Clinical Pharmacology   
Considerations for  
Human Radiolabeled 
Mass Balance Studies 
Guidance for Industry 
 
 
DRAFT GUIDANCE 
 
  This guidance document is being distributed for comment purposes only. 
 
Comments and suggestions regarding this draft document should be submitted within 90 days of 
publication in the Federal Register of the notice announcing the availability of the draft 
guidance.  Submit electronic comments to https://www.regulations.gov.  Submit written 
comments to the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 
Fishers Lane, Rm. 1061, Rockville, MD  20852.  All comments should be identified with the 
docket number listed in the notice of availability that publishes in the Federal Register. 
 
For questions regarding this draft document, contact (CDER) Office of Clinical Pharmacology 
Guidance and Policy Team at CDER_OCP_GPT@fda.hhs.gov. 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
May 2022 
Clinical Pharmacology 
 
   
Clinical Pharmacology 
Considerations for  
Human Radiolabeled 
Mass Balance Studies 
Guidance for Industry 
 
 
 
 
 
 
 
 
Additional copies are available from: 
Office of Communications, Division of Drug Information  
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov  
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs  
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
May 2022 
Clinical Pharmacology 
 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
TABLE OF CONTENTS 
   
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 1 
III.  RECOMMENDATIONS FOR HUMAN RADIOLABELED MASS BALANCE 
STUDIES ....................................................................................................................................... 2 
IV.  TIMING OF MASS BALANCE STUDIES .................................................................... 3 
V.  CONSIDERATIONS FOR DESIGNING MASS BALANCE STUDIES .................... 4 
A.  Study Design ................................................................................................................................... 4 
B.  Study Participants .......................................................................................................................... 4 
C.  Administered Radioactivity Dose and Radiolabel Position ........................................................ 4 
D.  Investigational Drug Dose ............................................................................................................. 4 
E.  Route of Administration and Formulation of the Investigational Drug ................................... 5 
F.  Determination of Absolute Bioavailability for Orally Administered Investigational Drugs in 
a Mass Balance Study .................................................................................................................... 5 
G.  Recovery ......................................................................................................................................... 5 
H.  Sample Collection and Handling .................................................................................................. 6 
I.  Parent and Metabolites.................................................................................................................. 6 
J.  Bioanalysis ...................................................................................................................................... 7 
VI.  REPORTING OF HUMAN RADIOLABELED MASS BALANCE STUDY ............ 7 
RESULTS ...................................................................................................................................... 7 
 
 
 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1  Clinical Pharmacology Considerations for Human Radiolabeled 
2  Mass Balance Studies  
3  Guidance for Industry1 
4   
5   
6  This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
7  Administration (FDA or Agency) on this topic.  It does not establish any rights for any person and is not 
8  binding on FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
9  applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff responsible 
10  for this guidance as listed on the title page.   
11   
12   
13   
14   
15  I.  INTRODUCTION  
16   
17  This guidance describes the FDAs recommendations regarding clinical pharmacology 
18  considerations for conducting human radiolabeled mass balance studies of investigational drugs, 
19  including:  (1) deciding whether and when to conduct the study, (2) designing the study, and (3) 
20  reporting results.2  This guidance does not cover animal mass balance studies, safety testing of 
21  drug metabolites, or recommendations for selecting the radioactive dose.   
22   
23  The contents of this document do not have the force and effect of law and are not meant to bind 
24  the public in any way, unless specifically incorporated into a contract.  This document is 
25  intended only to provide clarity to the public regarding existing requirements under the law.  
26  FDA guidance documents, including this guidance, should be viewed only as recommendations, 
27  unless specific regulatory or statutory requirements are cited.  The use of the word should in 
28  Agency guidance means that something is suggested or recommended, but not required. 
29   
30   
31  II.  BACKGROUND 
32   
33  A human radiolabeled (most commonly 14C and 3H) mass balance study is the single most direct 
34  method to obtain quantitative and comprehensive information on the absorption, distribution, 
35  metabolism, and excretion (ADME) of the drug in the human body.  The mass balance study can 
36  provide information to: 
37   
38    Determine the overall pathways of metabolism and excretion of an investigational drug. 
39   
40    Identify circulating metabolites. 
 
