Nonclinical Evaluation 

of Endocrine-Related 

Drug Toxicity 

Guidance for Industry 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER)
  
 
September 2015 

Pharmacology and Toxicology 

   
Nonclinical Evaluation 

of Endocrine-Related 

Drug Toxicity 

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  

Tel: 855-543-3784 or 301-796-3400;  Fax: 301-431-6353; Email:  druginfo@fda.hhs.gov  

http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Drug Evaluation and Research (CDER)
  
 
September 2015 

Pharmacology and Toxicology 

 
   
TABLE OF CONTENTS  

 
I.	   INTRODUCTION............................................................................................................. 1
  
II.	   BACKGROUND ............................................................................................................... 2
  
III. 	  OVERVIEW OF NONCLINICAL ASSESSMENTS.................................................... 3 
 
A. 	  Receptor-Binding Assays ............................................................................................................... 3
  
B. 	  Pharmacology Studies ................................................................................................................... 3
  
C. 	  Repeat-Dose Toxicity Studies ........................................................................................................ 4
  
D. 	  Developmental and Reproductive Toxicity Studies .................................................................... 4 
 
E. 	  Carcinogenicity Studies ................................................................................................................. 4
  
IV.  	 WHEN ADDITIONAL ASSESSMENT OF ADVERSE ENDOCRINE-RELATED  

EFFECTS MAY BE WARRANTED .............................................................................. 5 
 
A. 	  Are There Data Indicating Adverse Endocrine Activity? .......................................................... 5
  
B. 	  Is the Drug Being Used in a Population not Studied in Standard Toxicity Studies? ............... 5
  
C. 	  Is the Clinical Systemic Exposure to the Drug Near or Above the Exposure at the NOAEL 

for Endocrine Effects in Animals?  ............................................................................................... 6
  
V.  	 ADDITIONAL STUDIES FOR FURTHER ASSESSMENT OF ADVERSE 

ENDOCRINE-RELATED ACTIVITY .......................................................................... 6  

  Contains Nonbinding Recommendations 
Nonclinical Evaluation of Endocrine-Related Drug Toxicity 

1
 
Guidance for Industry  
 
 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic. It does not create any rights for any person and is not binding on FDA or the public.  You can 
use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  To 
discuss an alternative approach, contact the FDA staff responsible for this guidance as listed on the title 
page. 
 
 
 
 
I.  INTRODUCTION  
 
This guidance provides recommendations to sponsors of investigational new drug applications, 
new drug applications, and biologics license applications regulated by the Center for Drug 
Evaluation and Research (CDER) regarding nonclinical studies intended to identify the potential 
for a drug to cause endocrine-related toxicity.2    
 
The goals of this guidance are to: 
 
 	  Describe how endocrine-related toxicity is assessed using the standard battery of 

nonclinical tests 

 
 	  Identify situations in which additional studies should be considered to more fully 

characterize the endocrine-related toxicity of a drug 

 
This guidance focuses on nonclinical testing designed to assess the potential for a drug to cause 
endocrine effects that are unintentional and adverse.  It does not provide guidance relating to the 
development of drugs that are intended to interfere with the endocrine system to prevent or treat 
a particular disease or condition, and it does not provide detailed recommendations regarding 
clinical studies to investigate adverse endocrine-related activity.  Environmental assessment 
issues are not covered in this guidance. 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
                                                 
1  This guidance has been prepared by the Office of New Drugs in the Center for Drug Evaluation and Research 
(CDER) at the  Food and Drug Administration.  
 
2 For the purposes of this guidance, all references to drugs  include both human  drugs and therapeutic biological 
products  unless otherwise specified.  
1 
 Contains Nonbinding Recommendations 
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. 
 
 
II.  BACKGROUND 
 
Some compounds can interfere with the endocrine system of an organism or its progeny, 
resulting in adverse effects in one or more sensitive tissues.  Compounds can do this through 
multiple mechanisms, including:  (1) mimicking or enhancing the action of an endogenous 
hormone; (2) blocking a hormone receptor, thereby preventing the action of an endogenous 
hormone; or (3) affecting the synthesis, transport, metabolism, or excretion of an endogenous 
hormone.  These effects may be intended, as in the case of oral contraceptives, or they may be an 
unintended side effect. For biologically significant effects to be produced, exposure needs to be 
sufficiently high at the target site and the substance needs to be of sufficient potency.  It is noted 
that not all perturbations of the endocrine system are adverse.  For pharmaceuticals, only effects 
seen at clinically relevant exposures are of concern.  
 
