E&L Testing for Vial Closures: How the Study Programme Is Designed and Run

An extractables and leachables testing programme for a vial closure is a staged study, not a single test: a documented risk assessment sets the scope, a controlled extraction study maps everything that can come out of the stopper, seal, and coatings under exaggerated conditions, analytical screening identifies whatever exceeds a calculated reporting threshold, and a leachables study on the real drug product confirms what actually migrates over shelf life. Each stage produces evidence the drug manufacturer submits and defends.

For the underlying definitions, extractables and leachables in vial seals and closures explains what the two terms mean and which pharmacopoeial chapters apply. What follows covers study design, extraction conditions, analytical methods, thresholds, and re-testing.

Key takeaways

E&L work is a programme with a defined sequence: risk assessment, controlled extraction, analytical screening, leachables confirmation, toxicological assessment, report.
No pharmacopoeial chapter prescribes a fixed solvent set for vial closures. Extraction conditions are designed, justified, and documented case by case.
The reporting threshold is the analytical evaluation threshold (AET), derived from a safety concern threshold and adjusted by an analytical uncertainty factor.
Screening combines GC-MS, headspace GC-MS, LC-MS, and ICP-MS, because no single technique sees the whole profile.
ICH Q3E, the first harmonised E&L guideline, reached Step 2b on 1 August 2025 and is expected to be finalised around June 2027.
Re-testing is driven by change control, not a calendar: formulation, material, process, sterilisation, site, shelf-life, and container-size changes are all triggers.
A closure supplier contributes composition data, batch-to-batch consistency, and a Drug Master File the customer can reference.

What the programme has to cover

Scope is set by contact, not by component count: every material in direct or indirect contact with the drug or its headspace is in scope, which on a vial means the elastomeric stopper, the aluminium or aluminium-plastic seal, and every lacquer, adhesive, and ink on them.

Two scoping decisions get made early. The first is component-level or system-level testing: component testing gives interpretable data and lets a change to one part be assessed on its own, while testing the assembled container closure system reflects reality including interactions between parts, so most programmes do both, components first. The second is direct versus indirect contact: the stopper’s inner face touches the drug, and the seal is separated from it by the stopper, so the seal’s contribution is indirect and lower risk but not zero, because it shares headspace and its lacquer and plastic disc are further chemistries.

The rubber compound is usually the dominant source, which is why the elastomer family drives so much of the profile. That relationship is covered in rubber types used in vial closures.

Step 1: risk assessment and scoping

The programme starts with a documented risk assessment that decides how much testing is proportionate, using the quality risk management principles of ICH Q9(R1). Injectables sit at the high-risk end because the product is sterile, often aqueous, and administered directly.

The factors that move the assessment are the route of administration, the duration of contact (a two-year shelf life is a long extraction), the formulation’s leaching propensity (pH extremes, the surfactants common in biologic formulations, co-solvents, and lipid vehicles all pull more out of an elastomer), storage conditions, cumulative exposure, and the patient population. The output is a written justification for the study design, which is what a reviewer reads first.

Step 2: designing the controlled extraction study

A controlled extraction study deliberately over-stresses the material with several solvents of different polarity, at elevated temperature, for a defined time, so the resulting profile bounds the worst case rather than describing normal use. The design is a set of justified choices, not a recipe: neither USP nor ICH prescribes a solvent set for closures.

Design variable	Typical approach	Why
Solvent series	An aqueous solvent, pH-adjusted aqueous solvents, an alcohol/water mixture, and a non-polar organic solvent	One polarity misses whole compound classes
Temperature	Elevated above the real storage condition	Finishes the study in weeks, not years
Duration	Fixed intervals, often a kinetic profile	Shows whether extraction has plateaued
Surface-area-to-volume ratio	Matched to or exaggerated against the real package	Keeps the result relatable to the container
Technique	Reflux, sonication, sealed-vessel incubation, or Soxhlet	Chosen for the material and solvent

Two practical points shape the design. Extraction should be shown to approach exhaustion, usually by demonstrating that additional time or successive extractions stop yielding new compounds. And the study needs blanks and system suitability controls, because low-level artefacts from solvents, vessels, and the laboratory are otherwise indistinguishable from real extractables.

Step 3: choosing the analytical methods

No single technique sees the whole profile, so screening uses a panel: chromatography with mass spectrometry for organics across the volatility range, plus inductively coupled plasma mass spectrometry for elements.