1 This guidance has been prepared by the Office of Clinical Pharmacology, Office of Translational Sciences, in the 
Center for Drug Evaluation and Research at the Food and Drug Administration. 
 
2 21 CFR 201.57. 
  1  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
41   
42    Determine the abundance of metabolites relative to the parent or total drug-related 
43  exposure. 
44   
45  The results from mass balance studies help to: 
46   
47    Provide information on which metabolites should be structurally characterized and which 
48  metabolites should undergo nonclinical safety assessment or drug-drug interaction (DDI) 
49  evaluation.3,4 
50   
51    Assess whether renal or hepatic impairment studies or certain DDI studies are 
52  recommended for the investigational drug. 
53   
54    Assess the absolute bioavailability (see section V.F. Determination of Absolute 
55  Bioavailability for Orally Administered Drugs in a Mass Balance Study) and the extent of 
56  absorption of the investigational drug with additional data from other studies 
57  documenting the investigational drugs stability in the gastrointestinal tract. 
58   
59   
60  III.  RECOMMENDATIONS FOR HUMAN RADIOLABELED MASS BALANCE 
61  STUDIES     
62   
63  In general, mass balance studies should be conducted for all new molecular entities, as 
64  information obtained from the mass balance study helps inform the subsequent drug 
65  development program.5  When a human radiolabeled mass balance study is not conducted, the 
66  sponsor should provide adequate justification.  Unless clinical concerns suggest otherwise, a 
67  mass balance study might not be recommended in some circumstances, for example: 
68   
69    Drugs for which mass balance study results can be obtained from acceptable literature 
70  sources or FDA-approved product labeling.  
71   
72    Drugs such as monoclonal antibodies, endogenous substances, and analogs (e.g., 
73  peptides, hormones, oligonucleotide therapeutics) with known metabolism and 
74  elimination pathways based on basic pharmacology and nonclinical ADME information. 
75   
 
3 See the FDA guidance Safety Testing of Drug Metabolites (March 2020).  We update guidances periodically.  For 
the most recent version of a guidance, check the FDA guidance web page at https://www.fda.gov/regulatory-
information/search-fda-guidance-documents. 
 
4 See the FDA guidance In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated 
Drug Interactions (January 2020). 
 
5 For the purposes of this guidance, except where specifically indicated, references to drugs include drugs subject to 
approval under section 505(c) of the Federal Food, Drug, and Cosmetic Act (FD&C Act) (21 U.S.C. 355(c)) and 
biological products subject to licensure under section 351(a) of the Public Health Service (PHS) Act (42 U.S.C. 
262(a)) that are regulated as drugs. 
 
  2  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
76    Drugs with the majority of the dose (i.e., greater than or equal to 90 percent) recovered in 
77  the urine as the unchanged parent drug with minimum metabolism. 
78   
79    Drugs with no or negligible systemic exposures.  
80   
81  When a human radiolabeled mass balance study cannot be conducted (e.g., safety concerns 
82  because of the potential for radiolabeled moieties accumulating in critical organs), the sponsor 
83  should use alternative approaches, such as animal mass balance studies, metabolic profiling 
84  using qualitative techniques, urine collection in phase 1 trials, or in vitro assessments to 
85  characterize the ADME of the investigational drug.  Sponsors should consult with the 
86  appropriate FDA review division when alternative approaches are used. 
87   
88   
89  IV.  TIMING OF MASS BALANCE STUDIES 
90   
91  Mass balance studies should be conducted early in drug development, at the latest before 
92  initiating any late-phase clinical trials.  This timing allows information from the mass balance 
93  studies to be incorporated into the overall development program by: 
94   
95    Providing information on metabolism and excretion pathways.  This information, 
96  together with other in vitro and in vivo data, can inform the recommendation for and the 
97  design of DDI studies specific to the pathways involved in metabolism and excretion.  
98  For additional information on DDI studies, refer to the FDA guidances on drug 
99  interaction studies.6   
100   
101    Identifying metabolites for which nonclinical safety assessments should be performed.7    
102   
103    Guiding decisions for conducting renal and/or hepatic impairment studies.  For additional 
104  information on organ impairment studies, refer to the FDA guidances on renal and 
105  hepatic impairment.8 
106   
107    Avoiding unnecessary exclusions of patients with varying renal and/or hepatic function in 
108  the safety and efficacy clinical trials that support product approval. 
109   
110   
 
