Introduction:
TTC in Extractables and Leachables (E&L) provides an exposure-based approach for evaluating potential toxicological concerns from low-level chemical substances when complete compound-specific toxicity information may not be available. It is particularly relevant when trace-level organic leachables are detected from pharmaceutical packaging, manufacturing components, or drug-delivery systems.
Extractables are chemical substances that can be released from packaging, delivery systems, or manufacturing components under deliberately selected laboratory extraction conditions. Leachables are substances that actually migrate into the drug product under normal or simulated manufacturing, storage, and use conditions.
The distinction is important because an extractable represents a potential chemical migrant, whereas a leachable represents a substance that may contribute to actual patient exposure.
Modern analytical technologies such as LC-MS, GC-MS, high-resolution mass spectrometry, and ICP-MS can detect chemical substances at extremely low concentrations. However, analytical detection alone does not establish that a compound represents a meaningful patient safety risk.
TTC helps bridge this gap by providing an exposure-based framework for determining when a detected chemical requires further toxicological assessment.
For pharmaceutical manufacturers, CDMOs, CROs, and analytical laboratories, the appropriate application of TTC requires integration of:
- Chemical identification.
- Analytical concentration.
- Patient exposure.
- Route of administration.
- Treatment duration.
- Mutagenicity.
- Systemic toxicity.
- Local toxicity.
- Available toxicological data.
- Applicable regulatory thresholds.
For peptide and injectable products, specialized E&L programs may be required because packaging and delivery components can have direct contact with the drug product. A dedicated extractables and leachables testing program for peptide injectables can help characterize potential chemical migrants associated with peptide-based parenteral products.
Summary:
- TTC in Extractables and Leachables (E&L) is a risk-based toxicological concept used to evaluate very low chemical exposures when complete compound-specific toxicity data may not be available.
- For relevant mutagenic impurities, the ICH M7 framework provides a commonly used lifetime TTC of 1.5 µg/person/day.
- The draft ICH Q3E framework extends risk-based E&L assessment by considering TTC, Qualification Threshold (QT), Safety Concern Threshold (SCT), PDE, route of administration, exposure duration, and local toxicity.
- TTC should not be treated as a universal toxicological limit for every extractable or leachable.
- A compound-specific Permitted Daily Exposure (PDE) may be more appropriate when adequate toxicological information is available.
- The Analytical Evaluation Threshold (AET) connects toxicological exposure limits with analytical testing and helps establish an appropriate level for chemical characterization.
- A robust E&L program integrates material risk assessment, extractables testing, leachables testing, analytical identification, quantification, exposure assessment, toxicological evaluation, and risk management.
- USP <1663> and USP <1664> provide established frameworks for extractables and leachables assessment.
- The draft ICH Q3E guideline provides a developing international framework for risk-based assessment and control of E&L.
- TTC-based assessment is particularly valuable when E&L compounds are present at trace levels and compound-specific toxicity information is limited.
1: What Is the Threshold of Toxicological Concern?
The Threshold of Toxicological Concern (TTC) is an exposure-based concept used to identify a level of exposure below which a chemical is considered to represent a sufficiently low toxicological concern for a defined endpoint and applicable population, provided that the substance falls within the scope of the relevant TTC framework.
In pharmaceutical impurity assessment, TTC is particularly established for DNA-reactive or mutagenic impurities through ICH M7.
TTC should not be confused with a conventional analytical specification or a concentration at which toxicity suddenly begins.
A simplified E&L decision pathway is:
Analytical detection → Chemical identification → Exposure estimation → Toxicological classification → Appropriate threshold comparison → Risk decision
This approach prevents analytical detection alone from being interpreted as evidence of patient harm.
2: Why Is Threshold of Toxicological Concern Important in Extractables and Leachables Safety Assessment?
TTC is important because E&L studies can identify numerous chemical compounds for which complete compound-specific toxicological datasets are unavailable.
Without an exposure-based framework, every detected chemical could potentially require extensive toxicological testing. TTC provides a structured way to prioritize compounds according to exposure and toxicological concern.
The major benefits include:
- Establishing an exposure-based point of comparison.
