USP <1664>: Leachables Testing and the Role of Analytical Evaluation Threshold (AET)
While USP <1663> provides the framework for identifying potential extractables, USP <1664> focuses on verifying which of those compounds actually migrate into the drug product under real-world conditions. This phase is known as leachables testing.
In this article, we explore how leachables studies are conducted according to USP <1664> and explain the crucial role of the Analytical Evaluation Threshold (AET) in identifying compounds of concern.
What Are Leachables?
Leachables are substances that migrate from packaging or delivery systems into the drug product during manufacturing, storage, or use. Unlike extractables, which are identified under exaggerated conditions, leachables are measured under actual product-use conditions.
USP <1664> Overview
USP <1664> outlines the steps for designing, executing, and evaluating leachables studies based on product risk and previous extractables data.
Key Steps:
- Design a leachables study based on extractables profile (from USP <1663>)
- Choose drug-contacting components and dosage forms (e.g., inhalers, injectables)
- Define storage conditions (temperature, time, humidity)
- Use validated analytical methods to detect and quantify leachables
- Apply the AET to determine which compounds need further toxicological evaluation
The Analytical Evaluation Threshold (AET)
The AET is a calculated value that determines the minimum reporting level for leachables in analytical testing. Any compound found above the AET must be identified and evaluated for safety.
Formula for AET:
AET (µg/mL) = (Safety Concern Threshold × Dose) / Sample Volume
Where:
- Safety Concern Threshold (SCT): Typically 1.5 µg/day for mutagenic impurities (per ICH M7)
- Dose: Maximum daily drug dose (in mL or g)
- Sample Volume: Volume or mass of sample analyzed
Example:
SCT = 1.5 µg/day Daily dose = 5 mL Sample volume = 5 mL AET = (1.5 × 5) / 5 = 1.5 µg/mL
In this case, any compound above 1.5 µg/mL in the leachables study must be reported, identified, and evaluated toxicologically.
When Is Leachables Testing Required?
- For high-risk dosage forms (inhalation, injection, ophthalmic)
- For drug-device combination products (e.g., prefilled syringes)
- When new packaging materials are introduced
- For biologics and temperature-sensitive formulations
Link Between USP <1663> and <1664>
The extractables profile generated under USP <1663> forms the basis of leachables study design. Only compounds likely to leach into the drug product, based on worst-case conditions, are monitored in the USP <1664> study.
Example Workflow:
- Perform extractables testing on syringe barrel (USP <1663>)
- Identify potential leachables like DEHP, BPA
- Conduct real-time storage of filled syringe
- Analyze at intervals (e.g., 0, 3, 6 months)
- Quantify leachables and compare to AET
Conclusion
Leachables testing under USP <1664> provides assurance that packaging components do not release harmful compounds into drug products during real-world use. The AET plays a central role in determining which compounds must be identified and assessed for patient safety.
When extractables and leachables studies are used together, they form a powerful risk-based strategy to qualify packaging systems and meet regulatory expectations from agencies like the FDA, EMA, and WHO.