Extractables Leachables: Complete Guide to E&L Testing for Pharmaceutical Development

An extractables leachables study is a critical pharmaceutical testing program that identifies and quantifies chemical compounds migrating from product contact materials into drug products. E&L testing ensures patient safety by evaluating potential contaminants from container closure systems, manufacturing equipment, and delivery devices.

For CMC leads, analytical chemists, and packaging engineers, extractables and leachables testing represents one of the most complex and high-stakes aspects of pharmaceutical development. A single unidentified leachable can trigger FDA rejection letters, clinical holds, or post-market safety recalls that cost millions in delays and rework.

The stakes are particularly high because E&L programs directly impact:

• IND and NDA approval timelines
• Manufacturing process validation
• Container closure system selection
• Shelf-life stability claims
• Patient safety risk assessments

In this guide, you'll learn:

• What extractables and leachables are and why e&l testing is mandatory
• The complete extractables and leachables study workflow from planning to reporting
• FDA, EMA, and ICH regulatory requirements for e&l pharmaceutical programs
• How to design extraction studies and leachables testing protocols
• Common pitfalls in E&L studies and how to avoid costly mistakes
• How modern platforms streamline CMC documentation for E&L programs

What Are Extractables and Leachables?

Extractables are chemical compounds that can be extracted from product contact materials under controlled laboratory conditions using aggressive solvents, elevated temperatures, or extended contact times. These compounds represent the "worst-case" profile of what could potentially migrate from materials.

Leachables are chemical compounds that migrate from product contact materials into the drug product under normal manufacturing, storage, and use conditions. Leachables represent the real-world subset of extractables that actually appear in the final drug product.

Key differences between extractables and leachables:

• Extractables studies use forced extraction conditions (strong solvents, high temperatures) to identify all potential migrating compounds, creating a comprehensive chemical profile of materials
• Leachables testing uses actual drug product formulations under normal storage conditions to detect what actually migrates during shelf life
• Extractables data guides material selection and identifies compounds requiring toxicological assessment
• Leachables data demonstrates that migration under actual conditions remains within safe limits

The relationship between extractables and leachables follows a critical principle: all leachables should have corresponding extractables, but not all extractables become leachables. This is why extractables studies always precede leachables testing in E&L programs.

Why extractables and leachables matter for drug development:

• Regulatory requirement: FDA, EMA, and other health authorities require E&L data in Module 3 (CMC section) of regulatory submissions
• Patient safety: Unidentified leachables can cause adverse events, immunogenicity, or product efficacy loss
• Material qualification: E&L studies determine which packaging materials and manufacturing equipment are suitable for specific drug products
• Stability indication: Changes in leachables profiles during stability testing can indicate container closure system failures

E&L Testing Regulatory Requirements

FDA Guidance on Extractables and Leachables

The FDA requires extractables and leachables testing for multiple product types through various guidance documents:

Container Closure Systems for Injectable Drug Products (May 1999):

• Applies to all parenteral products (injectables, ophthalmic solutions, inhalation products)
• Requires extractables studies using multiple solvents with different polarities
• Mandates leachables testing in actual drug product formulation
• Expects toxicological assessment for all identified leachables above safety thresholds

Metered Dose Inhaler (MDI) and Dry Powder Inhaler (DPI) Drug Products (1998):

• Specific requirements for inhalation products due to direct lung exposure
• Requires both propellant-based and aqueous extractables studies for MDIs
• Mandates leachables monitoring throughout product lifecycle
• Lower safety thresholds due to inhalation route of administration

Nasal Spray and Inhalation Solution Drug Products (2002):

• Requirements for pump spray systems and plastic containers
• Extractables profiles for all product-contacting components
• Stability-indicating leachables testing

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Regulatory Timeline: E&L data is typically required for IND submissions when container closure system selection is finalized, and comprehensive E&L studies must be complete before NDA/BLA filing.