Test articles that affect the endocrine system include, but are not limited to, those that alter the 
function of the sex hormones (e.g., estrogen and androgen), the hypothalamic-pituitary-adrenal 
hormones, and thyroid hormone, and the hormones involved in feedback regulation of endocrine 
systems (e.g., the gonadotropin-releasing hormone, corticotropin).  The perturbation of endocrine 
systems potentially can cause a wide variety of adverse effects on development.  Appropriate 
hormonal exposure during the different developmental stages is critical to normal development.  
Increased or decreased estrogenic or androgenic activity at inappropriate times can produce 
transient or permanent alterations in end-organ structure and/or function.  For example, animal 
studies have shown that males exposed to antiandrogens during a specific perinatal period can be 
demasculinized in morphology, physiology, and behavior. 
 
Endocrine-active compounds also can affect the nervous system through receptors expressed on 
neuroendocrine cells or on neurons.  In rodents, for example, early developmental exposure to 
estradiol or testosterone establishes permanent sex-dependent brain morphology.  Sexual 
differentiation is also regulated by metabolism  of steroid hormones in the brain via enzymes 
controlling steroidogenesis such as CYP19A1 (P450 aromatase), which converts testosterone to 
estradiol. For example, in rodents, compounds that alter aromatase activity can cause an 
imbalance in sex hormones that result in altered sexual behavior.   
 
It is important to assess drugs being developed for human use for their potential effects on 
hormonal systems.  Nonclinical testing is appropriate for this assessment because endocrine 
systems are similar among vertebrates, and many species respond similarly with regard to their 
sensitivity to hormones.  Based on review of recent literature and experience with drugs being 
developed for human use, including those that target the endocrine systems, it is possible to 
identify potential endocrine effects in drugs not intended to possess those activities.  Endocrine 
effects in patient populations have been successfully predicted by standard nonclinical test 
batteries. The potential need for additional testing under specific circumstances is discussed in 
section IV. 
2 
 Contains Nonbinding Recommendations 
 
 
III.   OVERVIEW OF NONCLINICAL ASSESSMENTS 
 
 
As a part of the safety evaluation of drugs for human use, CDER recommends a standard battery 
of toxicity tests using study designs intended to identify potential toxicity (see the ICH guidances 
for industry M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and 
Marketing Authorization for Pharmaceuticals and S6(R1) Preclinical Safety Evaluation of 
Biotechnology-Derived Pharmaceuticals).3  A well-designed nonclinical testing program  
conducted using the recommended paradigm will detect clear adverse effects related to endocrine 
perturbation by drugs under development.  If endocrine effects are identified, other factors  
such as the indication; target population; and route, duration, and level of exposure relative to the 
expected clinical exposure  will determine how to proceed (e.g., whether further nonclinical 
testing or clinical monitoring is appropriate or not warranted). 
 
The typical nonclinical studies conducted during drug development are discussed below, with a 
focus on the information that the studies can provide about potential endocrine-related toxicity. 
 
A.  Receptor-Binding Assays 
 
Binding assays that include endocrine receptors can serve as an initial screen.  A lack of binding 
does not rule out a potential endocrine effect (e.g., a drug that does not bind to a receptor still 
could affect synthesis or metabolism of a hormone, resulting in an indirect adverse endocrine 
effect). Similarly, if binding does occur, this does not provide conclusive evidence that the 
interaction will lead to a significant biological change.  If a drug binds to an endocrine receptor, 
then additional testing  including in vitro studies and nonclinical in vivo functional assays  
can be used to characterize the interaction, if warranted.  
 
Enzyme assays also can be used to screen for the potential of a drug to interfere with the 
endocrine system by altering the activity of certain enzymes.  For example, some of the 
cytochrome P450 isozymes are involved in anabolism and catabolism of steroid hormones.  
These include CYP 11A1, 11B1, 11B2, 17A1, and 21A1, which act in steroid biosynthesis.  
Similarly, uridine diphosphate-glucuronosyltransferases in the liver metabolize circulating 
thyroid hormones. 
 
B.  Pharmacology Studies 
 
Sponsors typically conduct nonclinical studies to assess intended pharmacologic effects, 
including studies on the effects of the drug in in vitro and in vivo models of disease.  In some  
cases, the intended pharmacologic target of the drug can be within an endocrine-related pathway.  
The pharmacology studies can help characterize this endocrine activity.  
 
