Nonclinical Safety Evaluation of Pediatric Drug Products

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Guidance for Industry
Nonclinical Safety Evaluation
of Pediatric Drug Products
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
February 2006
Pharmacology and Toxicology

Guidance for Industry
Nonclinical Safety Evaluation
of Pediatric Drug Products
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 2006
Pharmacology and Toxicology

TABLE OF CONTENTS
I. 	INTRODUCTION............................................................................................................. 1
II. 	BACKGROUND ............................................................................................................... 2
III. 	GENERAL CONSIDERATIONS REGARDING THE NEED FOR STUDIES IN
JUVENILE ANIMALS .................................................................................................... 2
A. 	Differences in Drug Safety Profiles Between Mature and Immature Systems......................... 2
B. 	The Utility of Studies in Juvenile Animals................................................................................... 3
IV. 	GENERAL CONSIDERATIONS FOR EVALUATION OF PHARMACEUTICALS
IN JUVENILE ANIMALS ............................................................................................... 5
A. 	Scope of Nonclinical Safety Evaluation........................................................................................ 5
B. 	Timing of Juvenile Animal Studies in Relation to Clinical Testing........................................... 5
1. Long-Term Exposure in Pediatric Subjects ..................................................................................... 5
2. Short-Term Exposure in Pediatric Subjects..................................................................................... 6
3. Insufficient Clinical Data to Support Initiation of Pediatric Studies............................................... 6
C. 	Issues to Consider Regarding Juvenile Animal Studies ............................................................. 6
1. Developmental Stage of Intended Population.................................................................................. 7
2. Evaluating Data to Determine When Juvenile Animal Studies Should Be Used ............................. 7
3. Considering Developmental Windows When Determining Duration of Clinical Use ..................... 8
4. Timing of Exposure .......................................................................................................................... 8
5. Selection of Study Models ................................................................................................................ 8
V. 	GENERAL CONSIDERATIONS IN DESIGNING TOXICITY STUDIES IN
JUVENILE ANIMALS .................................................................................................... 9
A. 	Types of Studies ............................................................................................................................. 9
B. 	Animals ........................................................................................................................................... 9
1. Species ............................................................................................................................................. 9
2. Age ................................................................................................................................................. 10
3. Sex and Sample Size....................................................................................................................... 10
C. 	Exposure ....................................................................................................................................... 10
1. Route of Administration and Dosage Formulation........................................................................ 10
2. Frequency and Duration of Exposure............................................................................................ 11
3. Dose Selection................................................................................................................................ 11
D. 	Toxicological Endpoints and Timing of Monitoring................................................................. 11
VI. 	GENERAL CONSIDERATIONS FOR APPLICATION OF JUVENILE ANIMAL
DATA IN RISK MANAGEMENT................................................................................ 12
A. 	Use in Clinical Trials ................................................................................................................... 12
B. 	Use in Product Approval ............................................................................................................. 12
VII. 	HUMAN-TO-ANIMAL COMPARISONS OF DEVELOPMENTAL PERIODS.... 13
REFERENCES............................................................................................................................ 18

Contains Nonbinding Recommendations
Guidance for Industry1
Nonclinical Safety Evaluation of Pediatric Drug Products
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 document provides guidance on the role and timing of animal studies in the nonclinical
safety evaluation of therapeutics intended for the treatment of pediatric patients. The guidance
discusses some conditions under which juvenile animals can be meaningful predictors of toxicity
in pediatric patients and makes recommendations on nonclinical testing.
The scope of this guidance is limited to safety effects that cannot be adequately, ethically, and
safely assessed in pediatric clinical trials. Serious adverse effects that are irreversible are of
particular concern. The guidance also makes recommendations on the timing and utility of
juvenile animal studies in relation to phases of clinical development. Sponsors are encouraged to
communicate with the appropriate review division to determine whether a juvenile animal study
is needed for a particular drug product and to discuss protocol designs before study initiation.
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 Pediatric Subcommittee to the Pharmacology and Toxicology Coordinating
Committee within the Office of New Drugs (OND) in the Center for Drug Evaluation and Research (CDER) at the
Food and Drug Administration. It does not apply to pediatric products regulated by the Center for Biologics
Evaluation and Research (CBER). For information on products regulated by CBER, contact the appropriate CBER
office.
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II. 	BACKGROUND
Many therapeutics marketed in the United States and used in pediatric patients lack adequate
information in the labeling for use in that population. A survey conducted by the American
Academy of Pediatrics shows that the majority of the drugs listed in the Physician’s Desk
Reference lack information on safety and/or efficacy for pediatric use (Committee on Drugs,
American Academy of Pediatrics 1995). However, recent pediatric legislation, including the
Best Pharmaceuticals for Children Act (BPCA 2002) and the Pediatric Research Equity Act
(PREA 2003), have provided a mechanism to obtain the needed pediatric safety and efficacy
information in drug product labels.
