Guidance for Industry 

Safety Testing of Drug 

Metabolites 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER) 

February 2008 

Pharmacology and Toxicology 
Guidance for Industry

Safety Testing of Drug 

Metabolites 

Additional copies are available from: 

Office of Training and Communications 

Division of Drug Information, HFD-240 

Center for Drug Evaluation and Research 

Food and Drug Administration 

5600 Fishers Lane 

Rockville, MD 20857 

(Tel) 301-827-4573 

http://www.fda.gov/cder/guidance/index.htm 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER) 

February 2008 

Pharmacology and Toxicology 
TABLE OF CONTENTS 

I. INTRODUCTION............................................................................................................. 1

II. BACKGROUND ............................................................................................................... 2

III. GENERAL CONCEPTS IN METABOLITE SAFETY TESTING............................. 3

A. General Approaches for Assessing Metabolite Safety ................................................................4

B. Identification of Metabolites .........................................................................................................4

C. General Considerations for Nonclinical Study Design ...............................................................5

IV. RECOMMENDED STUDIES FOR ASSESSING THE SAFETY OF 

METABOLITES............................................................................................................... 5

A. General Toxicity Studies ...............................................................................................................6

B. Genotoxicity Studies ......................................................................................................................6

C. Embryo-Fetal Development Toxicity Studies..............................................................................6

D. Carcinogenicity Studies.................................................................................................................6

V. TIMING OF SAFETY ASSESSMENTS........................................................................ 7

GLOSSARY................................................................................................................................... 8

APPENDIX A: DECISION TREE FLOW DIAGRAM............................................................ 9

APPENDIX B: CASE EXAMPLES OF DRUG METABOLITES ........................................ 10
Contains Nonbinding Recommendations 

Guidance for Industry1

Safety Testing of Drug Metabolites 

This guidance represents the Food and Drug Administration’s (FDA’s) current thinking on this topic. It 

does not create or confer any rights for or on any person and does not operate to bind FDA or the public. 

You can use an alternative approach if the approach satisfies the requirements of the applicable statutes 

and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for 

implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate 

number listed on the title page of this guidance. 

I. INTRODUCTION

This guidance provides recommendations to industry on when and how to identify and 

characterize drug metabolites whose nonclinical toxicity needs to be evaluated. The safety of 

drug metabolites may need to be determined in nonclinical studies because these metabolites are 

either identified only in humans or are present at disproportionately higher levels in humans than 

in any of the animal species used during standard nonclinical toxicology testing.2

 

This guidance applies to small molecule nonbiologic drug products. This guidance does not 

apply to some cancer therapies where a risk-benefit assessment is considered. The Food and 

Drug Administration (FDA) is developing more specific guidance for the safety testing of drug 

metabolites in cancer therapies. 

FDA’s guidance documents, including this guidance, do not establish legally enforceable 

responsibilities. Instead, guidances describe the Agency’s current thinking on a topic and should 

be viewed only as recommendations, unless specific regulatory or statutory requirements are 

cited. The use of the word should in Agency guidances means that something is suggested or 

recommended, but not required. 

 

1

 This guidance has been prepared by the Pharmacokinetic Subcommittee of the Pharmacology and Toxicology 

Coordinating Committee (PTCC) in the Center for Drug Evaluation and Research (CDER) at the Food and Drug 

Administration. 

2

 See the Glossary for the definition of disproportionate drug metabolite. 

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Contains Nonbinding Recommendations 

II. BACKGROUND 

Nonclinical evaluation of drug safety usually consists of standard animal toxicology studies.3 

These studies usually include assessment of drug exposure, primarily parent drug plasma 

concentration. Generally, drug plasma concentration and systemic exposure in the nonclinical 

studies are compared with systemic exposure in humans to assess the potential risks suggested by 

nonclinical findings and guide monitoring in clinical trials. This testing paradigm usually is 

sufficient when the metabolic profile in humans is similar to that in at least one of the animal 

species used in nonclinical studies, but metabolic profiles can vary across species both 

quantitatively and qualitatively, and there are cases when clinically relevant metabolites have not 

been identified or adequately evaluated during nonclinical safety studies. This situation can 

occur if the metabolite is formed only in humans and is absent in the animal test species or if the 

metabolite is present at disproportionately higher levels in humans than in the animal species 

used in the standard toxicity testing with the parent drug. 