- Prioritizing potentially relevant leachables.
- Supporting toxicological decision-making.
- Connecting analytical findings with patient exposure.
- Supporting risk-based control strategies.
- Helping determine when additional toxicological data may be needed.
- Avoiding unnecessary testing when exposure is demonstrably very low.
The objective is not simply to determine whether a chemical is present. The objective is to determine whether the patient exposure associated with that chemical requires additional action.
3: Threshold of Toxicological Concern in Extractables and Leachables: How Does the Concept Work?
The practical application of TTC in Extractables and Leachables starts with determining the expected patient exposure to an identified or unidentified chemical.
For a liquid product, a simplified exposure calculation is:
Daily exposure (µg/day) = Leachable concentration (µg/mL) × Daily administered volume (mL/day)
For a solid dosage form:
Daily exposure (µg/day) = Leachable amount per unit × Number of units administered per day
For example, assume a leachable is present at 0.5 µg/mL and the patient receives 10 mL/day:
Daily exposure = 0.5 µg/mL × 10 mL/day = 5 µg/day
The calculated exposure can then be compared with the appropriate toxicological threshold.
However, this comparison should only be made after considering:
- Chemical identity.
- Mutagenic potential.
- Route of administration.
- Treatment duration.
- Systemic toxicity.
- Local toxicity.
- Patient population.
- Available toxicological data.
- Applicable TTC, QT, SCT, or PDE.
- Potential cumulative exposure.
4: TTC vs PDE vs QT vs SCT: What Is the Difference?
TTC, PDE, QT, and SCT serve different purposes within a risk-based E&L assessment.
| Concept | Primary purpose | Typical application |
|---|---|---|
| TTC | Exposure threshold for defined toxicological concern | Particularly relevant to certain mutagenic-risk assessments |
| PDE | Compound-specific permitted daily exposure | When adequate compound-specific toxicology is available |
| QT | Qualification threshold for systemic toxicity | Used for evaluating non-mutagenic systemic toxicity |
| SCT | Product-specific safety concern threshold | Trigger for safety assessment of relevant leachables |
| AET | Analytical Evaluation Threshold | Translates exposure-based concepts into analytical testing targets |
The draft ICH Q3E framework describes the SCT as a threshold below which a leachable exposure is considered sufficiently low to present negligible mutagenic and non-mutagenic toxicity concerns within the framework. It also provides separate TTC and QT values according to exposure duration and route.
Therefore, it is scientifically inappropriate to automatically apply “TTC” to every detected extractable or leachable.
5: What Is the 1.5 µg/day Threshold of Toxicological Concern in Pharmaceutical Impurity Assessment?
The 1.5 µg/person/day value is widely recognized in pharmaceutical impurity assessment as the TTC associated with relevant mutagenic impurities under a lifetime exposure framework.
The draft ICH Q3E table also lists 1.5 µg/day as the TTC for exposure durations greater than 10 years for oral and parenteral/dermal-transdermal/inhalation systemic exposure categories.
However, 1.5 µg/day is not a universal E&L acceptance limit.
The appropriate toxicological threshold depends on:
- Toxicological endpoint.
- Exposure duration.
- Route of administration.
- Chemical classification.
- Availability of compound-specific toxicity data.
- Applicable regulatory framework.
Consequently, an E&L assessor should not compare every detected compound against 1.5 µg/day without first establishing whether that threshold applies.
6: How Does Treatment Duration Affect Threshold of Toxicological Concern ?
Treatment duration is an important factor because the applicable systemic toxicity threshold can vary with the duration of patient exposure.
The draft ICH Q3E framework currently provides the following systemic toxicity thresholds:
| Exposure duration | Oral TTC | Parenteral, Dermal/Transdermal, Inhalation TTC |
|---|---|---|
| >10 years | 1.5 µg/day | 1.5 µg/day |
| >1 to 10 years | 10 µg/day | 10 µg/day |
| >1 month to 1 year | 20 µg/day | 20 µg/day |
| ≤1 month | 120 µg/day | 120 µg/day |
These are values from the draft ICH Q3E framework and should not be presented as final universally implemented regulatory requirements.