Drug development programs have used safety data from clinical studies in adults, supported by
nonclinical studies in adult animals, to support the use of a drug in pediatric patients. This
assumes that pediatric patients will exhibit similar disease progression, and respond similarly to
the intended therapeutic intervention. It is clear, however, that these studies may not always
assess possible drug effects on developmental processes specific to pediatric age groups.
Developmental processes in pediatric patients may differentially affect drug pharmacokinetics
and pharmacodynamics compared to adult therapeutic use. Some adverse effects may be very
difficult to detect in clinical trials or during routine postmarketing surveillance. Data obtained
from clinical pediatric initiatives have identified ineffective dosing and overdosing of effective
drugs as well as unnecessary exposure to ineffective therapies and identification of novel
pediatric adverse events. Juvenile animal studies may assist in identifying postnatal
developmental toxicities that are not adequately assessed in reproductive toxicity assessments
and that may not be adequately and safely tested in pediatric clinical trials.
III. 	GENERAL CONSIDERATIONS REGARDING THE NEED FOR STUDIES IN
JUVENILE ANIMALS
Considerations such as postnatal development and the utility of studies conducted using juvenile
animals are discussed in this section.
A. 	Differences in Drug Safety Profiles Between Mature and Immature Systems
Some therapeutics have shown different safety profiles in pediatric and adult patients. Inherent
differences between mature and immature systems introduce the possibility of drug toxicity, or
resistance to toxicity in immature systems that are not observed in mature systems. Several
factors contribute to these potential differences. Postnatal growth and development can affect
drug disposition and action. Examples include developmental changes in metabolism (including
the maturation rate of Phase I and II enzyme activities), body composition (i.e., water and lipid
partitions), receptor expression and function, growth rate, and organ functional capacity. These
developmental processes are susceptible to modification or disruption by drugs.
Although some age-dependent effects can be largely predicted by knowledge of the changes in
drug metabolic pathways during development, others cannot. There are several examples of
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drugs that exhibit differences in toxicity between adult and pediatric patients. These include the
following:
• 	Acetaminophen — Acute acetaminophen toxicity is a classic example of how maturation
can affect the toxicity profile of a drug. Young children are far less susceptible to acute
acetaminophen toxicity than adults because children possess a higher rate of glutathione
turnover and more active sulfation. Thus, they have a greater capacity to metabolize and
detoxify an overdose of acetaminophen when compared to adults (Insel 1996).
• 	Valproic acid — In contrast to acetaminophen, young children treated with valproic acid
appear disproportionately vulnerable to fatal hepatotoxicity (Dreifuss et al. 1987).
• 	Chloramphenicol — Chloramphenicol is associated with mortality in newborns because
exposure is increased due to a longer half-life (t½ = 26 h) compared to adults (t½ = 4 h)
(Kapusink-Uner et al. 1996).
• 	Inhaled corticosteroids — Inhaled corticosteroids have been found to decrease growth
velocity in children, an irrelevant endpoint in adults (FDA Talk Paper, Class Labeling for
Intranasal and Orally Inhaled Corticosteroid Containing Drug Products Regarding the
Potential for Growth Suppression in Children, 1998).
• 	Aspirin — Aspirin should not be used to treat children with influenza or varicella
infections because of their increased risk of developing Reye’s syndrome, a complication
not seen in adults (Belay et al. 1999).
• 	Lamotrigine — Children are at greater risk for developing hypersensitivity-type
reactions, including Stevens-Johnson syndrome, when treated with lamotrigine
(Guberman et al. 1999).
B. 	The Utility of Studies in Juvenile Animals
Adult clinical data can provide useful information regarding study design and dose selection for
further study in children in some circumstances. Nonclinical developmental toxicity studies
have traditionally focused on prenatal development, with only limited assessment of postnatal
developmental effects. Animals used in multiple-dose toxicity studies are usually peripubertal.
In some circumstances, data generated from these studies may provide sufficient information to
support pediatric clinical trials without additional animal studies, particularly if the intended use
includes adolescents but not younger children or infants. Since young animals in general exhibit
developmental characteristics similar to pediatric patients, they are considered appropriate
models for assessing drug effects in this population. The Agency believes that data from
juvenile animal studies can contribute to the assessment of potential drug toxicity in the pediatric
population, and can provide information that might not be derived from standard toxicology
studies using adult animals, or safety information from adult humans.
It is thought that organ systems at highest risk for drug toxicity are those that undergo significant
postnatal development. Thus, evaluation of postnatal developmental toxicity is a primary
concern. The structural and functional characteristics of many organ systems differ significantly
between children and adults as a result of the growth and development that takes place during
postnatal maturation. Examples include the following:
• 	Brain, where neural development continues through adolescence (Rice and Barone 2000)
• 	Kidneys, where adult levels of function are first reached at approximately 1 year of age
(Radde 1985)
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• 	Lungs, where most alveolar maturation occurs in the first 2 years of life (Burri 1997)
• 	Immune system, where adult levels of IgG and IgA antibody responses are not achieved
until about 5 and 12 years of age, respectively (Miyawaki et al. 1981)
• 	Reproductive system, where maturation is not completed until adolescence (Zoetis and
Walls 2003)
• 	Skeletal system, where maturation continues well into adulthood for 25-30 years (Zoetis
and Walls 2003)
• 	Gastrointestinal systems, which may have direct consequences on bioavailability,
clearance, and biotransformation of drugs are functionally mature by about 1 year of age
(Walthall 2005).