It is not standard practice for drug metabolites to be evaluated separately in a cross-species safety 

assessment. As a result, their specific contribution to the overall toxicity of the parent drug has 

often remained unknown. This lack of appreciation of the role of metabolites in drug toxicity 

may be partly because of the inadequate sensitivity of the analytical methods used to detect and 

characterize metabolites derived from the parent drug. Technological advances during the past 

decade have greatly improved the analytical capabilities to detect, identify, and characterize 

metabolites and allow for better understanding of the role metabolites play in drug safety 

assessment. 

Drugs entering the body undergo biotransformation via Phase I and Phase II metabolic pathways. 

Based on the nature of the chemical reactions involved, metabolites formed from Phase I 

reactions are more likely to be chemically reactive or pharmacologically active and, therefore, 

more likely to need safety evaluation. An active metabolite may bind to the therapeutic target 

receptors or other receptors, interact with other targets (e.g., enzymes, proteins), and cause 

unintended effects. This is a particularly important problem when such a metabolite is formed in 

humans and not in animals, but the occurrence of a metabolite only in humans and not in any 

animal test species is rare. A more common situation is the formation of a metabolite at 

disproportionately higher levels in humans than in the animal species used in safety testing of the 

parent drug. This disproportionality stems from the typical qualitative and/or quantitative 

differences in metabolic profiles between humans and animals. If at least one animal test species 

forms this drug metabolite at adequate exposure levels (approximately equal to or greater than 

human exposure), as determined during toxicology testing of the parent drug, it can be assumed 

that the metabolite’s contribution to the overall toxicity assessment has been established.4

 

3

 See the ICH guidances for industry S6 Preclinical Safety Evaluation of Biotechnology-Derived 

Pharmaceuticals and S7A Safety Pharmacology Studies for Human Pharmaceuticals 

(http://www.ich.org/cache/compo/276-254-1.html). 

4

 See Appendix A: Decision Tree Flow Diagram. This diagram describes which studies may be needed to determine 

safety of the drug metabolite. 

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Contains Nonbinding Recommendations 

Metabolites that form chemically reactive intermediates can be difficult to detect and measure 

because of their short half-lives. However, they can form stable products (e.g., glutathione 

conjugates) that can be measured and, therefore, may eliminate the need for further evaluation. 

Phase II conjugation reactions generally render a compound more water soluble and 

pharmacologically inactive, thereby eliminating the need for further evaluation. However, if the 

conjugate forms a toxic compound such as acylglucuronide, additional safety assessment may be 

needed.5

 

Demonstration that a metabolite is pharmacologically inactive at the target receptor does not 

guarantee that it is not toxic. Therefore, it may need to be tested in nonclinical toxicity studies.6

III. GENERAL CONCEPTS IN METABOLITE SAFETY TESTING 

We encourage the identification of differences in drug metabolism between animals used in 

nonclinical safety assessments and humans as early as possible during the drug development 

process.7 The discovery of disproportionate drug metabolites late in drug development can 

potentially cause development and marketing delays. 

Generally, metabolites identified only in human plasma or metabolites present at 

disproportionately higher levels in humans than in any of the animal test species should be 

considered for safety assessment. Human metabolites that can raise a safety concern are those 

formed at greater than 10 percent of parent drug systemic exposure at steady state.8

 The choice 

of a level of greater than 10 percent for characterization of drug metabolites reflects consistency 

with FDA and Environmental Protection Agency guidances.9

 

5

 Faed, EM, 1984, Properties of Acyl Glucuronides. Implications for Studies of the Pharmacokinetics and 

Metabolism of Acidic Drugs, Drug Metab Rev, 15, 1213-1249. 

6

 See Appendix B: Case Examples of Drug Metabolites. 

7

 Baillie, TA, MN Cayen, H Fouda, RJ Gerson, JD Green et al., 2002, Drug Metabolites in Safety Testing, Toxicol 

Appl Pharmacol, 182, 188-196. 

Hastings, KL, J El-Hage, A Jacobs, J Leighton, D Morse, R Osterberg, 2003, Drug Metabolites in Safety Testing, 

Toxicol Appl Pharmacol, 190(1), 91-92. 

8

 Exposure should be at steady state unless there is some justification for a different measure of exposure. 

Comparison between human and animal exposure generally is based on area under the curve, but sometimes it may 

be more appropriate to use Cmax. 

9

 U.S. Environmental Protection Agency, 1998, Health Effects Test Guidelines, OPPTS 870.7485, Metabolism and 

Pharmacokinetics. (http://www.epa.gov/epahome/research.htm) 

U.S. Food and Drug Administration, 2002, Guideline for Metabolism Studies and for Selection of Residues for 

Toxicological Testing, U.S. Food and Drug Administration, Center for Veterinary Medicine. 