This duration-dependent approach demonstrates why E&L toxicological assessment must consider the intended clinical use of the pharmaceutical product.
7: How Does Route of Administration Affect Threshold of Toxicological Concern in E&L Assessment?
Route of administration can substantially affect the toxicological interpretation of a leachable.
A chemical entering the body through an injectable, inhalation, ophthalmic, oral, or dermal route may have different systemic and local exposure characteristics.
The draft ICH Q3E framework therefore includes both systemic and local toxicity thresholds.
Examples of draft local toxicity thresholds include:
- Topical ophthalmic: 20 ppm
- Subcutaneous/intradermal: 50 ppm
- Dermal/transdermal: 500 ppm
- Inhalation: 5 µg/day
- Intracerebral, intrathecal, epidural, and intraocular: compound-specific evaluation
These values demonstrate why a single universal TTC cannot adequately represent every route of administration.
For injectable products, the toxicological evaluation should therefore consider both systemic exposure and route-specific considerations.
8: How Are Mutagenic and Non-Mutagenic Leachables Evaluated?
Mutagenic and non-mutagenic leachables require different toxicological assessment pathways.
Mutagenic or Potentially Mutagenic Leachables
Assessment may include:
- Chemical structure evaluation.
- ICH M7 classification.
- Ames testing or other appropriate experimental data.
- (Q)SAR assessment.
- Identification of structural alerts.
- Acceptable intake determination.
- Applicable TTC assessment.
- Exposure-duration considerations.
The purpose is to determine whether the compound presents a DNA-reactive or mutagenic concern at the estimated patient exposure.
Non-Mutagenic Leachables
For non-mutagenic substances, assessment may focus on:
- Systemic toxicity.
- Compound-specific PDE.
- QT.
- Local toxicity.
- NOAEL or LOAEL information.
- Read-across.
- Toxicological database quality.
- Exposure duration.
- Route-specific considerations.
Therefore, TTC should not replace compound-specific toxicology when adequate toxicological information is available.
9: How Does ICH Q3E Change the Approach to Threshold of Toxicological Concern in Extractables and Leachables?
The draft ICH Q3E Guideline for Extractables and Leachables represents an important development toward harmonizing E&L assessment internationally.
FDA published the Q3E draft guidance in November 2025 and identifies it as a draft, non-binding document that is not for implementation. The draft provides a holistic framework for assessment and control of E&L.
The draft framework can be viewed as:
Material assessment → Extractables characterization → Leachables assessment → Identification → Exposure assessment → Toxicological classification → SCT/QT/PDE/TTC assessment → Control strategy
The framework considers:
- Mutagenicity.
- Systemic toxicity.
- Local toxicity.
- Route of administration.
- Duration of exposure.
- Patient exposure.
- Chemical identity.
- Compound-specific toxicological information.
- Product-specific risk.
Because Q3E remains a draft regulatory framework as of September 2026, companies should verify its current status and applicable regional requirements when preparing regulatory submissions.
10: What Are Class 1, Class 2 and Class 3 Leachables?
The draft ICH Q3E framework categorizes leachables according to their toxicological characteristics and appropriate risk-management approach.
Class 1 – Leachables to Be Avoided
Class 1 includes certain high-concern substances, including certain mutagenic carcinogens and compounds with potent non-mutagenic toxicity concerns.
For Class 1 substances, the framework indicates that avoiding materials that may leach such compounds is generally the preferred approach where feasible. When use is unavoidable, a compound-specific safety limit should be established.
Class 2 – Leachables to Be Limited
Class 2 represents the intermediate category for compounds that require limitation according to the applicable toxicological threshold.
Depending on the compound, this may involve TTC, QT, or another scientifically justified safety threshold.
Class 3 – Leachables With Relatively Low Toxic Potential
Class 3 compounds are characterized by relatively low systemic toxicity.
The draft Q3E document describes Class 3 leachables as compounds with chronic parenteral PDEs of at least 1 mg/day under the specified methodology. It states that Class 3 leachables would not require further safety qualification when observed below 1 mg/day, subject to the framework’s conditions.
11: How Is Threshold of Toxicological Concern Converted Into an Analytical Evaluation Threshold?
