Human Radiolabeled Mass Balance Studies Supporting New Drugs Approved by FDA Between 2019 and 2023

1. Introduction

Human radiolabeled mass balance studies are essential for understanding the absorption, distribution, metabolism, and excretion (ADME) of investigational drugs. The Food and Drug Administration (FDA) guidance recommends conducting these studies for new molecular entities (NMEs) to inform drug development programs. Herein, we analyzed new drug applications (NDAs) approved between 2019–2023, and results showed continued alignment with previous 2014–2018 findings and with recommendations in the FDA guidance. These studies remain foundational to drug development but are evolving to incorporate scientific innovations.

2. Importance and Prior Experience with Human Radiolabeled Mass Balance Studies

Human radiolabeled mass balance studies, which characterize the ADME of investigational drugs, are critical components of clinical pharmacology programs [1, 2, 3, 4]. These studies provide essential information for understanding drug disposition, informing metabolite safety assessments, and guiding drug–drug interaction evaluations during drug development.

The regulatory relevance of mass balance studies has been increasingly recognized. The FDA published a final guidance titled “Clinical Pharmacology Considerations for Human Radiolabeled Mass Balance Studies” in July 2024 [5]. This guidance represents the Agency’s current thinking on designing, conducting, and reporting mass balance studies, emphasizing that such studies should generally be conducted for all NMEs to inform subsequent drug development programs. Similarly, the European Medicines Agency (EMA) has published a regulatory guideline for conducting and interpreting in vivo mass balance studies [6]. A review of NDAs submitted to the EMA highlighted common issues in mass balance study conduct (e.g., lack of study, incomplete sample collection) that led to suboptimal information generation, underscoring the need for continued evaluation of current practices [7].

The FDA guidance was informed, in part, by comprehensive analyses of mass balance studies supporting NME NDAs approved between 2014 and 2018, which demonstrated that about two‐thirds of approved new drugs relied on mass balance study findings to characterize pharmacokinetic properties and inform the subsequent development programs [8]. That survey also revealed consistent practices across approximately 100 radiolabeled mass balance studies, which typically enrolled on average six evaluable subjects, were predominantly conducted in healthy (90%) volunteers, men only (86%), utilized single‐dose design (97%), and employed the final approved route of administration (98%). The FDA guidance reinforces many of these practices while providing updated recommendations on study design, timing of study conduct, and reporting requirements for regulatory submissions.

3. Human Radiolabeled Mass Balance Studies Supporting New Drugs Approved Between 2019 and 2023

To provide updated insights into more recent mass balance study practices and their alignment with the FDA recommendations, we conducted a comprehensive analysis of human radiolabeled mass balance studies supporting NME NDAs approved between 2019 and 2023. Mass balance study information was collected from FDA clinical pharmacology reviews, original approved drug labeling from Drugs@FDA [9], and final clinical study reports included in the NDAs. Overall, 174 NME NDAs were approved during this period, of which 119 (68%) had information from mass balance studies that were conducted as part of the drug development program (Table 1). For these 119 drugs, a total of 124 mass balance studies were reported, including four drugs that had multiple mass balance studies. All studies utilized a radiolabeled mass balance design, except for one that used a ¹⁹F NMR methodology. For the remaining 55 drugs (32%), mass balance studies were not conducted due to well‐characterized ADME pathways, minimal systemic exposure (e.g., topical or ophthalmic products), radioactive diagnostic or therapeutic agents, or predominantly renal excretion as the unchanged parent drug. Of note, one drug had a mass balance study ongoing at the time of NDA submission. Some examples of specific study characteristics related to mass balance studies performed to support NME NDAs approved between 2019 and 2023 are provided in Table 2.

TABLE 1.

Overview of human radiolabeled mass balance studies supporting the initial approval of new drugs: 2019–2023 and 2014–2018.

Category	NME NDAs approved 2019–2023 a	NME NDAs approved 2014–2018 b	Overall NME NDAs approved 2014–2023 c
NME NDAs (%)
With MB studies	68%	66%	67%
Without MB studies	32%	34%	33%
With multiple MB studies	2%	2%	2%
Population (%) Healthy vs. Patient
Healthy	97%	90%	94%
Patients	2%	10%	5%
Healthy and Patients	1%	0	< 1%
Population (%) Sex
Male	90%	86%	88%
Female	3%	2%	2%
Male and Female	7%	12%	10%
Number of participants (n)
Mean	7	6	7
Median	6	6	6
Mode	6	6	6
Range	3–17	3–16	3–17
Dose (%)
Single	98%	97%	98%
Steady state	2%	3%	2%
MB route of administration (%)
Same route as approved	98%	98%	98%
Different route than approved	2%	2%	2%

^a 174 new molecular entity (NME) new drug applications (NDAs) were approved between 2019 and 2023 and included a total of 124 mass balance (MB) studies.

