OBJECTIVE:
The objective of this document is to define the mandatory requirements applying to Preservative Efficacy Test (Antimicrobial Effectiveness Test).
SCOPE:
Preservative Efficacy Test (Antimicrobial Effectiveness Test) as described in this Policy is applicable to the Pharmaceutical Preparation which contains added preservatives. The Preservative Efficacy Test (Antimicrobial Effectiveness Test) as described in the SOP does not intend to be used for routine control purposes.
RESPONSIBILITY:
In charge-Microbiology/ Head-Quality Control: for review and approval of SOP.
Microbiologist- to follow the procedure as per SOP.
ACCOUNTABILITY:
QA Head shall be Accountable for implementation of SOP.
PROCEDURE:
DEFINITION: The definitions listed here apply in the context of this SOP and may or may not be applicable in all other usage.
Antimicrobial Preservatives- Antimicrobial preservatives are substances added to the dosage forms in order to protect them from microbiological growth or from microorganisms that are introduced inadvertently during or subsequent to the manufacturing process.
Passage- One passage of culture is defined as the transfer of organisms from an established culture to fresh medium. Each cycle of freezing, thawing and revival in fresh medium is considered as a transfer.
Log- Logarithm is the exponent of 10. Log Reduction stands for a 10-fold (one decimal) or 90% reduction in numbers of live bacteria.
Principle:
Antimicrobial Effectiveness, inherent in the product or whether produced because of the addition of an antimicrobial preservative, must be demonstrated for injectable in multi – dose containers or for other products containing antimicrobial preservatives.
Requirement of Antimicrobial Preservatives to be added in the pharmaceutical preparations shall be evaluated at the product development stage. If a pharmaceutical product which does not itself possess adequate antimicrobial activity, antimicrobial preservatives needs to be added to prevent proliferation or to limit microbial contamination which might occur in the product and is hazardous to the patient and spoilage of the preparation during normal conditions of storage and use.
The antimicrobial preservative agents are generally toxic in nature. The concentration of the preservatives in the final packaged product should be below the toxic level.
Test requirements:
Following microorganisms, media and incubation conditions shall be used for Preservative Efficacy Testing (Antimicrobial Effectiveness testing).
| Micro-organism | Suitable medium | Incubation temperature | Inoculum incubation time |
| Pseudomonas aeruginosa (ATCC 9027; NCIMB 8626; CIP 82.118) | Casein soyabean digest agar (SCDA) | 32.5 ± 2.5 °C | 18 to 24 hrs |
| Staphylococcus aureus (ATCC 10231; NCTC 10788) | |||
| Escherichia coli (ATCC6538; NCIMB 8545; CIP 53.126) | |||
| Candida albicans ( ATCC 10231; NCPF 3179; IP 48.72) | Sabouraud Dextrose agar (SDA) without antibiotics | 22.5 ± 2.5 °C | 48 hours |
| Aspergillus brasiliensis ( ATCC 16404; IMI 149007; IP 1431.83) | 1 week or until good sporulation | ||
| * Zygosaccharomyces rouxii (NCYC 381; IP 2021.92) | 3 to 5 days |
*Zygosaccharomyces rouxii (NCYC 381; IP 2021.92) shall be used in case of oral preparations containing high sugar concentration as per Ph. Eur.
In case of sterile and aseptically manufactured products, preservative efficacy testing may include In-house isolates in addition to the above specified microorganisms
Culture suspensions shall be prepared by harvesting the microbial culture by washing the surface growth, collecting it in a suitable vessel and adding sufficient sterile saline test solution to obtain the cell population of about 1 x 10 8 colony forming units (cfu) / ml. The washing solution used for harvesting may be coupled with appropriate neutralizer.
Alternatively, the stock culture organisms shall be grown in a suitable liquid medium (i.e., Soybean–Casein Digest Broth or Sabouraud Dextrose Broth) and the cells shall be harvested by centrifugation and washing with sterilized saline solution.
Antimicrobial activity of the preservative and product should be eliminated by dilution, by filtration or by using specific inactivator/neutralizer which shall be demonstrated preferably at product development stage.
