Following on from our first blog post on this topic, we've expanded on our thoughts.

While many manufacturers will routinely subject their e-liquids (or even their aerosol condensates) to standard in vitro genotoxicity testing, for example for bacterial (Ames) mutagenicity and mammalian cell chromosomal damage, the FDA has noted that the results from these assays, while potentially insightful particularly if a positive result is achieved, are limited for such complex mixtures and do not allow for relative genotoxicity comparisons or for cancer risk assessments. The FDA memo therefore describes a component-based approach utilising a tiered weight-of-evidence (WoE) approach whereby , for each chemical constituent of the mixture, the available in vitro and in vivo genotoxicity and carcinogenicity data are taken into account in order to inform on their potential mutagenicity, clastogenicity and carcinogenicity, as well as any carcinogenicity classifications established by the US EPA or International Agency for Research on Cancer (IARC). Where data are lacking, computational tools like (Quantitative) Structure-Activity-Relationship ((Q)SAR) analysis may be used to predict the chemical’s genotoxic and carcinogenic properties. Following such a WoE analysis by the toxicologist, which may incorporate other supporting data like New Approach methodologies (NAMs), each chemical can then be categorised into the following tiers:

  • Tier 1 –Carcinogenic to humans (classified in Category 1 by IARC or Group A by the US EPA)
  • Tier 2 – Likely to be carcinogenic to humans (IARC 2A or US EPA B1/B2)
  • Tier 3 – Suggestive evidence of carcinogenic potential (IARC 2B or US EPA C)
  • Tier 4 – Potential carcinogenic hazard
    • Tier 4A - a positive finding from an in vivo carcinogenicity or genotoxicity study
    • Tier 4B - positive finding from in vitro or in silico Ames mutagenicity assay
    • Tier 4C - positive results from other (i.e., “non-Ames”) in vitro genotoxicity assays
    • Tier 4D - positive (“non-Ames”) in silico or read-across predictions for carcinogenicity or genotoxicity
    • Tier 4E - insufficient data available for Tier 5 classification
  • Tier 5 - Unlikely to contribute to carcinogenic risk of ENDS.

As the majority of chemicals will not have been evaluated by the US EPA or IARC, it is expected that most will fall within Tier 4 (for those with potential genotoxicity or carcinogenicity concerns) or Tier 5 (those considered unlikely to be genotoxic or carcinogenic). 

Whilst there is an abundance of discussion surrounding the bacterial mutagenicity (i.e. Ames) endpoint, the other “non-Ames” endpoints are not so well covered. However, the memo does indicate that, where genotoxicity and carcinogenicity data are lacking, computational predictions for chromosomal damage, chromosomal aberration, micronucleus formation, mouse lymphoma mutations and rodent carcinogenicity could be conducted. This framework suggests that if the chemical has been adequately tested for all genotoxicity and carcinogenicity endpoints, either by experimental or computational methods, and determined to be non-genotoxic and non-carcinogenic, categorisation into “Tier 5” (unlikely to contribute to carcinogenic risk of ENDS) is appropriate. For chemicals with either a genotoxicity or carcinogenicity concern identified, or those lacking adequate data, categorisation into “Tier 4” is appropriate.

According to the FDA, any chemical assigned to Tiers 1-4 may possess carcinogenic potential and therefore should be included in the Excess Lifetime Cancer Risk (ELCR) calculations and overall cancer risk assessment of the ENDS product. This is where the next memo comes in, also published in June 2024, entitled “Calculating Excess Lifetime Cancer Risk in ENDS Premarket Tobacco Product Applications”, in which another tiered hierarchy is described. Importantly, the FDA notes that the Threshold of Toxicological Concern (TTC) of 1.5 µg/day, applicable to a cancer risk management level of 1 additional cancer case in 100,000 consumers, is appropriate for the assessment of ENDS constituents. This means that for any chemical constituent with estimated consumer exposures levels below this TTC, no further evaluation is necessary and any potential carcinogens need not be included in the ELCR calculations. The focus of the cancer risk assessment will therefore be on the genotoxic and/or carcinogenic chemicals with exposures exceeding the TTC. For certain carcinogenic chemicals, Inhalation Unit Risk (IUR) values have already been derived by expert groups like the US EPA. IURs are an estimate of the increased cancer risk from inhalation exposure to a concentration of 1 µg/m3 over a 70-year lifetime. Following an adjustment to convert the IUR to a daily dose that corresponds to an excess cancer risk of 1 in 100,000, these values can be used in the ELCR calculation for that chemical. Where an IUR (or similar cancer potency value) is not available, but carcinogenicity studies have been conducted in laboratory animals, it may be possible to derive such a value (e.g. using the TD50 method). Otherwise, in the absence of such data, the TTC will need to be adopted in the cancer risk calculations. The ELCR for each individual chemical constituent is calculated by dividing the estimated consumer exposure (e.g. in µg/day) by its cancer potency value (either the adjusted IUR or TTC). Once this process has been conducted for all relevant Tier 1-4 chemicals of the ENDS product (e.g., any ingredients, leachables or other aerosol constituents with a potential carcinogenicity hazard), a cumulative ELCRc calculation can be performed by summing all of the individual ELCRs. This cumulative ELCR value ultimately informs on the overall cancer risk of the ENDS product. Although a crude and conservative method, it does allow for simple comparisons of carcinogenic potency to be made, for example with other ENDS or with comparator tobacco products on the market.

This overview demonstrates that the FDA CTP endorses pragmatic and conservative approaches to human health risk assessment as part of the PMTA submission process. The fact that they have released two detailed memos relating to genotoxicity and carcinogenicity assessment reinforces their focus on the assessment of cancer risk. We know that additional regulatory guidance and memorandums are in development by the FDA, and these will, when publicly released, provide further insights into the FDA’s thinking and expectations relating to other important toxicological considerations relevant to these product types, for example respiratory toxicity, cardiovascular risk, and computational toxicology.

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