                                                 
3  We  update  guidances periodically.  To make sure you  have the most recent version of a guidance, check the FDA  
Drugs guidance Web page  at  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.  
3 
 Contains Nonbinding Recommendations 
C.  Repeat-Dose Toxicity Studies  
 
Nonclinical safety assessments for most new drugs include repeat-dose toxicology studies in two 
species.4  For drugs intended to be used chronically, this typically means studies exposing two 
animal species to a wide range of doses of the test drug for up to 6 (rodent) or 9 (nonrodent) 
months. These studies generally include a variety of endpoints that can identify adverse 
endocrine-related activity. Changes in organ weight, gross organ pathology, clinical chemistry, 
and histopathology can all be indicative of particular endocrine effects.    
 
Organs typically collected for gross and histopathological evaluation include endocrine-sensitive 
organs such as the thyroid gland, adrenal glands, reproductive organs, and pituitary gland.  
Changes observed can indicate potential adverse endocrine effects.  For example, estrogenic 
drugs can produce effects such as increased ovarian and uterine weight, endometrial stimulation, 
mammary gland stimulation, decreased thymus weight and involution, and increased bone 
mineral density.  Other hormone activities produce characteristic histological changes in target 
tissues. Sometimes a carefully selected hormonal analysis can provide a biomarker for clinical 
monitoring and risk assessment. 
 
D.  Developmental and Reproductive Toxicity Studies  
 
For most drugs, sponsors routinely conduct a battery of tests designed to determine if a drug 
potentially impairs fertility or affects embryo/fetal and neonatal development.5  Some  
developmental stages (e.g., gestational, neonatal, peripubertal) are particularly sensitive to 
endocrine effects. Sensitive endpoints can include vaginal patency, preputial separation, and 
anogenital distance; for male offspring, sensitive endpoints can include nipple retention.  The 
reproductive performance of offspring also can be important for detecting neuroendocrine 
effects. Standard developmental and reproductive toxicity studies generally capture gestational 
developmental time points effectively, but might not be adequate for evaluation of effects on 
postnatal development unless the neonates were dosed directly during the lactation period.   
 
E.  Carcinogenicity Studies 
 
Carcinogenicity studies conducted in rats and mice are another important source of information 
about the effects of chronically used drugs.  Although the goal of these studies is to assess the 
drugs carcinogenic potential, organ changes assessed by histopathology also give information 
about potential endocrine effects. For example, persistent disruption of the hypothalamic-
pituitary adrenal/gonadal/thyroidal axis(es) can result in various neoplasms.  Histologic, organ 
weight, clinical chemistry, or hematologic data evaluated together can form recognized patterns 
of changes associated with hormonal effects that precede the neoplasm.  Often the available data 
are sufficient to identify the hormonal axes involved. 
                                                 
4 See the ICH guidances for industry M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials  
and Marketing Authorization for Pharmaceuticals and S6(R1) Preclinical Safety Evaluation of  Biotechnology-
Derived Pharmaceuticals. 
 
5 See the ICH guidance for industry  S5 Detection of Toxicity  to Reproduction for Medicinal Products.  
4 
 Contains Nonbinding Recommendations 
 
 
IV. 	 WHEN ADDITIONAL ASSESSMENT OF ADVERSE ENDOCRINE-RELATED  
EFFECTS MAY BE WARRANTED 
 
Standard nonclinical testing programs conducted in accordance with CDERs recommended 
paradigm generally are adequate to detect whether a drug has the potential to interfere with the 
endocrine system.  As a part of this overall assessment, there are specific factors to consider that 
could suggest a need for additional investigation. 
 
Sponsors should consider the following factors when deciding if the results of the standard 
nonclinical evaluation discussed above are adequate to evaluate the drugs adverse endocrine-
related effects or if additional assessment is warranted: 
 
	   Are there data on related compounds indicating that the drug or related compounds may 
have adverse endocrine-related effects? 
 
	   Is the drug proposed for use in a population not studied in standard toxicity studies? 
 
	   Is the clinical systemic exposure to the drug near or above the exposure at the no 

observed adverse effect level (NOAEL) for endocrine effects in animals?
   
 
These questions are further addressed below. 
 
