Lesson 10. Antimicrobial Effectiveness Testing with Paracetamol Oral Suspension

Antimicrobial Preservative

• Compounds included in water-based drug products to protect them from the growth of microorganisms that cause product spoilage.

• Added to sterile products packed in multiple-use containers to prevent growth of microorganisms inadvertently introduced during repeated product withdrawal

• May be added as single agents or used in combination

phenol, m-cresol, benzyl alcohol, chlorobutanol, phenoxyethanol, methylparaben, and propylparaben

common examples of antimicrobial preservatives

1% of the formulation

Concentration should be kept at a minimum (_________________) to prevent toxic effects in humans.

pH, presence of non-aqueous phase, absorption on suspended solids, or plastic packaging

Biocidal activity cannot be based on preservative concentration alone, since it may be affected by _____________________________________________________.

GMP (Good Manufacturing Practice)

Antimicrobial Preservative should not be used to replace ____________________, only to reduce viable microorganisms of non-sterile formulations, or to manage bioburden prior to product sterilization

preservative efficacy test or preservative challenge test

other name for Antimicrobial Effectiveness Testing (AET)

Antimicrobial Effectiveness Testing (AET)

• Used to assess the ability of preservative systems to protect the product formulation against microbial contamination during normal storage and use of product

• Performed during product development stage and as part of stability testing on sealed packages of the product

multi-dose parenteral and products with antimicrobial preservatives, like topical and oral dosage forms, ophthalmic, otic, nasal, irrigation, and dialysis fluids

Antimicrobial Effectiveness Testing (AET) is used for:

anhydrous products or for single-use injectable preparations only

Antimicrobial Effectiveness Testing (AET) is not used for:

inoculating separate containers

Antimicrobial Effectiveness Testing (AET)

a. Involves _________________________ of the product with known concentration of identified organisms

b. Challenge organisms used are based on _______________________________

c. USP requires inclusion of ___________________________________________

a = ?

likely product contaminants based on physical properties, formulation, and intended use

Antimicrobial Effectiveness Testing (AET)

a. Involves _________________________ of the product with known concentration of identified organisms

b. Challenge organisms used are based on _______________________________

c. USP requires inclusion of ___________________________________________

b = ?

bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus) and yeasts & molds (Candida albicans and/or Aspergillus niger)

Antimicrobial Effectiveness Testing (AET)

a. Involves _________________________ of the product with known concentration of identified organisms

b. Challenge organisms used are based on _______________________________

c. USP requires inclusion of ___________________________________________

c = ?

paracetamol oral suspension

Antimicrobial Effectiveness Testing (AET) Procedure:

1. Get four 60-mL original containers of __________________________________.

2. Inoculate the container with _________________________________________ of C. albicans, E. coli, P. aeruginosa, or S. Aureus and mix.

3. Label the containers and incubate at _______________________.

4. Calculate the ____________________________________ in each container based on the concentration of microorganisms in the microbial suspension and Sample containers on Days 7 and 14.

5. On day 7 and 14, Inoculate 0.1 mL of product from containers with E. coli, P. aeruginosa, and S. aureus to two separate SCDA plates each organism. Incubate at ___________________.

6. Inoculate 0.1 mL of product from the container with C. albicans to two separate SDA plates. Incubate at __________________.

7. Count the number of colonies and get the ________________________.

8. Determine the cfu/mL at Day 7 and Day 14 using: 𝐶𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿) = __________.

9. Calculate the log reduction at Day 7 and Day 14 using: ____________________.

1 = ?

0.2 mL of microbial suspension (1 x 10⁸ cfu/mL)

Antimicrobial Effectiveness Testing (AET) Procedure:

1. Get four 60-mL original containers of __________________________________.

2. Inoculate the container with _________________________________________ of C. albicans, E. coli, P. aeruginosa, or S. Aureus and mix.

3. Label the containers and incubate at _______________________.

4. Calculate the ____________________________________ in each container based on the concentration of microorganisms in the microbial suspension and Sample containers on Days 7 and 14.

