Nonclinical Evaluation of Endocrine-Related Drug Toxicity

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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
Guidance for Industry1
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, FDA’s guidance documents do not establish legally enforceable responsibilities.
Instead, guidances describe the Agency’s 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.
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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.
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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.
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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.
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Contains Nonbinding Recommendations
IV. 	WHEN ADDITIONAL ASSESSMENT OF ADVERSE ENDOCRINE-RELATED
EFFECTS MAY BE WARRANTED
Standard nonclinical testing programs conducted in accordance with CDER’s 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 drug’s 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
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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
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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.
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