6 See the FDA guidances In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated 
Drug Interactions (January 2020) and Clinical Drug Interaction Studies - Cytochrome P450 Enzyme- and 
Transporter-Mediated Drug Interactions (January 2020). 
 
7 See the FDA guidances Safety Testing of Drug Metabolites (March 2020) and M3(R2) Nonclinical Safety Studies 
for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (January 2010). 
 
8 See the FDA guidance Pharmacokinetics in Patients with Impaired Renal Function  Study Design, Data 
Analysis, and Impact on Dosing and Labeling (September 2020).  When final, this guidance will represent the 
Agencys current thinking on this topic.  See also the FDA guidance Pharmacokinetics in Patients with Impaired 
Hepatic Function: Study Design, Data Analysis, and Impact on Dosing and Labeling (March 2003). 
 
  3  Contains Nonbinding Recommendations 
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111  V.  CONSIDERATIONS FOR DESIGNING MASS BALANCE STUDIES    
112   
113  A.  Study Design 
114   
115    Generally, mass balance studies are non-randomized and open-label.   
116   
117  B.  Study Participants 
118   
119    Generally, mass balance studies can be conducted in healthy adult subjects.  If safety 
120  concerns preclude the enrollment of healthy subjects, mass balance studies can be 
121  conducted in the patient population of interest.   
122   
123    In general, a mass balance study should include at least six evaluable subjects who have 
124  completed the study procedures as detailed by the protocol.  Anticipated or known 
125  variability in pharmacokinetics and any relevant polymorphisms in genes coding for drug 
126  metabolizing enzymes or transporters should be considered when determining the number 
127  of subjects for enrollment.  
128   
129  C.  Administered Radioactivity Dose and Radiolabel Position 
130   
131    The absorbed dose of radioactivity should be estimated via dosimetry calculations based 
132  on data from animal studies.  Guidelines of other groups concerned with human safety 
133  (e.g., the International Commission on Radiological Protection (ICRP), Advisory 
134  Committee on Radiological Protection (ACRP)) should also be considered, as 
135  appropriate. 
136   
137    If the administered radioactivity dose is very low (less than 1,000 nCi), supporting data 
138  from dosimetry studies in animals might not be recommended.9  
139   
140    The position of the radioisotope should be chemically and metabolically stable such that 
141  the radionuclide is not lost during metabolism, and both the parent drug and metabolites 
142  can be detected and quantified.  Two separate labeling positions can be used if needed.   
143   
144  D.  Investigational Drug Dose 
145   
146    The dose of the non-radiolabeled investigational drug used in the mass balance study 
147  should be the final intended dose, or at least in the anticipated therapeutic dose range 
148  (taking into account the safety profile of the drug in the study population).  If the 
149  therapeutic range has not been identified at the time of conducting the mass balance 
150  study, the study should use a dose within the pharmacokinetic linearity range. 
151   
152    In general, a single-dose mass balance study is sufficient.  A multiple-dose study can be 
153  considered in some scenarios; for example, if the investigational drug and/or active 
 