5. On day 7 and 14, Inoculate 0.1 mL of product from containers with E. coli, P. aeruginosa, and S. aureus to two separate SCDA plates each organism. Incubate at ___________________.

6. Inoculate 0.1 mL of product from the container with C. albicans to two separate SDA plates. Incubate at __________________.

7. Count the number of colonies and get the ________________________.

8. Determine the cfu/mL at Day 7 and Day 14 using: 𝐶𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿) = __________.

9. Calculate the log reduction at Day 7 and Day 14 using: ____________________.

2 = ?

22.5 ± 2.50℃

Antimicrobial Effectiveness Testing (AET) Procedure:

1. Get four 60-mL original containers of __________________________________.

2. Inoculate the container with _________________________________________ of C. albicans, E. coli, P. aeruginosa, or S. Aureus and mix.

3. Label the containers and incubate at _______________________.

4. Calculate the ____________________________________ in each container based on the concentration of microorganisms in the microbial suspension and Sample containers on Days 7 and 14.

5. On day 7 and 14, Inoculate 0.1 mL of product from containers with E. coli, P. aeruginosa, and S. aureus to two separate SCDA plates each organism. Incubate at ___________________.

6. Inoculate 0.1 mL of product from the container with C. albicans to two separate SDA plates. Incubate at __________________.

7. Count the number of colonies and get the ________________________.

8. Determine the cfu/mL at Day 7 and Day 14 using: 𝐶𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿) = __________.

9. Calculate the log reduction at Day 7 and Day 14 using: ____________________.

3 = ?

initial concentration of viable microorganisms

Antimicrobial Effectiveness Testing (AET) Procedure:

1. Get four 60-mL original containers of __________________________________.

2. Inoculate the container with _________________________________________ of C. albicans, E. coli, P. aeruginosa, or S. Aureus and mix.

3. Label the containers and incubate at _______________________.

4. Calculate the ____________________________________ in each container based on the concentration of microorganisms in the microbial suspension and Sample containers on Days 7 and 14.

5. On day 7 and 14, Inoculate 0.1 mL of product from containers with E. coli, P. aeruginosa, and S. aureus to two separate SCDA plates each organism. Incubate at ___________________.

6. Inoculate 0.1 mL of product from the container with C. albicans to two separate SDA plates. Incubate at __________________.

7. Count the number of colonies and get the ________________________.

8. Determine the cfu/mL at Day 7 and Day 14 using: 𝐶𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿) = __________.

9. Calculate the log reduction at Day 7 and Day 14 using: ____________________.

4 = ?

30-35℃ for 3 days

Antimicrobial Effectiveness Testing (AET) Procedure:

1. Get four 60-mL original containers of __________________________________.

2. Inoculate the container with _________________________________________ of C. albicans, E. coli, P. aeruginosa, or S. Aureus and mix.

3. Label the containers and incubate at _______________________.

4. Calculate the ____________________________________ in each container based on the concentration of microorganisms in the microbial suspension and Sample containers on Days 7 and 14.

5. On day 7 and 14, Inoculate 0.1 mL of product from containers with E. coli, P. aeruginosa, and S. aureus to two separate SCDA plates each organism. Incubate at ___________________.

6. Inoculate 0.1 mL of product from the container with C. albicans to two separate SDA plates. Incubate at __________________.

7. Count the number of colonies and get the ________________________.

8. Determine the cfu/mL at Day 7 and Day 14 using: 𝐶𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿) = __________.

9. Calculate the log reduction at Day 7 and Day 14 using: ____________________.

5 = ?

20-25℃ for 5 days

Antimicrobial Effectiveness Testing (AET) Procedure:

1. Get four 60-mL original containers of __________________________________.

2. Inoculate the container with _________________________________________ of C. albicans, E. coli, P. aeruginosa, or S. Aureus and mix.

3. Label the containers and incubate at _______________________.

4. Calculate the ____________________________________ in each container based on the concentration of microorganisms in the microbial suspension and Sample containers on Days 7 and 14.

5. On day 7 and 14, Inoculate 0.1 mL of product from containers with E. coli, P. aeruginosa, and S. aureus to two separate SCDA plates each organism. Incubate at ___________________.