^b 154 NME NDAs were approved between 2014 and 2018 and included a total of 104 mass balance studies (see reference [8] for more details).

^c 328 NME NDAs were approved between 2014 and 2023 and included a total of 228 mass balance studies.

TABLE 2.

Select examples of study characteristics of mass balance studies supporting the initial approval of new drugs between 2019 and 2023.

Characteristic	Example	Description
Population	Tazemetostat	Mass balance information from cancer patients
	Lurbinectedin	Mass balance information from cancer patients
	Difelikefalin	Mass balance information from patients with end stage renal disease on hemodialysis and in healthy subjects
Route of administration	Zavegepant	Approved as nasal spray and a different route was utilized for the mass balance study because of practical considerations (a single‐dose study to assess the mass balance after intravenous dosing of radiolabeled drug)
Long half‐life	Oteseconazole	Median terminal half‐life is 138 days. Subjects remained at the clinical study site for 17 days post‐dosing, returning for 5 in‐patient follow‐up visits at prespecified timepoints through ~10 months post‐dosing. Recovery of radioactivity in urine and feces was interpolated between scheduled study visits
Multiple dose	Voxelotor	Multiple doses of the drug to achieve steady state followed by a single oral dose administration of radiolabeled drug
Two‐part mass balance and absolute bioavailability studies	Tazemetostat	Absolute bioavailability was evaluated using intravenous radiolabeled (< 1 μCi) microdose along with the mass balance study for the radiolabeled oral drug. Radioactivity bioanalysis was performed by accelerator mass spectrometry (AMS)
	Rimegepant
	Abrocitinib
Alternative approaches	Nirmatrelvir	Mass balance and metabolite profiling were performed via 19F‐NMR and LC–MS/MS using unlabeled nirmatrelvir (coadministered with ritonavir)

Note: For further information refer to the drug reviews available from Drugs@FDA or other publicly available sources (e.g., clinicaltrials.gov).

Generally, mass balance studies can be conducted in healthy adult participants [5]. Consistent with prior reports [7, 8], the majority (97%) of the mass balance studies in our analysis were performed in healthy participants, while 2% were conducted in cancer patients and 1% in both healthy participants and the intended patient population (subjects undergoing hemodialysis). Similarly, consistent with prior reports [7, 8], the majority (90%) of the mass balance studies enrolled (i.e., based on the study inclusion and exclusion criteria) only male participants, 7% of studies allowed for inclusion of either men or women, and 3% of studies specified enrollment of only women (for drugs with sex‐specific indication). Though the sample size of a mass balance study is dependent on the drug (accounting for factors such as PK variability) and the study objectives, in general, it is recommended that a mass balance study include at least six evaluable participants as having fewer evaluable participants may limit the interpretability of the data [5]. In this analysis, the mean, median, and mode for the sample size were 7, 6, and 6, respectively, while the range was 3–17 participants.

For mass balance studies, the route of administration should include the final intended routes of administration, unless precluded by practical considerations [5]. Consistent with this recommendation, 98% of studies utilized the same route of administration as the approved route. For 2% of studies, a different route (intravenous injection) was utilized because of practical considerations (e.g., inhalation product).

To obtain mass balance information, a single‐dose study is typically sufficient [5]. When a single‐dose study is not feasible or if the drug and/or active metabolite exhibit time‐dependent pharmacokinetics, a multiple‐dose study can be considered. For a drug that exhibits time‐dependent pharmacokinetics, study participants receive a single radiolabeled dose of the drug after achieving steady state with non‐radiolabeled doses of the drug. Because this method evaluates the clearance pathway of the radiolabeled drug, concurrent bioanalytical evaluation of the non‐radiolabeled moieties of interest can help understand the accumulation of the parent drug and relevant metabolites at steady‐state when interpreting the study results [5]. In this analysis, consistent with the recommendation, 98% of the studies used a single dose. For the remaining 2%, in one instance, a single‐dose study was infeasible because it was conducted in patients and in the other instance, the sponsor deemed it suboptimal to conduct a single‐dose study because it would lead to incomplete identification and quantification of metabolites due to time‐dependent pharmacokinetics of the parent drug and its metabolites.