(http://www.fda.gov/cvm/Guidance/guideline3pt1.html) 

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Contains Nonbinding Recommendations 

A. General Approaches for Assessing Metabolite Safety 

A metabolite identified in animals that is not present in humans can mean that a toxicity 

observed in that animal species, attributed to the metabolite, may not be relevant to humans. 

Conversely, a drug metabolite identified during clinical development that is not present in animal 

test species or is present at much lower levels in animals than in humans can suggest the need for 

further studies in animals to determine the potential toxicity of the metabolite. In such cases, two 

approaches can be considered to assess the drug metabolite. The first approach is to identify an 

animal species routinely used in toxicity studies that forms the metabolite at adequate exposure 

levels (equivalent to or greater than the human exposure), and then investigate the drug’s toxicity 

in that species. The second approach, if a relevant animal species that forms the metabolite 

cannot be identified, is to synthesize the drug metabolite and directly administer it to the animal 

for further safety evaluation. In this approach, analytical methods that are capable of identifying 

and measuring the metabolite in nonclinical toxicity studies should be developed. 

The difficulties associated with synthesizing a specific metabolite as well as the inherent 

complexities that accompany its direct administration are acknowledged. Direct dosing of a 

metabolite to animals may lead to subsequent metabolism that may not reflect the clinical 

situation and thus may complicate the toxicity evaluation. Moreover, new and different 

toxicities may arise from administration of the metabolite that were not observed with the parent 

drug. However, notwithstanding these possible complications, identification and evaluation of 

the potential toxicity of the drug metabolite is considered important to ensure clinical safety, and 

the decision to conduct direct safety testing of a metabolite should be based on a comprehensive 

evaluation of the data on the parent drug and any information available for the metabolite. 

Appendix B provides three case examples when drug metabolites were formed at 

disproportionately higher levels in humans than in test animals used in the nonclinical studies 

and how the safety evaluation was approached. In Case 1, testing of the drug metabolite was not 

needed because the metabolite was adequately characterized in nonclinical toxicity studies with 

the parent drug. However, in Cases 2 and 3, the drug metabolites had to be tested in toxicity 

studies by direct administration to the animal. In Case 3, the drug metabolite was 

pharmacologically inactive at the therapeutic target receptor but showed a unique toxicity not 

observed with the parent molecule. 

B. Identification of Metabolites 

Metabolite concentrations cannot be inferred by measurement of parent drug concentrations. 

The metabolic profile of the drug should be identified during the drug development process. 

This identification can be accomplished at different stages of development using in vitro and in 

vivo methods. In vitro studies can use liver microsomes, liver slices, or hepatocytes from 

animals and humans and generally should be conducted before initiation of clinical trials. In 

vivo metabolism study results in nonclinical test species generally should be available early in 

drug development, and their results will either confirm the results obtained from the in vitro 

studies or reveal quantitative and/or qualitative differences in metabolism across species. It is 

the latter situation that may pose a safety concern. Human in vivo metabolism studies usually 

have been performed relatively later in drug development, but we strongly recommend in vivo 

metabolic evaluation in humans be performed as early as feasible. 

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Contains Nonbinding Recommendations 

Adequacy of exposure to drug metabolites that are present at disproportionately lower levels in 

animals used in nonclinical studies should be considered on a case-by-case basis. Generally, 

systemic exposure is assessed by measuring the concentration of the parent drug at steady state, 

in serum or plasma. However, when measurements cannot be made in plasma of the test species 

for any reason, verification of adequate exposure can be made in other biological matrices such 

as urine, feces, or bile.10 We encourage contacting the FDA early in drug development to 

discuss these issues. 

C. General Considerations for Nonclinical Study Design 

When designing a nonclinical study for a disproportionate drug metabolite, it is important to 

consider the following factors: 

• Similarity of the metabolite to the parent molecule 

• Pharmacological or chemical class 

• Solubility 

• Stability in stomach pH 

• Phase I versus Phase II metabolite 

• Relative amounts detected in humans versus the amounts detected in animals 

Additional factors to consider include the proposed drug indication and patient population (e.g., 

nonclinical studies can be abbreviated for serious indications such as amyotrophic lateral 

sclerosis (ALS)). The proposed duration of use (short term, intermittent use, chronic) and levels 

of exposure at the therapeutic dose also should be considered when designing the nonclinical 

studies for the drug metabolite. 

IV. RECOMMENDED STUDIES FOR ASSESSING THE SAFETY OF 

METABOLITES 

Good laboratory practice guidelines apply to the nonclinical studies with the drug metabolite 

designed to evaluate safety (21 CFR part 58). The following studies may need to be performed 

to assess the safety of the disproportionate drug metabolite. 