Technique	What it covers
Headspace GC-MS	Volatile organics, including residual monomers and process solvents
GC-MS	Semi-volatile organics such as antioxidants, plasticisers, and cure-system residues
LC-MS, with UV or diode-array detection	Non-volatile and thermally labile organics, oligomers, degradation products
ICP-MS	Elemental impurities, assessed against ICH Q3D

Screening is normally semi-quantitative, with responses estimated against a small set of representative internal standards rather than an authentic standard for every peak. That is why an uncertainty factor is applied to the reporting threshold. Compounds that matter are re-measured against their own reference standards.

The framework for justifying this work sits in USP informational chapters <1663> (extractables) and <1664> (leachables), while material-level requirements remain in USP <381> and the USP <661> series.

Step 4: setting the acceptance thresholds

The threshold that governs the whole programme is the analytical evaluation threshold: the concentration at or above which a compound must be reported, identified, and assessed, and below which it can be justified as negligible. The analytical burden follows from where that line sits.

The AET is derived from a safety concern threshold expressed as a daily intake, then converted into a concentration using the number of container units a patient could receive per day and the fill volume. The Product Quality Research Institute recommended a safety concern threshold of 0.15 µg/day for orally inhaled and nasal drug products, and 1.5 µg/day in its later work on parenteral and ophthalmic products. ICH M7 separately sets a threshold of toxicological concern of 1.5 µg/day for mutagenic impurities, which controls for any compound with a structural alert.

An analytical uncertainty factor is applied to the calculated value, giving the final AET, because semi-quantitative estimates can under-report. Elemental findings are handled separately, against the permitted daily exposures in ICH Q3D.

Step 5: the leachables study

The leachables study asks the only question that matters clinically: what actually ends up in the drug product, at what concentration, by the end of shelf life. It runs on the real formulation in the real container closure system at the real storage condition, normally aligned with the stability protocol so samples are pulled at the same time points.

The target list comes from the extractables profile, so the two studies are sequential rather than parallel, and where the product is analytically intractable a justified simulating solvent is used instead. Orientation is often overlooked: a vial stored inverted or on its side puts liquid against the stopper’s sealing face, which upright storage does not, so the study orientation should match the label.

Step 6: toxicological assessment and reporting

Every compound reported above the AET receives a documented toxicological assessment against its estimated patient exposure. Compounds with adequate safety data are assessed against a permitted daily exposure; those without are handled by read-across from structurally related substances, or by the threshold of toxicological concern where that is defensible. The report then carries the study justification, the extraction and analytical conditions, method suitability data, the AET calculation with its assumptions shown, an identification and concentration for every peak above the AET, and the toxicological conclusion.

What ICH Q3E will change

**ICH Q3E, *Guideline for Extractables and Leachables*, is the first harmonised international guideline for this work; it reached Step 2b on 1 August 2025 and, following public consultation, is anticipated to reach Step 4 around June 2027.** Until then it is a draft and not binding, but it signals where expectations are heading.

The draft applies an ICH Q9-aligned risk management framework to manufacturing systems as well as packaging and delivery systems, and proposes a three-class scheme for leachables: a high-concern class to be avoided or controlled to compound-specific limits, a default class controlled within threshold limits, and a low-toxicity class with supporting monographs. It also formalises the extractables-to-leachables correlation as the basis for replacing routine leachables testing on stability. The draft is published by ICH and the European Medicines Agency.

When to repeat E&L testing

E&L work is not re-run on a schedule; it is re-run when something changes that could alter the profile, which makes change control the real control mechanism. The triggers that require at least a re-assessment, and often new testing, are:

A change to the elastomer compound, lacquer, ink, adhesive, or resin, including a change of the supplier’s own raw-material source.
A change to the moulding, curing, coating, washing, or siliconisation process, or a site transfer or new production line.
A change of sterilisation method or dose, since irradiation and moist heat generate different degradation products.
A formulation change that alters extraction behaviour: pH, surfactant, co-solvent, or vehicle.
A change in fill volume or container size, which moves the surface-area-to-volume ratio, or an extension of shelf life or change of storage orientation.
A new route, patient population, or dosing regimen that raises cumulative exposure, or new toxicological data on a compound already known to be present.

This is why the change-notification clause in a supplier quality agreement carries so much weight. A supplier that changes a lacquer without notifying its customers can invalidate a filed E&L package; the obligations behind that clause are covered in cGMP for vial closure suppliers.