9 See the Radioactive Drug Research Committee (RDRC) Program web page at https://www.fda.gov/drugs/science-
and-research-drugs/radioactive-drug-research-committee-rdrc-program. 
  4  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
154  metabolite exhibits time-dependent pharmacokinetics or when the study will be 
155  conducted in patients and a single dose study is not feasible.  In such instances, the 
156  subjects would receive a single radiolabeled dose of the drug after reaching steady state 
157  with non-radiolabeled doses.  Because this approach only evaluates the clearance 
158  pathway of the radiolabeled drug, bioanalysis of the non-radiolabeled moieties at steady 
159  state can help interpret the results (see section J for Bioanalysis). 
160   
161  E.  Route of Administration and Formulation of the Investigational Drug 
162   
163    The routes of administration for the mass balance study should include the final intended 
164  routes of administration, unless precluded by practical considerations (e.g., inhalation 
165  products).   
166   
167    The formulation used in the mass balance study contains both radiolabeled and non-
168  radiolabeled drug materials, and this fit-for-purpose formulation is different from the 
169  final intended formulation. 
170   
171    Although formulation differences may cause some changes in ADME parameters (e.g., 
172  absorption), the formulation used in the study should still provide sufficient information 
173  to fulfill the objectives of the mass balance study.   
174   
175  F.  Determination of Absolute Bioavailability for Orally Administered 
176  Investigational Drugs in a Mass Balance Study  
177   
178    Information on the absolute bioavailability of the investigational drug can help interpret 
179  mass balance data and understand the overall drug elimination pathways.   
180   
181    When only the oral formulation is being developed, an absolute bioavailability study can 
182  be combined with the mass balance study in a single protocol in a two-part study.  For 
183  example, Part A can be the human radiolabeled mass balance study for the orally 
184  administered investigational drug.  Part B can determine the absolute bioavailability of 
185  the investigational drug administered as an oral non-radiolabeled dose (see section D for 
186  dose) and an intravenous radiolabeled microdose (without the need for an intravenous 
187  toxicology program if the existing oral toxicity studies provide adequate exposure 
188  margins).10  Part A and Part B of the study should be conducted with an adequate 
189  washout period.  For drugs with long elimination half-lives, a parallel study design might 
190  be more practical.  
191   
192  G.  Recovery 
193   
194    Ideally, total recovery of radioactivity in urine and feces should be at least 90 percent.  
195  Adequate justification should be provided when recovery is less than 90 percent.  
196   
 
10 See the FDA guidance M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and 
Marketing Authorization for Pharmaceuticals (March 2013). 
  5  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
197  H.  Sample Collection and Handling 
198   
199    Plasma, urine, feces, and other matrices as applicable, should be collected for quantitative 
200  analysis of total radioactivity and for metabolite profiling.  
201   
202    Ideally, sample collection should continue until:  (1) the cumulative radioactivity exceeds 
203  90 percent of the administered dose in urine and feces; and (2) the radioactivity in the 
204  urine and feces is less than 1 percent of the administered dose over a 24-hour period on 2 
205  consecutive sample collection days.  
206   
207    For drugs with a long half-life (parent or metabolites), when an extended stay in the clinic 
208  becomes impractical to achieve greater than 90 percent recovery, alternative sample 
209  collection strategies should be considered to get an estimate of the final recovery.   
210   
211    Plasma, urine, and feces samples should be properly stored and handled after sample 
212  collection and before analysis.  The stability of the investigational drug in the 
213  corresponding matrices should be assessed to avoid misinterpretation of metabolite 
214  profiling results due to interference by degradation products. 
215   
216    For quantitative profiling in plasma, urine, and feces, samples should be analyzed 
217  separately for each subject and not pooled across subjects. 
218   
219    Identification of metabolites is usually done after pooling of samples in the matrix of 
220  interest (plasma, urine, or feces) within each subject.  In certain cases (e.g., low levels of 
221  metabolites), it may be helpful to pool samples across subjects.   
222   
223    If scientifically warranted, the sponsor should collect a pre-dose blood sample for 
224  prospective/retrospective pharmacogenetic analysis. 
225   
226  I.  Parent and Metabolites 
227   
228    In addition to the parent drug, metabolite profiling should be performed in plasma, urine, 
229  and feces samples.  
230   
231    The ratio of plasma metabolite to parent exposure can provide information on whether 
232  and which metabolites should be considered for further DDI evaluation.11  
233   
234    The ratio of plasma metabolite to total drug-related exposure can provide information on 
235  whether and which metabolites should be considered for further nonclinical safety 
 
