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EP 2.6.1

Nano-Test® > Research > Standards > EP 2.6.1

EP 2.6.1

EP 2.6.1, the European Pharmacopoeia (Ph. Eur.) Section 2.6 (Biological Tests) contains the general method ‘Sterility’. This section defines reference methods for the detection of living micro-organisms in pharmaceutical products which must be supplied in a sterile state.

Harmonisation and relevant standards: This section is harmonised with USP <71> (Sterility Tests) and JP 4.06; the principles of the methods, the culture media and the evaluation criteria are largely identical. Sterility testing of medical devices is covered within the scope of ISO 11737-2 and in the context of sterilisation validation; EP 2.6.1 serves, in particular, as the basis for routine release testing of pharmaceutical products. The appropriate framework must be selected depending on the product type and regulatory requirements.

Basic principle

The sterility test is based on determining whether living micro-organisms are present in a specific quantity of the product. Test E does not prove that the product is sterile; rather, it assesses the likelihood of contamination on the basis of a specific sampling plan and method. The results should therefore be interpreted in the context of production process control, bioburden data and sterilisation validation.

Methods

EP 2.6.1 defines two basic methods:

  • Membrane filtration process: The product (or its solution) is filtered through a membrane with a pore size of 0.45 µm; micro-organisms are retained in the membrane, and the membrane is transferred to a suitable culture medium, or the culture medium is passed through the membrane. This is the preferred method for clear liquid products, water-soluble products, hollow-body products and washable products. For products containing antibiotics, it also enables the removal of inhibitory substances.
  • Direct inoculation method: The product is transferred directly into the culture medium and incubated. This method is used for products where membrane filtration is not possible (e.g. ointments, creams, certain suspensions).

There are additional guidelines for certain types of products: oily products, ointments, creams, aerosol and inhalation products, medical devices, transdermal systems, gases, etc. For these products, sample preparation (dissolving, emulsifying, neutralising, washing) is crucial to the applicability of the method.

Culture media

  • Tryptic Soy Broth (TSB) / Soybean-Casein Digest Broth: For aerobic bacteria and fungi; incubate at 30–35 °C
  • Sabouraud dextrose broth (SDB) / Sabouraud liquid medium: For fungi and yeasts; incubated at 20–25 °C
  • Where necessary, culture media suitable for anaerobic products (e.g. thioglycolate medium) and appropriate conditions are used
  • The performance of the culture media must be confirmed by a growth promotion test

Incubation

  • For the membrane filtration method: usually 14 days; TSB at 30–35 °C, SDB at 20–25 °C
  • For the direct inoculation method: usually 14 days; however, this may vary depending on the product’s characteristics and the requirements of the monograph
  • Regular visual inspections are carried out during incubation; cloudiness is taken as an indicator of growth

Method Suitability

The validity of the sterility test depends on demonstrating the suitability of the method. A key component of this is the bacteriostasis/fungistasis test:

  • It must be demonstrated that the product does not inhibit the growth of a small number (usually ≤100 CFU) of microorganisms on the selected culture medium
  • The following microorganisms are typically used as test organisms: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Clostridium sporogenes, Candida albicans, Aspergillus brasiliensis
  • If inhibition is observed, the procedure must be rectified by appropriate neutralisation (e.g. penicillinase, beta-lactamase), dilution or flushing

Checks and evaluation

  • Negative control: Monitoring the sterility of the culture medium and the test environment
  • Positive control: Confirmation that the test system is functioning correctly (usually in conjunction with verification of the method’s suitability)
  • Interpretation: If no growth is observed at the end of the incubation period, the product has passed the sterility test; if growth is observed, it has failed the test and the microorganism that has grown must be identified
  • Where applicable, the provisions of the Pharmacopoeia regarding a repeat test (re-test) shall apply; however, a repeat test does not replace the root cause analysis

The key parameters reported in the study are as follows:

  • Product description, monograph requirements and sampling plan
  • Sterility testing method used (membrane filtration or direct inoculation)
  • Culture media used (TSB, SDB or others) and incubation conditions (temperature, duration)
  • Sample size and sample preparation method
  • Results of the method suitability assessment / bacteriostasis and fungistasis test
  • Test microorganisms used and recovery data
  • Negative and positive controls and their results
  • Visual assessment and detection of bacterial growth following incubation
  • Results of microbial growth identification
  • Test result: Sterility test passed / failed
  • Edition of the Ph. Eur. used and monograph reference
  • Data on measurement uncertainty and repeatability

Why is it carried out?

EP 2.6.1 is used for the following scientific and regulatory purposes:

  • Compliance with the Pharmacopoeia: Carrying out the legally required sterility testing for products in accordance with the relevant monographs and ensuring their suitability for placing on the EU market
  • Ensuring patient safety: Assessment of contamination with living micro-organisms in sterile injectable preparations, ophthalmic preparations, infusion solutions and medical devices
  • Product release: Assessment of the sterility of production batches prior to placing on the market
  • Integration into sterilisation validation: Demonstration of the efficacy of sterilisation processes (in conjunction with ISO 11737-2, ISO 11135, ISO 11137, ISO 17665, ISO 14937)
  • Raw material and process control: Monitoring the microbiological status of raw materials, intermediate products and packaging components
  • Quality control and testing: Demonstration of compliance with the pharmacopoeia and of good manufacturing practice during regulatory inspections
  • Root cause analysis: A contribution to identifying the source of contamination in the event of a sterility failure
  • Evidence-based approach: Contribution to the comprehensive assurance of product sterility in conjunction with data on bioburden (ISO 11737-1), endotoxins (ISO 11737-3, EP 2.6.14), sterilisation validation and packaging (ISO 11607)

EP 2.6.1 does not, in itself, prove the sterility of the product; sterility is a measure of probability and is assessed in conjunction with the sterility assurance level (SAL). For pharmaceutical products, the sterility test should be carried out in conjunction with the control of the manufacturing process and sterilisation validation.

As NanoTest, we carry out sterility tests in accordance with EP 2.6.1 with scientific rigour, utilising our accredited laboratory facilities and quality management system.

Please feel free to contact NanoTest for information on the selection of methods, the suitability of methods, sample preparation, incubation conditions, the test schedule and the reporting procedures for your sterility tests in accordance with EP 2.6.1.

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