USP <85> Bacterial Endotoxins Test (BET)

USP General Chapter <85> establishes the harmonized compendial methods for detecting and quantifying Gram-negative bacterial endotoxins in articles where an endotoxin limit applies (e.g., parenteral drug substances/products and certain aqueous materials). Because endotoxins are pyrogenic lipopolysaccharides capable of causing severe febrile reactions, BET is a critical release and control test. The chapter describes lysate-based methods using horseshoe crab amebocyte lysate (LAL/TAL). Animal-free recombinant methods are addressed separately in USP <86> (official May 1, 2025).

Scope: Applies to raw materials, in-process samples, and finished parenteral products with an established endotoxin limit. For medical devices, testing limits and extraction procedures are defined outside <85> (e.g., USP <161>, FDA/AAMI standards).

Principle: Endotoxin activates a zymogen cascade in the amebocyte lysate, yielding a measurable gel, turbidity, or chromophore.

Test methods (harmonized).
• Method A—Gel-clot (limit test; presence/absence at the labeled sensitivity).
• Method B—Turbidimetric techniques (kinetic or end-point; quantitative via standard curve).
• Method C—Chromogenic techniques (kinetic or end-point; quantitative via standard curve).
Sensitivity/precision depend on reagent and method configuration; selection must be justified and suitable for the article.

Preparatory test—Test for Interfering Factors (product suitability). Demonstrate that the sample does not inhibit or enhance the lysate reaction by testing Positive Product Controls (PPCs) (product spiked with a known endotoxin level) against controls without product. Acceptance: PPC recovery typically 50–200% of the spiked value; if interference is observed, mitigate (e.g., dilution, validated neutralizers) and re-establish suitability.

Endotoxin limit calculation (drug products). The allowable limit L is dose-dependent and calculated as:
L = K / M (in EU/mL or EU/mg), where K is the threshold pyrogenic dose per kg body weight and M is the maximum dose per kg per hour of the product. K = 5.0 EU/kg for routes other than intrathecal; K = 0.2 EU/kg for intrathecal products. (Device limits are set separately; e.g., USP <161> includes limits such as NMT 20 EU/device and NMT 2.15 EU/device for cerebrospinal-fluid-contacting devices.)

Regulatory/harmonization notes:

• <85> is a PDG-harmonized chapter aligned with EP 2.6.14 and JP 4.01;
• ICH Q4B Annex 14 confirms use of these harmonized texts across ICH regions to avoid redundant testing.
• FDA also lists USP <85> as a recognized consensus standard.