11 See the FDA guidances In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated 
Drug Interactions (January 2020) and Clinical Drug Interaction Studies - Cytochrome P450 Enzyme- and 
Transporter-Mediated Drug Interactions (January 2020). 
 
  6  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
236  evaluation.  Generally, if a metabolite accounts for more than 10 percent of the total 
237  drug-related exposure in plasma, the metabolite should be structurally characterized.12 
238   
239    Ideally, more than 80 percent of the radioactivity recovered in the excreta should be 
240  identified.  Adequate justification should be provided in instances when less than 80 
241  percent of the recovered radioactivity is identified.  
242   
243  J.  Bioanalysis 
244   
245    The choice of bioanalytical techniques and any associated method validation depends on 
246  the objective of the mass balance study.  Typically, both radiolabeled and non-
247  radiolabeled analytical techniques are used.  
248   
249    For the bioanalysis of radioactivity, detection and quantification of radioactivity should 
250  be performed in all applicable biological matrices using radioactivity counting techniques 
251  (e.g., liquid scintillation counting (LSC), accelerator mass spectrometry (AMS), high-
252  performance liquid chromatography (HPLC) with radio-detection).  
253   
254    For the bioanalysis of the non-radiolabeled moiety, quantification of the unchanged 
255  parent drug and metabolites should be performed in all applicable biological matrices 
256  using a sensitive analytical technique such as liquid chromatography with tandem mass 
257  spectrometry (LC-MS/MS).  Validated bioanalytical methods should be used for the 
258  matrices that are sampled.13  
259   
260   
261  VI.  REPORTING OF HUMAN RADIOLABELED MASS BALANCE STUDY  
262  RESULTS    
263   
264    The mass balance clinical study report should include the following: 
265   
266    Plasma and whole blood concentration versus time profiles of total radioactivity. 
o
267   
268    Plasma concentration versus time profiles for the non-radiolabeled moieties 
o
269  including the parent drug and, if possible, metabolites (refer to section V.J. 
270  Bioanalysis). 
271   
272    Descriptive statistics of pharmacokinetic parameters for total radioactivity, the 
o
273  parent drug, and if possible, metabolites in plasma (e.g., the area under the 
274  concentration time curve (AUC), the maximum concentration (C ), the time to 
max
275  maximum concentration (T ), terminal half-life). 
max
276   
 
12 See the FDA guidances Safety Testing of Drug Metabolites (March 2020) and M3(R2) Nonclinical Safety Studies 
for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (January 2010). 
 
13 See the FDA guidance Bioanalytical Method Validation (May 2018). 
 
  7  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
277    The cumulative percentage of the administered radioactive dose recovered in 
o
278  urine, feces, and total excreta (urine and feces combined) versus time profiles. 
279   
280    Quantitative information on the radioactivity associated with the parent drug and 
o
281  each identified metabolite in collected matrices (e.g., plasma, urine, feces). 
282   
283    A biotransformation scheme with the structures or descriptions of the metabolites, 
o
284  if available. 
285   
286    Results from mass balance studies are generally included in Subsection 12.3 
287  Pharmacokinetics of the approved prescribing information.14,15 
 
14 See the FDA guidance Clinical Pharmacology Labeling for Human Prescription Drug and Biological Products - 
Content and Format (December 2016). 
 
15 21 CFR 201.57. 
  8