6. Inoculate 0.1 mL of product from the container with C. albicans to two separate SDA plates. Incubate at __________________.

7. Count the number of colonies and get the ________________________.

8. Determine the cfu/mL at Day 7 and Day 14 using: 𝐶𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿) = __________.

9. Calculate the log reduction at Day 7 and Day 14 using: ____________________.

6 = ?

average of two plates

Antimicrobial Effectiveness Testing (AET) Procedure:

1. Get four 60-mL original containers of __________________________________.

2. Inoculate the container with _________________________________________ of C. albicans, E. coli, P. aeruginosa, or S. Aureus and mix.

3. Label the containers and incubate at _______________________.

4. Calculate the ____________________________________ in each container based on the concentration of microorganisms in the microbial suspension and Sample containers on Days 7 and 14.

5. On day 7 and 14, Inoculate 0.1 mL of product from containers with E. coli, P. aeruginosa, and S. aureus to two separate SCDA plates each organism. Incubate at ___________________.

6. Inoculate 0.1 mL of product from the container with C. albicans to two separate SDA plates. Incubate at __________________.

7. Count the number of colonies and get the ________________________.

8. Determine the cfu/mL at Day 7 and Day 14 using: 𝐶𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿) = __________.

9. Calculate the log reduction at Day 7 and Day 14 using: ____________________.

7 = ?

𝑚𝑒𝑎𝑛 𝑜𝑓 2 𝑝𝑙𝑎𝑡𝑒𝑠/ 0.2 𝑚𝐿

Antimicrobial Effectiveness Testing (AET) Procedure:

1. Get four 60-mL original containers of __________________________________.

2. Inoculate the container with _________________________________________ of C. albicans, E. coli, P. aeruginosa, or S. Aureus and mix.

3. Label the containers and incubate at _______________________.

4. Calculate the ____________________________________ in each container based on the concentration of microorganisms in the microbial suspension and Sample containers on Days 7 and 14.

5. On day 7 and 14, Inoculate 0.1 mL of product from containers with E. coli, P. aeruginosa, and S. aureus to two separate SCDA plates each organism. Incubate at ___________________.

6. Inoculate 0.1 mL of product from the container with C. albicans to two separate SDA plates. Incubate at __________________.

7. Count the number of colonies and get the ________________________.

8. Determine the cfu/mL at Day 7 and Day 14 using: 𝐶𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿) = __________.

9. Calculate the log reduction at Day 7 and Day 14 using: ____________________.

8 = ?

𝐿𝑜𝑔 𝑅𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 = 𝑙𝑜𝑔 10 𝑎𝑡 𝑠𝑡𝑎𝑟𝑡 𝑐𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿) − 𝑙𝑜𝑔 10 𝑜𝑓 𝑓𝑖𝑛𝑎𝑙 𝑐𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿)

Antimicrobial Effectiveness Testing (AET) Procedure:

1. Get four 60-mL original containers of __________________________________.

2. Inoculate the container with _________________________________________ of C. albicans, E. coli, P. aeruginosa, or S. Aureus and mix.

3. Label the containers and incubate at _______________________.

4. Calculate the ____________________________________ in each container based on the concentration of microorganisms in the microbial suspension and Sample containers on Days 7 and 14.

5. On day 7 and 14, Inoculate 0.1 mL of product from containers with E. coli, P. aeruginosa, and S. aureus to two separate SCDA plates each organism. Incubate at ___________________.

6. Inoculate 0.1 mL of product from the container with C. albicans to two separate SDA plates. Incubate at __________________.

7. Count the number of colonies and get the ________________________.

8. Determine the cfu/mL at Day 7 and Day 14 using: 𝐶𝑜𝑛𝑐 (𝑐𝑓𝑢/𝑚𝐿) = __________.

9. Calculate the log reduction at Day 7 and Day 14 using: ____________________.

9 = ?

no longer increase

Expected result of AET:

• Initially, there will be growth of microorganisms, but after several days, their number should ________________ because of the presence of preservatives.