EMA Requirements for E&L Pharmaceutical Programs

The European Medicines Agency addresses extractables and leachables through multiple guidelines:

Guideline on Plastic Immediate Packaging Materials (CPMP/QWP/4359/03):

• Comprehensive framework for plastic materials in direct drug contact
• Requirements for composition documentation and extractables testing
• Toxicological risk assessment methodology
• Change control requirements when altering materials

Requirements for all pharmaceutical products include:

• Complete compositional information for all product contact materials
• Systematic extractables studies using relevant extraction conditions
• Leachables testing under accelerated and real-time stability conditions
• Safety qualification using ICH Q3C, Q3D, and M7 guidelines
• Documentation in Module 3.2.P.7 (container closure system) of eCTD submissions

ICH Guidelines Relevant to E&L Studies

ICH Q3D is particularly critical for E&L programs because metal leachables from stainless steel processing equipment, rubber stoppers, and glass containers frequently exceed permitted daily exposure limits, requiring either material changes or toxicological justifications.

The Complete E&L Study Workflow

Phase 1: E&L Program Planning

Before conducting any laboratory testing, comprehensive planning determines program scope and success criteria.

Step 1: Identify All Product Contact Materials

• Container closure system components (vials, stoppers, seals, caps)
• Manufacturing equipment surfaces (tanks, tubing, filters, gaskets)
• Delivery device components (syringes, pumps, valves, actuators)
• Processing aids (tubing, O-rings, filter membranes)

Step 2: Conduct Risk Assessment

Each material is assessed for:

• Duration of product contact (minutes, days, years)
• Contact temperature (refrigerated, ambient, elevated)
• Surface area to volume ratio (higher ratio = greater extraction potential)
• Material composition (known vs. proprietary formulations)
• Historical extractables data (if available for same materials)

Step 3: Define Testing Strategy

Step 4: Establish Safety Thresholds

The Analytical Evaluation Threshold (AET) determines which leachables require identification:

For a single-dose injectable product (1 mL):

• AET = 1.5 µg/day ÷ 1 dose/day = 1.5 µg/mL

For a chronic inhalation product (2 actuations × 4 times daily):

• AET = 1.5 µg/day ÷ 8 actuations/day = 0.19 µg/actuation

Phase 2: Extractables Studies

Extractables studies create a comprehensive chemical profile of all materials under aggressive conditions.

Controlled Extraction Study Design:

Example Controlled Extraction Protocol:

1. Cut material samples to known surface area
2. Extract in 5 different solvents (water, 50% ethanol, hexane, 0.1N HCl, 0.1N NaOH)
3. Incubate at 60°C for 72 hours
4. Analyze extracts using multiple orthogonal analytical techniques
5. Quantify all peaks above detection limits
6. Identify compounds above 10% of AET

Analytical Techniques for Extractables Detection:

Extractables Study Deliverables:

• Comprehensive list of all extracted compounds
• Quantification data for each compound
• Structural identification (or proposed structures) for compounds >10% AET
• Material safety assessment based on extractables profile
• Recommendation for leachables monitoring strategy

Phase 3: Leachables Testing

Leachables testing demonstrates that migration under actual storage conditions remains within safe limits.

Leachables Study Design Principles:

Use actual drug product formulation, not surrogate solvents. The drug product's pH, ionic strength, surfactants, and cosolvents significantly affect leaching rates.

Test at multiple time points and conditions:

• Time zero (baseline)
• Accelerated stability (40°C/75%RH): 1, 3, 6 months
• Long-term stability (25°C/60%RH): 3, 6, 12, 18, 24 months (through shelf life)
• Stressed conditions if relevant (light exposure, freeze-thaw)

Sample from actual container closure system configuration:

• Same vendor and lot of materials
• Same manufacturing assembly process
• Same drug product fill volume
• Same primary packaging orientation (upright vs. inverted)

Analytical approach:

1. Targeted analysis: Quantify known extractables in drug product
2. Non-targeted screening: Search for unexpected leachables not found in extractables studies
3. Identification: Structurally elucidate any leachable >AET
4. Safety assessment: Calculate patient exposure and compare to toxicological limits

Leachables Acceptance Criteria:

Example Leachables Testing Scenario:

A biologic drug product in a 10 mL glass vial with rubber stopper for intravenous injection:

• Dose: 10 mL once weekly
• AET: (1.5 µg/day × 10 mL) ÷ (10 mL/dose × 1/7 doses/day) = 10.5 µg/10 mL = 1.05 µg/mL
• Testing: Analyze drug product at release and all stability time points
• Finding: 2-mercaptobenzothiazole (rubber accelerator) detected at 0.8 µg/mL at 6 months/40°C
• Action Required: Identify compound (done - known rubber additive), calculate exposure (0.8 µg/mL × 10 mL × 1 dose/week = 5.6 µg/week = 0.8 µg/day), assess safety (below most PDE values for this compound, but formal tox assessment needed)

Common Product Contact Materials and Their Extractables Profiles

Elastomeric Components (Rubber Stoppers, Gaskets, O-Rings)

Rubber formulations are the most common source of leachables in pharmaceutical products due to their complex compositions.