The Analytical Evaluation Threshold (AET) connects toxicological exposure assessment with analytical testing.
A simplified relationship is:
AET (µg/mL) = Safety Threshold (µg/day) ÷ Daily Drug Product Volume (mL/day)
For example:
Safety threshold = 1.5 µg/day
Daily product volume = 10 mL/day
Therefore:
AET = 1.5 ÷ 10 = 0.15 µg/mL
An analytical program should then be designed to detect and characterize compounds around the relevant threshold.
The actual AET calculation may also consider:
- Maximum daily dose.
- Number of units.
- Extraction volume.
- Surface area.
- Concentration factors.
- Analytical response factors.
- Analytical uncertainty.
- Study-specific considerations.
The draft Q3E document specifically discusses analytical uncertainty factors when semi-quantitative methods are used, recognizing that analytes can have different detector response factors compared with reference standards.
12: Why Is Analytical Sensitivity Critical in TTC-Based E&L Programs?
TTC-based risk assessment is only useful when analytical methods can detect and characterize compounds around the relevant analytical threshold.
E&L programs commonly use complementary analytical technologies such as:
- GC-MS for volatile and semi-volatile organic compounds.
- Headspace GC-MS for volatile compounds.
- LC-MS/MS for non-volatile and semi-volatile compounds.
- High-resolution LC-MS for unknown screening and accurate-mass characterization.
- ICP-MS for elemental and metal analysis.
- FTIR for material characterization.
- NMR for structural elucidation of challenging compounds.
No single analytical technique can characterize every potential extractable or leachable. Method selection should therefore reflect the chemical characteristics of the materials, formulation, packaging system, and expected migrants.
A detailed GMP-compliant extractables and leachables E&L study can help demonstrate how analytical characterization and study design can be integrated into a structured E&L program.
13: What Happens When a Leachable Exceeds the TTC?
Exceeding a TTC does not automatically mean that a drug product is unsafe. Instead, it indicates that further toxicological evaluation or a more specific safety assessment may be necessary.
Potential next steps include:
- Confirming the analytical result.
- Confirming compound identity.
- Reviewing possible analytical artifacts.
- Calculating patient exposure.
- Assessing mutagenicity.
- Conducting (Q)SAR evaluation.
- Reviewing available toxicological literature.
- Performing read-across where scientifically justified.
- Establishing a compound-specific PDE.
- Conducting additional toxicological testing when necessary.
- Investigating the source of the leachable.
- Implementing appropriate controls.
Therefore:
TTC is a decision point, not automatically a product specification.
The ultimate assessment should be based on the complete scientific and toxicological context.
14: TTC and Unknown Leachables: Why Identification Matters
Unknown leachables represent one of the most challenging areas of E&L safety assessment.
A chromatographic peak without structural identification provides limited information for toxicological assessment.
A typical unknown-identification workflow can involve:
Accurate mass → Molecular formula → Isotope pattern → MS/MS fragmentation → Database searching → Structural assignment → Reference standard confirmation
High-resolution mass spectrometry can provide accurate-mass information that helps narrow potential molecular formulas, while MS/MS fragmentation provides additional structural information.
For volatile compounds, GC-MS can provide complementary structural information.
When a compound remains unidentified, its assessment may consider:
- Analytical evidence.
- Estimated concentration.
- Patient exposure.
- Potential chemical class.
- Structural information.
- Applicable toxicological thresholds.
- Uncertainty associated with identification.
Unknown compounds above the applicable analytical threshold should not simply be dismissed because their structures are unavailable.
15: How Does TTC Support Risk-Based E&L Control?
A risk-based approach prevents E&L programs from becoming purely analytical exercises.
The complete process can be summarized as:
1. Material Risk Assessment
Identify polymers, elastomers, adhesives, coatings, lubricants, processing aids, filters, tubing, connectors, and other materials that may contribute chemical substances.
2. Extractables Study
Characterize substances released from the materials under controlled laboratory extraction conditions.
3. Leachables Study
Determine which compounds migrate into the drug product under relevant storage and use conditions.
4. Analytical Identification
Identify and quantify relevant compounds using appropriate analytical platforms.