A. 	 Are There Data Indicating Adverse Endocrine Activity?  
 
When data (e.g., class effects, receptor binding) show a potential adverse endocrine-related 
effect, the sponsor should consider whether additional investigation is warranted.  If the 
endocrine effect was expected based on the primary pharmacology or class of the drug and those 
class effects have been well characterized previously, additional endocrine-specific investigation 
might not be necessary.  However, if it is not known whether under conditions of use the 
endocrine-related finding will be relevant to humans, additional studies sometimes can be 
warranted.  
 
B. 	 Is the Drug Being Used in a Population not Studied in Standard Toxicity 
Studies? 
 
Standard nonclinical studies are adequate for detecting adverse effects following in utero 
exposures and exposures to adults, but might not be adequate for detecting effects following 
exposures during the early postnatal period. Direct dosing during the early postnatal period 
should be conducted to ensure exposure during the postnatal period when insufficient exposure 
occurs during the lactation period.  The endocrine changes experienced by juvenile animals can 
have long-term consequences not adequately demonstrated by studies conducted only in adult 
animals.  When only adult animals have been tested, additional studies designed specifically to 
evaluate toxicity in juvenile animals can be important   
 
5 
 Contains Nonbinding Recommendations 
C. 	 Is the Clinical Systemic Exposure to the Drug Near or Above the Exposure at 
the NOAEL for Endocrine Effects in Animals? 
 
Sponsors should assess the exposure-response relationship of any potential endocrine activity 
identified in standard nonclinical studies.  Those endocrine activities that occur in animals only 
at exposures substantially above (e.g., greater than 50 times) the human exposure usually do not 
warrant additional investigation.  Additional assessment is likely to be appropriate only if human 
exposure is comparable to (or exceeds) the exposure level at which endocrine-related activity is 
observed in standard nonclinical studies and relevance to humans is not understood.  Note that 
assessment of human exposure should take into consideration possible exposure to multiple 
drugs containing the same active agent (e.g., over-the-counter monograph active ingredients) or 
exposure to multiple agents having similar endocrine effects.  This potentially can increase the 
risk compared to that of a single drug.  
 
 
V. 	 ADDITIONAL STUDIES FOR FURTHER ASSESSMENT OF ADVERSE 
ENDOCRINE-RELATED ACTIVITY   
 
As previously discussed, the FDA has concluded that the standard battery of nonclinical tests for 
new drugs detects relevant adverse endocrine-related effects in almost all cases.  Based on a 
thorough evaluation of the totality of data from  the standard battery of nonclinical tests and the 
factors identified in section IV, sponsors should determine whether the assessment of adverse 
endocrine-related activity is adequate or whether additional studies should be conducted to 
further characterize potential endocrine effects.  Sponsors should consider whether risk for 
adverse endocrine-related activity can be expected at clinical exposures.  In situations where 
there is a large exposure margin, no additional studies may be necessary.  
 
If further studies are warranted, sponsors can consider the following additional assessments: 
 
Mechanistic Studies.  Results of standard studies demonstrating evidence of adverse 
endocrine effects may trigger questions on mode of action and human relevance that could be 
addressed by mechanistic studies.  For example, if changes in endocrine tissues are noted in a 
standard study, the role of inhibition or induction of P450 isozymes might be appropriate to 
explore, because such inhibition or induction can be a mechanism by which drugs alter 
hormone levels in animals.  These mechanisms  might not be relevant for the drug in humans.  
If the relevance to humans of an endocrine signal is important for a regulatory decision, the 
sponsor can consider whether mechanistic studies in alternative animal models may be 
informative.  
 
Nonclinical Juvenile Studies.  Juvenile animal studies can be helpful in addressing the long-
term endocrine effects from acute or continuous exposure for those drugs administered to 
neonates and pediatric patients, when these effects cannot be adequately predicted from adult 
animals.   
 
Clinical Studies.  As an alternative to, or in addition to, supplemental nonclinical assessment 
of endocrine toxicity, inclusion of endocrine endpoints (e.g., hormone levels) in clinical 
6 
 Contains Nonbinding Recommendations 
studies can be important to clarify the relevance of adverse endocrine activity identified in 
nonclinical studies. Planning for these assessments should be discussed with the FDA.  
 
In summary, the standard comprehensive test battery is generally sufficient to identify endocrine-
related toxicity. Depending on the outcome of a standard battery of nonclinical tests, additional 
nonclinical studies may be warranted to more fully characterize the endocrine-related toxicity 
potential of a drug. For particular situations, the review division should be contacted for further 
guidance. 
 
7