 

10 See the ICH guidance for industry S3A Note for Guidance on Toxicokinetics: The Assessment of Systemic 

Exposure in Toxicity Studies (http://www.ich.org/cache/compo/276-254-1.html). 

Although the drug metabolite of toxicological concern usually is one circulating in plasma at greater than 10 percent 

of parent systemic exposure, other metabolites also can elicit safety concern. For example, a drug metabolite 

representing greater than 10 percent of urinary excretion relative to the bioavailable dose, or a human fecal 

metabolite in cases where biliary elimination is the predominant route of excretion in humans, may reflect potential 

localized renal or bile duct toxicity, respectively. Further characterization in these instances should be addressed on 

a case-by-case basis with the review division. 

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Contains Nonbinding Recommendations 

A. General Toxicity Studies 

The potential toxicity of a disproportionate drug metabolite should be evaluated to enable 

comparisons between the metabolite and its parent drug. The duration of the general toxicity 

study with direct dosing of the metabolite should follow the recommendations listed in the ICH 

guidance for industry M3(R1) Nonclinical Safety Studies for the Conduct of Human Clinical 

Trials for Pharmaceuticals.

11 The toxicity of the drug metabolite should be investigated at 

multiples of the human exposure or at least at levels comparable to those measured in humans. 

We also recommend using the parent drug’s intended clinical route of administration. However, 

with justification, other routes can be used to achieve sufficient exposure to the disproportionate 

metabolite. If the clinical route is oral, it is important to verify the stability of the metabolite in 

the stomach environment. It is crucial to gather toxicokinetic data from this type of study to 

ensure adequate exposure. 

B. Genotoxicity Studies 

The potential genotoxicity of the drug metabolite should be assessed in an in vitro assay that 

detects point mutations and in another assay that detects chromosomal aberrations. It is 

important that these assays be conducted according to the recommendations in the ICH 

guidances for industry S2A Guidance on Specific Aspects of Regulatory Genotoxicity Tests for 

Pharmaceuticals and S2B Genotoxicity: A Standard Battery for Genotoxicity Testing of 

Pharmaceuticals.

12 If one or both of the in vitro tests are equivocal and/or positive, performance 

of the complete standard battery of genotoxicity studies may be warranted (ICH S2A). 

C. Embryo-Fetal Development Toxicity Studies 

When a drug is intended for use in a population that includes women of childbearing potential, 

embryo-fetal development toxicity studies should be performed with the drug metabolite. We 

may ask for other reproductive toxicity studies on a case-by-case basis, depending on results of 

the general toxicity and embryo-fetal development studies. Reproductive toxicity studies should 

be conducted in accordance with the ICH guidance for industry S5(R2) Detection of Toxicity to 

Reproduction for Medicinal Products and Toxicity to Male Fertility.

13 Sometimes the conduct 

of an embryo-fetal development toxicity study in only one species that forms the drug metabolite 

can be justified. 

D. Carcinogenicity Studies 

Carcinogenicity studies should be conducted on metabolites of drugs that are administered 

continuously for at least 6 months, or that are used intermittently in the treatment of chronic or 

recurrent conditions when the carcinogenic potential of the metabolite cannot be adequately 

evaluated from carcinogenicity studies conducted with the parent drug. A single carcinogenicity 

 

11 See http://www.ich.org/cache/compo/276-254-1.html. 

12 See http://www.ich.org/cache/compo/276-254-1.html. 

13 See http://www.ich.org/cache/compo/276-254-1.html. 

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Contains Nonbinding Recommendations 

study or an alternative bioassay should be conducted and the studies should be conducted in 

accordance with the ICH guidances for industry S1A Need for Carcinogenicity Studies of 

Pharmaceuticals, S1B Testing for Carcinogenicity of Pharmaceuticals, and S1C(R1) Dose 

Selection for Carcinogenicity Studies of Pharmaceuticals and Limit Dose.

14 

V. TIMING OF SAFETY ASSESSMENTS 

Early identification of disproportionate drug metabolites can provide clear justification for 

nonclinical testing in animals, assist in interpreting and planning clinical studies, and prevent 

delays in drug development. If toxicity studies of a drug metabolite are warranted, studies 

should be completed and study reports provided to the FDA before beginning large-scale clinical 

trials. 

To optimize and expedite drug development for serious or life-threatening diseases other than 

cancer (e.g., ALS, stroke, human immunodeficiency virus), the number and type of nonclinical 

studies for the drug metabolites can be modified on a case-by-case basis for those drugs with 

major beneficial therapeutic advances, and for drugs for illnesses that lack an approved effective 

therapy. Sponsors should contact the appropriate review division to discuss such situations. 