5. Exposure Assessment
Calculate potential patient exposure using concentration and dosing information.
6. Toxicological Assessment
Apply the appropriate TTC, QT, PDE, local toxicity threshold, or other scientifically justified approach.
7. Risk Characterization
Determine whether additional toxicological qualification, investigation, or control is required.
8. Control Strategy
Implement appropriate material, supplier, packaging, manufacturing, or formulation controls.
For products manufactured using single-use technologies, this assessment becomes particularly important because polymers, tubing, bags, filters, connectors, and other components may contribute potential extractables.
ResolveMass provides additional information on single-use systems and GMP extractables and leachables testing and the analytical considerations involved in managing leachables risk in biopharmaceutical manufacturing.
16: How Do Single-Use Systems Affect E&L Safety Assessment?
Single-use systems can introduce additional E&L considerations because pharmaceutical manufacturing may involve multiple polymeric and elastomeric components that come into contact with process streams.
Potential components include:
- Single-use bags.
- Tubing.
- Filters.
- Connectors.
- Sampling systems.
- Mixing components.
- Storage assemblies.
- Processing assemblies.
The E&L risk assessment should consider the material composition, contact time, temperature, surface-area-to-volume ratio, process conditions, and nature of the contacting solution.
For a more detailed discussion of the regulatory and analytical considerations, see extractables and leachables requirements for single-use bioprocessing.
This is particularly relevant for biologics and other complex pharmaceutical manufacturing processes where multiple single-use components may be used across the production workflow.
17: What Are Common Mistakes in Applying TTC to E&L Assessment?
1. Treating TTC as a Universal Limit
TTC is not a universal acceptance criterion for every chemical, route, or product.
2. Automatically Applying 1.5 µg/day
The 1.5 µg/day value should not automatically be applied to every leachable.
The applicable toxicological category must first be established.
3. Ignoring Treatment Duration
Exposure duration can materially affect the applicable threshold.
4. Ignoring Local Toxicity
Systemic exposure alone may not capture risks associated with ophthalmic, inhalation, dermal, or other routes.
5. Applying Generic TTC When a PDE Is Available
When adequate compound-specific toxicological information exists, a PDE may provide a more appropriate basis for risk assessment.
6. Using an AET Without Considering Actual Dosing
The AET should be connected to the product’s maximum daily exposure and applicable toxicological threshold.
7. Assuming Detection Equals Toxicity
Detection establishes the presence of a chemical; it does not automatically establish a clinically meaningful safety risk.
8. Failing to Investigate Unknowns
Unknown compounds above the applicable analytical threshold require scientifically justified evaluation.
9. Ignoring Packaging or Manufacturing Changes
Changes to formulation, packaging, manufacturing processes, suppliers, or delivery systems can alter the E&L profile and may require reassessment.
Regulatory Landscape for TTC and E&L Assessment
ICH Q3E
The draft ICH Q3E Guideline for Extractables and Leachables provides a dedicated framework for E&L assessment and control.
FDA published the draft guidance in November 2025 and identifies it as a Draft Level 1 Guidance containing non-binding recommendations and marked “Not for implementation.”
Therefore, as of September 2026, the article should refer to ICH Q3E as a draft framework unless and until a final guideline becomes effective.
ICH M7
ICH M7 provides a framework for assessing and controlling DNA-reactive or mutagenic impurities and is an important reference when evaluating mutagenic concerns associated with pharmaceutical impurities.
USP <1663>
USP <1663> provides a framework for designing and executing extractables assessments associated with pharmaceutical packaging and delivery systems.
It emphasizes scientifically justified study design rather than prescribing one universal extraction protocol.
USP <1664>
USP <1664> addresses the assessment of drug-product leachables associated with pharmaceutical packaging and delivery systems.
FDA Container-Closure Considerations
FDA’s current container-closure guidance framework emphasizes evaluating packaging systems in the context of the specific drug product, dosage form, and intended use. FDA also published a new draft guidance on Container Closure Systems for Human Drugs and Biological Products in August 2026, which remains a draft and is not for implementation.
For generic injectables and other parenteral products, container-closure considerations can therefore form an important part of the broader E&L strategy.
