EDQM has issued draft revisions to two key general chapters – 2.6.14. Bacterial Endotoxins and 5.1.13. Pyrogenicity – as published in Pharmeuropa 37.2. These proposed updates introduce a new recombinant Factor C (rFC) bacterial endotoxin test method and refine pyrogen testing guidance to reflect modern, sustainable practices.
The draft proposals in Pharmeuropa highlight the rationale and scope of these changes. According to the European Directorate for the Quality of Medicines & HealthCare (EDQM), the aim is to formally integrate rFC as an official endotoxin test in the Ph. Eur. and ensure the pyrogen testing guidance aligns with this integration.
Integration of Recombinant Factor C (rFC) in Chapter 2.6.14
Chapter 2.6.14, “Bacterial Endotoxins,” is undergoing a notable update with the addition of Method G: Fluorimetric end-point method using rFC. The chapter’s introduction has been edited to reflect that the bacterial endotoxins test (BET) can be performed using either the traditional Limulus amoebocyte lysate (LAL) from horseshoe crab blood or recombinant Factor C derived from the horseshoe crab gene sequence.
In practical terms, the rFC assay is a fluorescent readout method: it measures the fluorescence generated after the enzymatic cleavage of a fluorogenic substrate by rFC, analogous to how LAL-based methods detect endotoxin via clotting or color change.
This fluorimetric rFC method – previously described in detail in the dedicated chapter 2.6.32 – is now being folded into 2.6.14 as Method G without changes in technical principle.
All six existing BET methods (A through F, which include gel-clot, turbidimetric, and chromogenic techniques) are retained, and the rFC-based Method G joins this list as a fully equivalent compendial method.
In case of any doubt or dispute in results, the chapter continues to designate the classic gel-clot (Method A) as the referee test, unless otherwise specified by a monograph. This ensures continuity with the existing framework – laboratories can adopt rFC where appropriate, but the historical reference standard (LAL gel-clot) remains the final arbiter if needed.
Updates to Chapter 5.1.13 on Pyrogenicity Testing
The Pyrogenicity chapter (5.1.13) is being revised with the endotoxin chapter to ensure consistency and reinforce best practices in pyrogen testing. One key change is the removal of any references to chapter 2.6.32.
Previously, 5.1.13 had acknowledged the existence of the separate rFC endotoxin test chapter; with rFC moving into 2.6.14, those mentions are no longer needed.
Going forward, any guidance in 5.1.13 regarding bacterial endotoxin testing will point solely to chapter 2.6.14 (which now encompasses all recognized BET methods A–G). This edit is largely editorial but important for clarity – users of the Pharmacopoeia will have a single, consolidated chapter to consult for endotoxin tests.
More substantively, chapter 5.1.13 is gaining new language to encourage consideration of sustainability when selecting an endotoxin test method. In practice, this means that when deciding among the BET methods (gel-clot, turbidimetric, chromogenic, or rFC), pharmacopoeia users are reminded to weigh their choice’s environmental and ethical impact.
This aligns with the broader industry trend towards the 3Rs (Reduction, Refinement, Replacement of animal-based tests) and reflects the Ph. Eur. Commission’s awareness of sustainability in quality control.
Implications for Quality Control and Regulatory Compliance
These proposed revisions carry several implications for pharmaceutical quality control (QC) labs and regulatory compliance strategies. First and foremost, including the rFC fluorimetric method in chapter 2.6.14 means that for European Pharmacopoeia users, recombinant Factor C will become an official, pharmacopeial method for endotoxin testing.
Laboratories would be able to employ rFC as Method G and cite compliance with Ph. Eur. 2.6.14, without the need to reference an alternative chapter or validate a completely new method externally. This should simplify the adoption of rFC assays across the industry: as a compendial method, rFC is considered to be validated following accepted scientific practice.
In practical terms, a company switching from an LAL-based method to the rFC method would still perform necessary method suitability tests – e.g. checking for interfering factors in their product matrix – but would not need to conduct extensive validation studies to prove the basic efficacy of the rFC assay, since the Pharmacopoeia already establishes that.
Quality control units should also note the editorial nature of some changes. For example, any internal SOPs or documentation that previously mentioned chapter 2.6.32 would need updating once the revisions take effect, as the standalone rFC chapter will be dropped. Instead, references should point to 2.6.14 (Method G) for the rFC test.
Similarly, the phrasing in filings or Certificates of Analysis might be standardized to reflect that all BET are done per Ph. Eur. 2.6.14, even if using rFC.
From a sustainability perspective, the official recognition of rFC marks a milestone. The pharmaceutical industry has been increasingly pressured to adopt more sustainable and animal-free testing methods.
Horseshoe crab populations, especially the Asian species, have faced threats partly due to demand for LAL. By transitioning to rFC, the industry can reduce its impact on these species.
Broader Industry Impact and Harmonisation Outlook
The proposed Ph. Eur. revisions reflect a broader momentum in the pharmaceutical sector toward modernizing and harmonizing pyrogen testing. In recent years, major pharmacopeias and regulators have been converging on the acceptance of recombinant endotoxin testing reagents.
The European Pharmacopoeia was an early mover by introducing chapter 2.6.32 for rFC in 2020, and it is now taking the next step by fully integrating rFC into the core BET chapter. The United States Pharmacopeia, as noted, has developed USP Chapter <86> “Bacterial Endotoxins Test Using Recombinant Reagents” to provide an equivalent avenue for using rFC and other recombinant cascade reagents alongside the official LAL-based chapter <85>.
SEE ALSO: USP Introduces Chapter <86> Bacterial Endotoxins Test Using Recombinant Reagents for Early Adoption
While the exact approaches differ (Ph. Eur. embedding rFC into the main chapter vs. USP creating a parallel chapter), the end result is greater flexibility and endorsement of animal-free endotoxin testing methods globally.
For pharmaceutical professionals, these changes underscore the importance of staying up-to-date with pharmacopoeial developments. Firms should examine how the rFC method compares with their current LAL tests regarding performance and ease of implementation.
Many may opt to perform side-by-side comparisons or validation bridging studies in anticipation of the official adoption, even if not strictly required, to ensure a smooth transition. Additionally, companies should review their pyrogen testing strategies in light of the clarified guidance: products containing components from Gram-positive bacteria or other sources of fever reactions should incorporate the MAT sooner rather than later.
Final Thoughts
By proactively adapting to the revised chapters, QC laboratories will not only remain compliant but can also improve the robustness of their safety testing and contribute to industry-wide goals of animal welfare and environmental sustainability.
The Ph. Eur.’s proposed revisions in Pharmeuropa 37.2 mark a progressive shift in how endotoxin and pyrogen testing are addressed in compendial standards. The integration of recombinant Factor C as Method G in chapter 2.6.14 elevates the rFC assay to an equal footing with traditional LAL methods, signaling strong confidence in this technology.
Concurrently, the updates to chapter 5.1.13 reinforce a comprehensive approach to pyrogen detection – combining the best of modern in vitro methods (rFC and MAT) to protect patients, while encouraging the pharmaceutical sector to embrace more sustainable practices. Once adopted and implemented, these changes will influence quality control testing, regulatory submissions, and even R&D decisions globally.
