectant solution on the challenge test organisms.
11. After 30 minutes, take a swab and detect the bacterial count as per SOP for swab testing.
12. Repeat the same procedure, steps no. 9 to 12 to determine the efficacy of 70% IPA solution after 24 hrs, 48 hrs, 72 hrs and 96 hrs at room temperature.
Result Calculation
Calculate the reduction in microbial count using following formula.Log Reduction = log10(N0) − log10(N)
Where:
N0 = number of organisms before disinfection
N = number of organisms after disinfection
Acceptance Criteria
- The reduction in microbial account should be ≥ 90% (1 log reduction) at all time intervals.
- There should not be any significant change in efficacy between 0 hr and 96 hrs samples.
Method 2: Settle Plate Method
Microorganisms present in the environmental air are allowed to settle on agar plates before and after spraying the 70% IP solution in the air. The reduction in the colony count after IPA application indicates the efficacy of 70% IPA solution.Procedure
1. Expose the pre-incubated sterile Soybean casein digest agar plate for 2 hours.2. After plate exposure time, immediately clean the floor with IPA 70% v/v solution.
3. After proper cleaning, spray the area with IPA 70 % v/v solution and immediately close the room for 30 minutes to facilitate the action of disinfectant solution.
4. After 30 minutes of contact time, immediately expose the plate as per latest SOP for plate exposure and detect the bacterial count per location per 2 hours.
5. Repeat the same procedure, steps no. 1 to 4 to determine the efficacy of IPA 70% v/v solution after 24 hrs, 48 hrs, 72 hrs and 96 hrs at room temperature
Acceptance Criteria
- Colony count on “After IPA” plates should be reduced by at least 90% compared to “Before IPA” plates.
- No increase in microbial count should be observed in intervals like 24 hrs, 48 hrs, 72 hrs and 96 hrs.
Regulatory Expectations
Regulatory agencies like USFDA, WHO, EUGMP expect manufacturers to validate the stability and shelf life of all disinfectant solution used in GMP facilities. The key references of regulatory guidelines are:- WHO Technical Report Series 961, Annex 6 – GMP for sterile pharmaceutical products.
- USP <1072> – Disinfectants and antiseptics.
- EU GMP Annex 1 (2023 update) – Cleanroom cleaning and disinfection validation.
Limitations of IPA Disinfection
70% IPA solution is effective against most vegetative bacteria and viruses but not against spores. Some spore forming organisms like Bacillus subtilis or areas requiring sporicidal actions, additional disinfectants or sterilization methods must be used. FDA ref. Page 34 - Disinfection Efficacy.Validation of shelf life of 70% IPA is a critical GMP requirement in pharmaceutical manufacturing facilities. By performing validation using both the surface web method and settle plate method, manufacturers can confirm that the 70% IPA maintains its antimicrobial property throughout its use.
Generally, a freshly prepared 70% IPA solution remains effective for 96 hours if it is stored in a clean and closed container at controlled temperature. Some pharmaceutical facilities assign longer or shorter shelf lives based on their own validation data.
Q1. Why is 70% IPA used instead of 99%?
Answer: 70% IPA solution contains 30% water that allows the alcohol to penetrate microbial cell wall and slows IPA evaporation that ensures the proper destruction of microbes.Q2. What is the purpose of shelf life validation for IPA?
Answer: IPA solution validation is required to confirm that it maintains microbial effectiveness and chemical stability during storage.Q3. What are the acceptance criteria for shelf life validation?
Answer: IPA solution should maintain at least 90% microbial reduction and a significant change in efficacy.Q4. What is the typical validated shelf life for 70% IPA?
Answer: Most manufacturing facilities validate 70% IPA up to 96 hours after preparation.Q5. What containers are suitable for storing 70% IPA?
Answer: HDPE bottles, stainless steel cans and spray bottles with the tight closures are used store 70% IP solutionQ6. Is 70% IPA effective against spores?
Answer: No, 70% IPA solution is effective against bacteria and viruses but not spores. Some other disinfectants must be used for sporicidal action.Q7. Can IPA lose potency over time?
Answer: Yes, alcohol in the 70% IPA solution can evaporate or absorb moisture from the environment. Due to this change in concentration the effectiveness can be reduced.Q8. What happens if microbial reduction drops below 90%?
Answer: If microbial reduction is below 90% then IPA solution fails in validation. In such conditions shelf life should be reduced or revalidation with improved container control should be conducted.Q9. Can I extend IPA shelf life beyond 96 hours?
Answer: Yes, the shelf life of IBA can be extended beyond 96 hours but validation studies must prove the consistent microbial reduction and stability of IPA solution.Q10. How can I minimize IPA evaporation during storage?
Answer: By storing the IPA solution in tightly closed containers and keeping away from direct heat and air flow, evaporation of IPA can be minimized during storage.
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