For an orally administered drug, it is difficult to ascertain the route of elimination without knowing the absolute bioavailability of the drug, especially for drugs excreted extensively in feces as unchanged parent drug. When only the oral formulation is being developed, it is possible to conduct an absolute bioavailability study using an intravenous radiolabeled microdose along with the mass balance study for the radiolabeled oral drug as a two‐part study in a single protocol. Overall, 22 studies utilized a 2‐part approach adding an absolute bioavailability assessment to the mass balance study.

The radioactivity dose administered in humans is typically estimated from nonclinical dosimetry studies, and should follow appropriate regulations and applicable guidelines [5]. In this analysis, the radioactivity dose range was 200 nCi to 610 μCi. The most common radioactivity dose was 1 μCi to < 100 μCi (36%), 100 μCi (32%), > 100 μCi (28%), and less frequently 0.1 to < 1 μCi (4%). Accelerator mass spectrometry (AMS) is often used for radioactivity bioanalysis in studies with low radioactivity doses (< 1 μCi), given its higher sensitivity compared to conventional liquid scintillation counting. Low radioactivity doses may be driven by safety considerations regarding radioactivity exposure, or the incorporation of an absolute bioavailability assessment.

The total recovery of radioactivity in the urine and feces should exceed 90% of the administered dose [5]. This high level of recovery ensures that elimination pathways are fully understood and rules out drug accumulation in the body. When recovery is low, it is essential to investigate potential reasons to ensure result reliability. In this analysis, recovery of ≥ 90% was reported in 54% of the studies, 85% to < 90% was reported in 22%, recovery of 80% to < 85% was reported in 10%, and < 80% recovery was reported in 14% of the studies. For some drugs, mass balance studies were repeated due to low recovery. Potential reasons for incomplete recovery in some studies included loss via expired air and long half‐life of the drug or its metabolites. In some cases, no reason was identified; however, excretion routes were clear (e.g., primarily fecal, < 1% renal), and supporting nonclinical studies showed no tissue accumulation.

The general recommendation is that the dose used in the mass balance study should be the final intended dose [5]. If the final intended dose is not known at the time of conducting the mass balance study, a dose in the anticipated therapeutic dose range or in the pharmacokinetic linearity range can be used to allow for extrapolation of mass balance data. In this analysis, 64% of the studies used the same approved dose, while 11% used a lower dose, and 25% used a higher dose. Of those that used a dose different from the approved dose, most of the studies used a dose in the PK linearity range.

4. Conclusion and Looking Forward

Mass balance studies remain the cornerstone of drug development programs, providing a comprehensive understanding of drug ADME properties in humans. These studies continue to be fundamental for fulfilling regulatory requirements and supporting final drug approval, as evidenced by their consistent use in two‐thirds of approved NME NDAs. Our analysis of NDAs approved between 2019 and 2023 showed similar results to our previous analysis of NDAs approved between 2014 and 2018 (Table 1) and demonstrated alignment with the FDA final guidance on mass balance studies [5, 8].

Our analyses identified emerging trends that reflect the ongoing evolution of mass balance study practices as sponsors incorporate new scientific approaches and innovations. Notable developments include increased incorporation of absolute bioavailability assessments along with the mass balance study (18% vs. 13% in 2014–2018) and the use of ¹⁹F NMR methodology as an alternative approach to the traditional radiolabeled mass balance study (Table 2). The expansion of methodological options, including ¹⁹F NMR spectroscopy and AMS, is broadening the ADME toolkit and offering alternatives to conventional radiolabeled approaches. These observations underscore the dynamic nature of the field and point toward several key areas of future development.

Streamlined study designs that combine mass balance with absolute bioavailability assessments maximize information yield while minimizing subject burden and development timelines. These advances, coupled with enhanced in silico modeling capabilities and physiologically based pharmacokinetic approaches, are expected to further enhance the generation of comprehensive human ADME information, ultimately supporting the success of drug development programs [4].

Funding

The authors have nothing to report.

Disclosure

Disclaimer: This manuscript reflects the views of the author and should not be construed to represent FDA's views or policies.

Conflicts of Interest

The authors declare no conflicts of interest.

Ramamoorthy A., Chen A., Chow E. C. Y., et al., “Human Radiolabeled Mass Balance Studies Supporting New Drugs Approved by FDA Between 2019 and 2023,” Clinical and Translational Science 19, no. 8 (2026): e70679, 10.1111/cts.70679.