Typical rubber extractables include:

Risk mitigation for rubber components:

• Use pharmaceutical-grade elastomers with disclosed formulations
• Select materials with history of use in similar products
• Consider coated stoppers (Teflon-coated, film-coated) to reduce leaching
• Conduct extractables studies early to inform material selection
• Request certificates of analysis showing extractables profiles from vendors

Plastic Materials (Bottles, Bags, Tubing, Syringes)

Different plastic polymers have characteristic extractables based on their synthesis and additives.

Extractables by plastic type:

PVC extractables deserve special attention because di(2-ethylhexyl) phthalate (DEHP) plasticizer can leach at levels of concern, particularly for neonatal and pediatric populations. The FDA has issued warnings about DEHP exposure from medical devices, driving industry shift to DEHP-free or PVC-free alternatives.

Glass Containers

Glass is generally considered the safest primary packaging, but it is not extractables-free.

Glass extractables include:

• Elemental leachables: Sodium, silicon, boron, aluminum (from glass matrix delamination)
• Surface treatments: Ammonium sulfate (from sulfur treatment processes)
• Washing residues: Detergents, buffers if not properly rinsed

Type I borosilicate glass (highest quality pharmaceutical glass) has the lowest leaching potential, but delamination can occur with certain formulations:

• High pH solutions (>8) accelerate glass dissolution
• Alkaline degradation products from APIs
• Autoclaving or heat sterilization stress
• Long-term storage (multi-year shelf life products)

Glass delamination appears as:

• Visible flakes or particles in solution
• Increased silicon and boron levels in ICP-MS testing
• pH shifts due to alkali extraction
• Cloudiness or turbidity development

Designing E&L Studies for Different Product Types

Injectable Products (Parenterals)

Injectable products have the most stringent E&L requirements due to direct systemic exposure.

Container closure system components to test:

• Glass vial (Type I borosilicate preferred)
• Elastomeric stopper (butyl rubber most common)
• Aluminum overseal and flip-off cap
• Syringe components if pre-filled (barrel, plunger, tip cap, needle shield)

Extractables conditions for injectables:

• Aqueous extraction (water for injection or physiological saline)
• Organic extraction (ethanol, isopropanol, or ethanol-water mixtures)
• Acidic/basic extraction if product pH is extreme
• Temperature: 40°C minimum, 60°C for accelerated
• Duration: 14 days minimum, often 28+ days

Leachables testing for injectables:

• Test drug product at intended storage temperature
• Include stress condition (typically 40°C/75%RH for 6 months)
• Analyze at multiple time points (0, 3, 6, 12, 18, 24 months)
• Test for both extractables-derived leachables AND unexpected compounds
• Pay special attention to rubber-derived compounds (most common leachables source)

Safety thresholds for injectables:

• AET typically 0.15 µg/day (based on 1.5 µg/day SCT ÷ 10 doses/day assumption)
• Lower thresholds for neonatal/pediatric products
• Consider cumulative exposure for multi-dose vials or chronic therapies

Inhalation Products (MDIs, DPIs, Nebulizers)

Inhalation products face unique E&L challenges due to lung exposure and complex delivery devices.

Product contact materials in MDIs:

• Aluminum canister (usually coated)
• Metering valve (multiple elastomers, plastics, metal parts)
• Actuator (plastic housing, nozzle)
• Internal coatings (fluoropolymer linings)

Extractables study design for MDIs:

• Propellant-based extraction: Use actual propellant (HFA-134a or HFA-227ea) as extraction solvent
• Aqueous extraction: Simulate lung fluid contact (phosphate buffer, surfactant solutions)
• Component-level testing: Test each device component separately, then complete device
• Temperature: Accelerated conditions (40°C) and stress (60°C)
• Duration: Minimum 14 days, often 28 days

Leachables testing for inhalation products:

• Test drug product in complete device configuration
• Analyze throughout shelf life and in-use period
• Consider both "shot-weighted" (per actuation) and cumulative exposure
• Monitor for dose delivery changes (leachables can affect valve performance)
• Test under multiple orientations if relevant

Unique considerations for inhalation:

• Lower safety thresholds: Direct lung exposure is often considered higher risk than oral
• Valve performance impact: Leachables can affect spray pattern, plume geometry, delivered dose
• Coating degradation: Fluoropolymer coatings can degrade, creating particles or extractables
• Multiple material interactions: Complex devices have 10-20+ components interacting

Oral Solid Dosage Forms (Tablets, Capsules)

Oral solid dosage forms generally have lower E&L risk but still require evaluation.

Primary packaging materials:

• Blister packaging (PVC/PVDC/Aclar film, aluminum foil)
• HDPE bottles with induction seal and desiccant
• Aluminum tubes (for effervescent tablets)

E&L considerations for oral solids:

• Lower moisture content = reduced leaching potential compared to liquids
• Indirect contact: Blister films don't directly touch tablets (minimal migration)
• Accelerated conditions matter more: Elevated temperature and humidity drive migration
• Focus on volatile compounds: Small molecules migrate more readily through polymer films

Typical E&L approach for oral solids:

• Extractables studies on blister films or bottles using food simulants (water, ethanol solutions)
• Leachables testing: Store tablets in actual packaging at ICH stability conditions
• Analyze tablet cores for migrated compounds at 3, 6, 12, 24 months
• Focus analytical methods on compounds known to migrate through plastics

Common oral solid leachables:

• Plasticizers from PVC blisters (if used)
• Antioxidants from HDPE bottles
• Volatile aldehydes from PVC degradation
• Printing ink components (if in direct contact)

CMC Documentation Requirements for E&L Studies

Module 3.2.P.7: Container Closure System

All E&L data must be documented in the eCTD Module 3.2.P.7 section, which describes and justifies the container closure system.

Required content for P.7:

3.2.P.7.1 Description of Container Closure System:

• Complete list of all components (primary, secondary, tertiary packaging)
• Material composition for each component (polymer type, additives, coatings)
• Supplier information and material specifications
• Dimensions, thickness, and configurations
• Sterilization method if applicable

3.2.P.7.2 Suitability of Container Closure System:

• E&L study summaries (extractables and leachables results)
• Safety assessment of identified leachables
• Functional testing (seal integrity, moisture barrier, light protection)
• Compatibility with drug product (stability data)
• Justification for material selection

E&L documentation structure within P.7:

Cross-references to other modules:

• Module 2.3.P (Drug Product Summary): High-level E&L summary
• Module 3.2.P.8 (Stability): Leachables data from stability studies
• Module 2.6.3 (Pharmacology): Toxicology assessment of leachables
• Module 2.6.6 (Toxicology Summary): Safety qualification conclusions

Stability Data Integration

Leachables testing is always integrated with ICH stability studies.

Stability protocol requirements:

Time points for leachables analysis:

• Long-term (25°C/60%RH): 0, 6, 12, 18, 24, 36 months (or shelf life)
• Accelerated (40°C/75%RH): 0, 3, 6 months
• Intermediate (30°C/65%RH): If accelerated shows significant change

Stability-indicating approach:

• Include leachables quantification in stability-indicating methods
• Set specifications for known leachables (typically NMT limits)
• Monitor for new or unexpected leachables appearing over time
• Trend leachables levels to assess shelf life acceptability

Example stability specification for leachables:

Common E&L Program Pitfalls and How to Avoid Them

Pitfall 1: Starting E&L Studies Too Late

The problem: Many development teams delay E&L studies until preparing for NDA filing, only to discover unacceptable leachables that require changing materials, re-manufacturing clinical supplies, and repeating stability studies.

The consequence: 6-12 month delays to submission timelines, millions in wasted drug substance, repeated clinical manufacturing campaigns.

The solution:

• Conduct extractables screening during Phase 1 for all proposed container closure materials
• Include leachables testing in first formal stability study (Phase 2)
• Build 3-6 months into project timelines for potential material changes
• Maintain alternative material options as backup plans

Pitfall 2: Inadequate Analytical Method Sensitivity

The problem: Analytical methods cannot detect leachables at AET levels (typically 0.15 µg/mL or lower), leading to "not detected" results that don't satisfy regulatory requirements.