What the closure supplier contributes

The supplier cannot run the drug manufacturer’s E&L programme, but it supplies the three things that make it tractable: controlled composition, batch-to-batch consistency, and referenceable regulatory documentation. Those three are also most of what separates a premium closure from a commodity one. Composition data narrows the search space; consistency is what allows a profile generated on three batches to represent future supply; and a Type III Drug Master File lets the customer cite the supplier’s confidential material and process detail through a letter of authorisation instead of re-deriving it.

How this works in practice at Autofits

Autofits manufactures the aluminium and aluminium-plastic seals that sit above the stopper, so its contribution to a customer’s E&L package is documentary and material rather than analytical. The relevant levers are the composition of the aluminium, the lacquers and inks applied to it, and the plastic used for the disc or button, all held under specification and traceable by batch. Production runs under an ISO 15378:2017 quality system alongside ISO 9001:2015 and ISO 14001:2015 certification, with a Drug Master File customers can reference in their own submissions, in a 75,000 sq ft Nashik facility with an ISO Class 8 cleanroom. The range is on the products page and the certification set on the quality page.

Frequently asked questions

What does an E&L testing programme for a vial closure involve?

Six stages: a risk assessment that scopes the work, a controlled extraction study that over-stresses the stopper, seal, and coatings with several solvents at elevated temperature, analytical screening by GC-MS, headspace GC-MS, LC-MS, and ICP-MS, a leachables study on the real drug product across shelf life, a toxicological assessment of everything above the reporting threshold, and a report justifying each choice.

What extraction conditions are used for extractables studies on closures?

There is no prescribed set. Studies typically use several solvents of differing polarity, including an aqueous solvent, pH-adjusted aqueous solvents, an alcohol and water mixture, and a non-polar organic solvent, at a temperature above the real storage condition, with multiple time points and a defined surface-area-to-volume ratio. The conditions must be justified in writing and shown to approach exhaustion.

What is the analytical evaluation threshold (AET)?

The AET is the concentration at or above which an extractable or leachable must be reported, identified, and toxicologically assessed. It is calculated from a safety concern threshold expressed as a daily intake, converted using the daily number of container units and the fill volume, then reduced by an analytical uncertainty factor. Compounds below it can be justified as negligible without individual identification.

When does E&L testing have to be repeated?

Whenever a change could alter the profile: a change to the elastomer compound, lacquer, ink, or resin; to the moulding, curing, or washing process; to the sterilisation method; a site transfer; a formulation change affecting pH or surfactant content; a change in fill volume or container size; a shelf-life extension; or new toxicological data. It is driven by change control, not a fixed interval.

What is ICH Q3E and is it in force?

ICH Q3E is the draft *Guideline for Extractables and Leachables*, the first harmonised international guideline on the subject. It reached Step 2b on 1 August 2025, with Step 4 finalisation anticipated around June 2027, so it is not yet binding. It proposes a risk management framework aligned with ICH Q9, a three-class scheme for leachables, and a formal role for the extractables-to-leachables correlation.

Does the aluminium seal need its own E&L data?

The seal is separated from the drug by the stopper, so its contribution is indirect and lower risk, but it is assessed rather than excluded by assumption: it shares the vial headspace, and its lacquers, inks, and plastic disc are distinct chemistries. In practice it is covered through the supplier’s composition data and Drug Master File.

Sources

ICH: Quality Guidelines, including the Q3E draft *Guideline for Extractables and Leachables* (Step 2b, 1 August 2025), Q3D(R2) Elemental Impurities, and Q9(R1) Quality Risk Management (https://www.ich.org/page/quality-guidelines)
European Medicines Agency: draft ICH Q3E guideline for extractables and leachables (https://www.ema.europa.eu/en/documents/scientific-guideline/draft-ich-q3e-guideline-extractables-leachables_en.pdf)
ICH: M7 Assessment and Control of DNA Reactive (Mutagenic) Impurities (https://www.ich.org/page/multidisciplinary-guidelines)
USP-NF: General Chapters <381>, <661.1>, <661.2>, <1663> Assessment of Extractables, and <1664> Assessment of Leachables (https://www.usp.org/)
Product Quality Research Institute (PQRI): recommendations on safety thresholds for extractables and leachables (https://pqri.org/)

*Last updated: 2026-07-31. This article is general technical information, not regulatory or compliance advice; confirm current standard editions and requirements with the issuing bodies.*