 

14 See http://www.ich.org/cache/compo/276-254-1.html. 

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Contains Nonbinding Recommendations 

GLOSSARY 

Disproportionate drug metabolite — A metabolite present only in humans or present at higher 

plasma concentrations in humans than in the animals used in nonclinical studies. In general, 

these metabolites are of interest if they account for plasma levels greater than 10 percent of 

parent systemic exposure, measured as area under the curve (AUC) at steady state. 

Metabolite — A compound derived from the parent drug through Phase I and/or Phase II 

metabolic pathways. 

Pharmacologically active metabolite — A metabolite that has pharmacological activity at the 

target receptor. The activity may be greater than, equal to, or less than that of the parent drug. 

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Contains Nonbinding Recommendations 

APPENDIX A: 

DECISION TREE FLOW DIAGRAM 

Disproportionate Drug Metabolite 

<10% parent 

systemic exposure 

(AUC)

No further testing needed 

to evaluate metabolite 

>10% parent 

systemic exposure 

(AUC)

Formed in any 

animal test species? 

Yes 

No How much? 

Exposure in animal 

studies does not approach 

human exposure 

Exposure in animal 

studies does approach 

human exposure 

No further testing needed 

to qualify metabolite 

Nonclinical testing with 

the drug metabolite 

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Contains Nonbinding Recommendations 

APPENDIX B: 

CASE EXAMPLES OF DRUG METABOLITES 

Case 1. 

From an initial mass balance study, a metabolite represented 1 to 2 percent of total 

radioactive dose in rat plasma, 5 percent in dogs, and 20 percent in humans (radioactivity 

of this metabolite in urine and/or feces was minimal). Based on the up-to-20 fold greater 

exposure in humans than in animals, nonclinical safety testing was recommended. 

However, the data generated in the general toxicology studies with the parent drug in the 

rat and dog suggested that the maximum doses tested produced metabolite exposures that 

represented at least the therapeutic exposure at the maximum recommended human dose. 

Also, the plasma concentrations of this metabolite measured in the in vivo genetic 

toxicity study, embryo-fetal development toxicity study, and carcinogenicity studies 

conducted with the parent drug provided adequate exposure and characterization of the 

metabolite. Therefore, no additional testing with the metabolite was needed. 

Case 2. 

Two primary hydroxylated metabolites, M1 and M2, were shown to undergo further 

oxidation to form secondary metabolites M3 and M4 using hepatic microsomes and 

hepatocytes from human, monkey, rat, dog, rabbit, and mouse. This metabolic profile 

was later confirmed by in vivo data. The results showed the following: 

• M1 and M4 were the predominant metabolites in human, monkey, and dog 

microsomes, whereas rat, mouse, and rabbit formed M2 and M3. 

• M4 was formed in humans at 4 fold higher levels than parent drug, but M4 was 

formed at very low levels in rodents and only represented one-third of the parent 

exposure in monkey (see Table 1). 

Table 1: AUC0-24hr at the Maximum Dose 

 Human (MRHD) Monkey Rat 

Parent 1,800 15,000 12,500 

M4 7,700 5,000 135 

• Severe drug-related and novel target organ toxicities were observed with the 

parent drug in monkeys but not in rats. 

• M4 was pharmacologically inactive at the drug target receptors. 

The following additional studies were done with M4: 

• Subchronic toxicity study: 3 months in the rat 

• Embryo-fetal development study in the rat 

• In vitro genotoxicity testing: M4 was positive for point mutation and 

chromosomal aberration; the parent drug was negative 

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Contains Nonbinding Recommendations 

• Because of the positive genotoxicity, a carcinogenicity study that included M4 

was recommended 

Case 3. 

M2 is a Phase I oxidative metabolite that formed up to 50 percent of parent drug exposure 

in humans, 10 percent of parent drug exposure in mice, 15 percent of parent drug 

exposure in dogs, and only trace amounts in rats. In vitro metabolism studies in these 

species supported the in vivo findings. Based on structure activity relationship analyses, 

there was no reason to anticipate any difference or exaggeration in toxicity of the 

metabolite compared to parent molecule. The parent drug showed no significant toxicity 

or identifiable target organ of toxicity in any of the animal species tested in safety 

assessment studies. Because disproportionate human exposure was identified, further 

safety testing was needed. When M2 was tested in a short-term tolerance study in the 

dog, it produced unexpected and significant cardiotoxicity at all doses and in all of the 

dogs. M2 was pharmacologically inactive at the therapeutic target receptor. 

11