Frequently Asked Questions About TTC in Extractables and Leachables
What is TTC in Extractables and Leachables?
TTC in Extractables and Leachables is an exposure-based toxicological concept used to evaluate potential risks from low-level chemical substances when complete compound-specific toxicity information may not be available.
What is the difference between TTC and PDE?
TTC is an exposure-based threshold used for defined toxicological situations. PDE is a compound-specific permitted daily exposure derived from available toxicological data.
What is SCT in E&L assessment?
SCT, or Safety Concern Threshold, is a product-specific threshold used in the draft ICH Q3E framework to identify leachables whose exposure requires toxicological safety assessment.
Does a leachable above TTC mean the drug product is unsafe?
Not automatically. Exceeding an applicable threshold indicates that further toxicological evaluation or a more specific safety assessment may be necessary.
How is AET related to TTC?
AET converts an exposure-based safety threshold into an analytical concentration target using product dosing information and other relevant study factors.
Can TTC be used for unknown leachables?
TTC may contribute to the assessment of compounds with limited toxicological information, but unknowns require careful consideration of analytical evidence, exposure, structural information, and applicable toxicological criteria.
Conclusion: The Future of TTC in Extractables and Leachables
TTC in Extractables and Leachables provides an important bridge between highly sensitive analytical detection and scientifically meaningful toxicological risk assessment.
Its value is greatest when it is used as part of a structured decision-making framework rather than as a universal pass/fail limit.
The emerging ICH Q3E framework strengthens this approach by integrating TTC, QT, PDE, local toxicity, exposure duration, route of administration, leachable classification, and product-specific SCT concepts into a comprehensive E&L risk-management strategy.
For pharmaceutical developers, the central principle is:
Detect → Identify → Quantify → Calculate Exposure → Assess Toxicological Hazard → Compare With the Appropriate Threshold → Control Where Necessary
A scientifically robust E&L program therefore combines advanced analytical capabilities with toxicological interpretation and risk-based decision-making.
This becomes especially important for:
- Peptide injectables.
- Generic injectable drugs.
- Biologics.
- Single-use bioprocessing systems.
- Complex container-closure systems.
- Prefilled syringes.
- Drug-delivery devices.
- Long-term pharmaceutical products.
ResolveMass Laboratories Inc. supports pharmaceutical and biopharmaceutical development programs with analytical characterization and E&L testing strategies designed to generate actionable data for product development, quality assessment, and regulatory support.
Frequently Asked Questions:
No. The 1.5 µg/day value is associated with the applicable mutagenic impurity framework and long-term exposure. E&L assessment may require TTC, QT, PDE, local toxicity thresholds, or compound-specific assessment depending on the chemical and product.
Yes. Injectable products can involve direct systemic administration, making assessment of potential migrants from container-closure systems, delivery components, and manufacturing-contact materials particularly important.
Yes. Route of administration can influence both systemic exposure and local toxicity. Therefore, oral, parenteral, inhalation, ophthalmic, dermal, and other routes require appropriate consideration.
Single-use systems contain multiple polymeric and elastomeric components that can potentially contribute extractables and leachables to pharmaceutical manufacturing processes. Their material composition, contact conditions, and intended use should therefore be considered in the E&L risk assessment.
Reference
- Hayashi T, Fukushima A, Hirose A. P31-11 Threshold of toxicological concern (TTC) for E&L with less-than-lifetime exposure. Toxicology Letters. 2025 Sep 1;411:S415.https://www.sciencedirect.com/science/article/pii/S037842742502538X
- Dewhurst I, Renwick AG. Evaluation of the threshold of toxicological concern (TTC)–challenges and approaches. Regulatory Toxicology and Pharmacology. 2013 Feb 1;65(1):168-77.https://www.sciencedirect.com/science/article/pii/S0273230012000542
- Zaleski R, Embry M, McKee R, Teuschler LK. Exploring the utility of the threshold of toxicological concern (TTC) as a screening approach for complex substances. Regulatory Toxicology and Pharmacology. 2021 Dec 1;127:105051.https://www.sciencedirect.com/science/article/pii/S0273230021001926
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