The consequence: FDA information requests, delayed approvals, requirement for method re-development and sample re-testing.

The solution:

• Establish method detection limits at least 10x below AET during method development
• Use multiple orthogonal techniques (GC-MS AND LC-MS) to cover all compound types
• Validate sensitivity using spiked samples at AET levels
• Document detection limits clearly in validation reports

Target method sensitivity for common product types:

Pitfall 3: Incomplete Extractables Studies

The problem: Extractables studies use only water or only one temperature, missing compounds that would extract under different conditions.

The consequence: Leachables appear in drug product that weren't in the extractables profile, requiring retrospective identification and safety assessment under time pressure.

The solution:

• Use minimum 3 solvents covering polarity range (aqueous, polar organic, non-polar)
• Test at elevated temperature (minimum 40°C, preferably 60°C)
• Include stressed pH conditions if drug product is acidic or basic
• Test duration should exceed shelf life at accelerated timescale
• Use actual container closure configuration, not coupon samples when possible

Pitfall 4: Missing Toxicological Assessment

The problem: Leachables are identified and quantified but no toxicological safety assessment is provided, or assessment lacks depth.

The consequence: FDA complete response letter requiring safety data before approval can proceed.

The solution:

• For all leachables >AET: Conduct formal safety assessment using ICH M7 (genotoxicity) and Q3C/Q3D (general toxicity) frameworks
• Calculate patient exposure (µg/day) based on maximum leachable level and dosing regimen
• Compare exposure to established PDE values or conduct read-across to analogous compounds
• Document assessment in Module 2.6 or provide standalone toxicology report
• Engage toxicologist early in E&L program planning

Toxicological assessment framework:

Pitfall 5: Ignoring Manufacturing Equipment Leachables

The problem: E&L programs focus only on container closure system, ignoring stainless steel tanks, tubing, filters, and other manufacturing equipment.

The consequence: Metal leachables (chromium, nickel, iron) from equipment appear in drug product, requiring post-hoc qualification.

The solution:

• Map all product contact surfaces in manufacturing process
• Sample drug product after each unit operation (post-filtration, post-filling, etc.)
• Test for elemental impurities using ICP-MS per ICH Q3D
• Include equipment extractables in overall E&L program scope
• Qualify cleaning procedures to remove organic extractables from reusable equipment

Common manufacturing equipment leachables:

How Assyro Streamlines E&L Documentation for CMC Submissions

Extractables and leachables studies generate massive amounts of analytical data, toxicology assessments, and cross-referencing requirements across eCTD modules. Managing this documentation complexity is a major pain point for CMC leads.

Common documentation challenges in E&L programs:

• Cross-referencing E&L data across Module 2.3, 3.2.P.7, and 3.2.P.8
• Ensuring consistency between summary tables and raw data appendices
• Tracking which leachables have completed safety assessments
• Updating stability data with leachables results as new time points complete
• Version control when container closure systems change during development

How Assyro helps with E&L CMC documentation:

Assyro's AI-powered platform validates eCTD submissions against FDA, EMA, and ICH requirements, including comprehensive checks for E&L documentation completeness:

• Automated cross-reference validation: Ensures every leachable mentioned in Module 3.2.P.7 has corresponding safety assessment in Module 2.6.3
• Completeness checking: Verifies extractables studies included required solvents, temperatures, and analytical methods per guidance
• Consistency validation: Confirms leachables levels in stability tables (Module 3.2.P.8) match safety assessment exposure calculations (Module 2.6)
• Template-based structure: Pre-built eCTD templates for E&L sections ensure no required elements are missing
• Change tracking: When container closure systems change, Assyro flags all modules requiring updates

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Time savings: CMC teams using Assyro for E&L documentation report 40-60% reduction in time spent cross-checking references and finding documentation gaps before submission.

Key Takeaways

Next Steps

Managing extractables and leachables studies creates one of the most complex documentation challenges in pharmaceutical development, with E&L data spanning multiple eCTD modules and requiring precise cross-referencing.

Organizations managing regulatory submissions benefit from automated validation tools that catch errors before gateway rejection. Assyro's AI-powered platform validates eCTD submissions against FDA, EMA, and Health Canada requirements, providing detailed error reports and remediation guidance before submission.

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