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EP 2 June 2014

Nano-Test® > Research > Standards > EP 2 June 2014

EP 2 June 2014

EP 2.6.14, European Pharmacopoeia (Ph. Eur.) Section 2.6 (Biological Tests) sets out the general method for ‘Bacterial Endotoxins’. This section describes test procedures using Limulus amoebocyte lysate (LAL) for the determination of lipopolysaccharides (endotoxins) derived from the cell walls of Gram-negative bacteria.

Harmonisation and relevant standards: This section is harmonised with USP <85> (Bacterial Endotoxins Test) and JP 4.01; the principles of the methods, the validation requirements and the acceptance criteria are largely identical. References outside the pharmacopoeia include ISO 10993-11 (Evaluation of material-related pyrogenicity in medical devices) and USP <161> (blood/solvent contact sets); these standards are taken into account when detecting endotoxins in medical devices.

Basic principle

LAL is a lysate derived from the amoebocytes of the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus), which triggers an enzymatic coagulation cascade in the presence of endotoxins. Depending on the method used, this cascade is measured in terms of gel formation, a change in turbidity or colour development. The accuracy of the test is ensured by specifying the sensitivity of the lysate (λ, EU/mL), the standard endotoxin calibration (CSE/RSE) and the interference control.

Methods

EP 2.6.14 defines four basic groups of methods:

  • Gel-Clot method: threshold test and semi-quantitative method; visual assessment of gel formation in a lysate-endotoxin mixture. This is the most widely used method and is regarded as the reference method.
  • Turbidimetric method: Photometric measurement of the change in turbidity resulting from coagulation; kinetic or end-point mode
  • Chromogenic method: Photometric measurement of the coloured product formed by the breakdown of a synthetic substrate via the coagulation cascade; kinetic or end-point mode
  • Semi-quantitative gel clot method: an extended form of the threshold test using multiple dilutions

Key calculations and terms

  • Endotoxin limit (EL): Limit defined in the monograph (e.g. K/M formula for parenteral products; K = pyrogenic threshold dose, M = maximum human dose per kg per hour)
  • Maximum Valid Dilution (MVD): MVD = (EL × sample concentration) / λ
  • λ (lambda): Specified sensitivity of the lysate (EU/mL)
  • CSE (Control Standard Endotoxin): A control standard for endotoxins calibrated against the RSE (Reference Standard Endotoxin)
  • PPC (Positive Product Control): Confirmation of the detection of a known quantity of endotoxin in the sample; assessment of interference (inhibition/potentiation)
  • Interference test (Test for Interfering Factors): Must be carried out for each product during the validation phase; elimination of interference through appropriate dilution or adjustment of the method

The key parameters reported in the study are as follows:

  • Product description, endotoxin limit (EL) and its basis (monograph/formula)
  • LAL method used (gel aggregate, turbidimetric, chromogenic) and mode
  • Lysate batch, λ value and manufacturer’s specifications
  • CSE/RSE calibration and validation data
  • MVD and applied dilutions
  • Results of the interference test (PPC recovery, acceptance range)
  • Results of the negative and positive controls
  • Test results and comparison with EL
  • Edition of the Pharmacopoeia used (Ph. Eur. Edition) and chapter version
  • Measurement uncertainty and repeatability data

What is this for?

EP 2.6.14 is used for the following scientific and regulatory purposes:

  • Safety of parenteral products: Evidence that endotoxin contamination in injectable preparations, infusion solutions, eye drops and other pharmaceutical products is within the specified limits
  • Pharmacopoeia compliance: Carrying out the legally required testing for products for which an endotoxin test is specified in the monographs of the European Pharmacopoeia
  • Evaluation of medical devices: Assessment of material-related pyrogenicity and endotoxins in products that come into contact with blood or solutions (e.g. infusion sets, catheters, implants) (in conjunction with ISO 10993-11 and USP <161>)
  • Raw Material and Process Control: Monitoring the endotoxin load in water (purified water, water for injection), raw materials and packaging components in pharmaceutical manufacturing
  • Sterilisation and process validation: Demonstration of the efficacy of depyrogenation processes
  • Quality control and release: Assessment of production batches for endotoxins prior to market launch
  • Market surveillance and inspection: Demonstration of compliance with the Pharmacopoeia during inspections by the competent authorities
  • Animal welfare (3Rs): Use of the LAL test as an alternative to the pyrogen test on rabbits (EP 2.6.8)
  • Approach to evidential weight: contribution to the holistic safety assessment of the product in conjunction with data on sterility, biocompatibility and pyrogenicity

EP 2.6.14 covers bacterial endotoxins exclusively; material-related pyrogenicity (non-endotoxic pyrogens) and microbial contamination do not fall within the scope of this test and must be assessed using the appropriate methods (EP 2.6.8 rabbit pyrogen test, sterility test).

As NanoTest, we carry out bacterial endotoxin tests in accordance with EP 2.6.14 with scientific rigour, within the framework of our accredited laboratory infrastructure and our quality management system.

Please feel free to contact NanoTest for information on method selection, validation and run-in testing, MVD calculation, sample acceptance criteria, the test plan and the reporting procedures for your bacterial endotoxin tests in accordance with EP 2.6.14.

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