Inactivator/Neutralizer shall be validated for their ability to support the growth of the test organisms (Efficacy and Toxicity).
The initial cfu per mL in each culture suspension shall be determined, using the conditions of media and microbial recovery incubation times. This value serves to calibrate the size of inoculum used in the test.
The bacterial and yeast suspensions shall be used within 24 hrs. and fungal suspension may be stored under refrigerator for up to 7 days, OR as per established hold time.
NOTE: The microorganisms used in the test should not be more than five passages removed from the original ATCC culture.
Estimation of inoculum concentration may be performed using Turbidimetric measurements of the challenge micro-organisms and refrigerate the suspension, if it is not used within 2 hours.
Methodology:
Product Categories Pharmaceutical preparations to be tested for Antimicrobial Effectiveness test are divided into following categories. The criteria of antimicrobial effectiveness are based on the route of administration.
| Category | Product description |
| 1 | Parenteral preparations, eye preparations, intrauterine preparations and intramammary preparations. |
| 2 | Topical preparations, Ear preparations, nasal preparations, preparations for cutaneous applications and preparations for inhalation. |
| 3 | *Oral preparations, or mucosal preparations and rectal preparations. |
*As per USP, Oral preparations are separately categorized as –
| 1 | Oral products other than antacids, made with aqueous bases or vehicles. |
| 2 | Antacids made with an aqueous base. |
Test Procedure:
The test for Antimicrobial Effectiveness Test of Preservatives shall be performed based on the Product Category at specific intervals as given in the compendial chapters.
The test shall be performed either in original containers, if sufficient volume of product is available in each container. The product container shall be entered aseptically (i.e., needle and syringe through an elastomeric rubber stopper or pipetting aseptically, as appropriate), or in sterile, capped bacteriological containers of suitable size into which a sufficient volume of product has been transferred.
The inoculated containers shall be stored at prescribed temperature and sample shall be withdrawn from the container at specific interval of time for analysis and counting the organisms in the sample, so removed, from the container.
Each container shall be inoculated with one of the prepared and standardized inoculum and shall be mixed thoroughly for uniform distribution.
The volume of inoculum suspension used should be between 0.5 to 1.0 % of the volume of the product.
The inoculum concentration shall be varied based on the product category as per SOP.
The test shall be performed using either pour plate method or by membrane filtration, as appropriate and incubated at appropriate incubation by giving maximum incubation period, as given in the compendial chapter for Antimicrobial Effectiveness Test.
The change in log 10 values of the concentration of cfu per ml of each microorganism shall be calculated by using the calculated concentrations of cfu per ml present at the start of the test and express the changes in terms of log reductions.
If no neutralizing method is found suitable to neutralize the antimicrobial property, then it shall be considered that the failure to isolate the inoculated organism is attributable to the microbiocidal activity of the product. However, for monitoring proposes, the testing should be carried out using all microorganisms specified in the test.
Note: The inoculated sample containers shall be maintained at 22.5 ± 2.5°C protected from light. Where applicable, the pH shall be verified on withdrawn sample during suitability study. If necessary, pH shall be adjusted to 6 – 8 using suitable buffer or dilute solution of weak acids/base, as appropriate.
Criteria for Antimicrobial Effectiveness Test:
The acceptance criteria, in terms of decrease in the number of microorganisms with time, vary for different type of preparations/category, according to the degree of protection intended as per SOP.
No increase shall be defined as not more than 0.5 log10 units higher than the previous value measured.
REFERENCES:
Not Applicable
ANNEXURES:
Not Applicable
ENCLOSURES: SOP Training Record.
DISTRIBUTION:
Master Copy : Quality Assurance Department
Controlled Copy No. 01 : Head Quality Assurance
Controlled Copy No. 02 : Head QC (Micro)
ABBREVIATIONS:
| QC | : | Quality control |
| No. | : | Number |
| SOP | : | Standard Operating Procedure |
| EM | : | Environment Monitoring |
REVISION HISTORY:
CHANGE HISTORY LOG
| Revision No. | Details of Changes | Reason for Change | Effective Date |
| 00 | New SOP | Not Applicable | To Be Written Manual |