FDA Genotoxicity And Carcinogenicity Assessment Of ENDS: A Regulatory Deep Dive For PMTA Success

Electronic Nicotine Delivery Systems (ENDS) remain under intense regulatory scrutiny by the U.S. Food and Drug Administration (FDA) Centre for Tobacco Products (CTP). Among the most critical scientific pillars of a Premarket Tobacco Product Application (PMTA) is the assessment of genotoxicity and carcinogenicity, particularly in relation to long-term cancer risk. 

In 2024, the FDA released two pivotal internal memoranda that provide unprecedented insight into how regulators expect manufacturers to evaluate, categorize, and quantify carcinogenic risk arising from ENDS ingredients, leachable, and aerosol constituents. 

These memoranda clearly demonstrate that traditional in vitro testing alone is no longer sufficient. Instead, FDA endorses a component-based, tiered Weight-of-Evidence (WoE) approach, combined with Excess Lifetime Cancer Risk (ELCR) calculations. 

This blog by Maven Regulatory Solutions provides a detailed, practical interpretation of FDA expectations and what ENDS manufacturers must do to align their PMTA submissions with current regulatory thinking. 

Limitations of Traditional Genotoxicity Testing for ENDS 

Many ENDS manufacturers routinely conduct standard in vitro genotoxicity assays, including: 

• Bacterial reverse mutation (Ames) tests 
• Mammalian chromosomal aberration assays 
• Micronucleus assays 

While the FDA acknowledges the scientific value of these tests particularly when positive results are observed it has emphasized key limitations when applied to complex mixtures such as e-liquids and aerosols: 

FDA-Identified Limitations 

• Results cannot support relative genotoxicity comparisons 
• Results cannot be used for quantitative cancer risk assessment 
• Whole-mixture testing does not identify individual chemical drivers of risk 

As a result, FDA has moved decisively toward a chemical-by-chemical risk assessment framework. 

FDA’s Component-Based Weight-of-Evidence (WoE) Framework 

The FDA memorandum outlines a tiered classification system for each chemical constituent in an ENDS product. This approach integrates: 

• In vitro genotoxicity data 
• In vivo carcinogenicity studies 
• Human epidemiological evidence (where available) 
• IARC and U.S. EPA classifications 
• Computational toxicology ((Q)SAR) 
• New Approach Methodologies (NAMs) 

Carcinogenic Risk Classification – Tiered Framework

Beyond Ames: FDA’s Expectations for Non-Ames Endpoints 

Although bacterial mutagenicity remains widely discussed, FDA’s memo clarifies that non-Ames endpoints are equally important, especially when data gaps exist. 

Computational & Experimental Endpoints Encouraged by FDA 

• Chromosomal aberrations 
• Micronucleus formation 
• Mouse lymphoma mutation assays 
• Rodent carcinogenicity 
• In silico carcinogenicity prediction models 

If a chemical is adequately assessed across all relevant endpoints and shows no concern, it may be classified as Tier 5. Otherwise, Tier 4 classification is expected. 

FDA ELCR Framework: Translating Hazard into Quantified Risk 

Threshold of Toxicological Concern (TTC) 

In its second 2024 memorandum, FDA confirms that a TTC of 1.5 µg/day is appropriate for ENDS cancer risk assessment. This TTC corresponds to a risk level of: 

1 additional cancer case per 100,000 lifetime users 

Key Regulatory Implications 

• Constituents below TTC require no further cancer risk evaluation 
• Constituents above TTC must be included in ELCR calculations 

Cancer Potency Metrics Used by FDA 

For Tier 1–4 chemicals exceeding the TTC, FDA expects use of: 

ELCR Calculation Formula 

ELCR = Estimated Daily Exposure ÷ Cancer Potency Value 

Once calculated for all relevant chemicals, FDA expects a cumulative ELCR (ELCRc) to be derived by summing individual risks. 

Why FDA’s Approach Matters for PMTA Strategy 

Although conservative, this framework allows: 

• Transparent comparisons across ENDS products 
• Benchmarking against combustible tobacco products 
• Consistent regulatory decision-making 

FDA’s publication of multiple toxicology memoranda confirms a clear regulatory priority: 

Cancer risk assessment is central to PMTA authorization decisions 

Looking Ahead: Expanding FDA Toxicology Expectations 

FDA has indicated that additional guidance is in development, likely addressing: 

• Respiratory toxicity 
• Cardiovascular risk 
• Computational toxicology 
• Systems toxicology and NAMs 

Manufacturers that proactively align now will be far better positioned for regulatory success. 

How Maven Regulatory Solutions Supports ENDS & PMTA Programs 

Maven Regulatory Solutions provides specialist FDA-focused toxicology and regulatory support, including: 

• ENDS ingredient toxicological assessments 
• Genotoxicity & carcinogenicity WoE frameworks 
• ELCR modelling and documentation 
• PMTA toxicology module preparation 
• FDA engagement and regulatory intelligence 

Our experts translate complex FDA expectations into clear, defensible submissions. 

Frequently Asked Questions (FAQ) 

Is Ames testing alone sufficient for PMTA submissions? 

No. FDA considers it informative but insufficient for cancer risk assessment. 

Are in silico tools acceptable to FDA? 

Yes, when used appropriately as part of a WoE approach. 

What happens if a chemical exceeds TTC? 

It must be included in ELCR calculations. 

Does Tier 4 mean automatic rejection? 

No, but it triggers quantitative risk assessment requirements. 

Conclusion: Building Defensible PMTAs in a Risk-Focused FDA Landscape 

FDA’s ENDS memoranda clearly signal a shift toward data-integrated, conservative, and transparent cancer risk assessment. Companies that rely on outdated testing paradigms risk delays or denial of PMTA authorization. 

By embracing FDA’s tiered WoE and ELCR frameworks early, manufacturers can reduce regulatory uncertainty, strengthen scientific credibility, and accelerate approval pathways. 

Maven Regulatory Solutions stands ready to support you at every step.