Studies in juvenile animals may be useful in the prediction of age-related toxicity in children, as
shown in the following examples:
• 	The effects of phenobarbital on cognitive performance in children were predicted by
experimental studies examining the effects of this drug on the developing rodent nervous
system (Farwell et al. 1990; Fonseca et al. 1976; Diaz et al. 1977)
• 	The vulnerability of human neonates to hexachlorophene neurotoxicity was modeled in
developing rats and monkeys (Towfighi 1980)
• 	The increased susceptibility of infants to verapamil-induced cardiovascular complications
would be expected based on animal studies demonstrating a greater sensitivity of the
immature heart to calcium channel blockade (Skovranek et al. 1986; Boucek et al. 1984)
• 	An increased risk of convulsions in young children treated with theophylline was
predicted by studies of the preconvulsant effects of this agent in developing rodents
(Mares et al. 1994; Yokoyama et al. 1997)
Examples of drug-induced postnatal developmental toxicity demonstrated in animals include the
following:
• 	Neurobehavioral impairment in adult rats following early postnatal exposure to
methamphetamine (Vorhees et al. 1994)
• 	The effects of methylphenidate on growth and endocrine function in young rats (Greeley
and Kizer 1980; Pizzi et al. 1987)
• 	Apoptotic neurodegeneration in neonatal rats treated with NMDA receptor antagonists
(Ikonomidou et al. 1999)
• 	Decreased myelination and axonal damage induced in preweanling rats by vigabatrin
(Sidhu et al. 1997)
• 	Long-term changes in serotonergic innervation in rats exposed to fluoxetine during early
juvenile life (Wegerer et al. 1999)
• 	Chondrotoxicity in immature animals treated with fluoroquinolones (Stahlmann et al.
1997)
Although the significance of these findings for humans is uncertain, there is evidence that
some of these effects can be relevant to growing children, notably those of methylphenidate
(Mattes and Gittelman 1983; Croche et al. 1979) and fluoroquinolones (Chang et al. 1996; Le
Loet et al. 1991).
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IV. 	GENERAL CONSIDERATIONS FOR EVALUATION OF PHARMACEUTICALS
IN JUVENILE ANIMALS
A. 	Scope of Nonclinical Safety Evaluation
The nonclinical safety evaluation of pediatric therapeutics should primarily focus on their
potential effects on growth and development that have not been studied or identified in previous
nonclinical and clinical studies. Juvenile animal testing may be useful in assessing potential
developmental age-specific toxicities and differences in sensitivity between adult and immature
animals. Although the toxicological assessment should focus primarily on the active moiety,
testing the inactive ingredients in the clinical formulation can also be important, particularly
when a drug’s pharmacodynamics or distribution are altered by the inactive ingredients or when
uncharacterized excipients are present. Additional recommendations on testing excipients can be
found in the guidance for industry Nonclinical Studies for the Safety Evaluation of
Pharmaceutical Excipients.2 The toxicological assessment should include local and systemic
analyses of effects on postnatal growth and development in the anticipated pediatric population.
The known pharmacological and toxicological properties of the drug relative to the proposed
patient population should be considered. Any concerns for postnatal developmental toxicity can
be addressed either in juvenile animal studies or by modified study designs (e.g., modification of
segment III reproductive toxicity studies to include animals of similar developmental status as
the pediatric population of concern). Juvenile animal studies are especially relevant when a
known target organ toxicity occurs in adults in tissues that undergo significant postnatal
development. The extent and timing of nonclinical safety studies will depend on the available
safety information for a particular product. For example, the information needed to support a
new pediatric indication for an approved product used in adults may be quite different from the
information needed to support pediatric use of a new molecular entity because of the postnatal
developmental safety concerns in the later population. These concerns will be considered for
their particular clinical indications on a case-by-case basis within the drug review divisions.
B. 	Timing of Juvenile Animal Studies in Relation to Clinical Testing
Specific recommendations regarding the timing of nonclinical toxicology studies are available in
the ICH guidance for industry M3 Nonclinical Safety Studies for the Conduct of Human Clinical
Trials for Pharmaceuticals (ICH M3 safety studies guidance). The recommendations presented
here for juvenile animal studies may assist in identifying postnatal developmental toxicities that
are not adequately assessed in general toxicity studies with mature animals and that may not be
adequately and safely tested in pediatric clinical trials.
1. 	Long-Term Exposure in Pediatric Subjects
Most clinical studies in pediatric subjects do not involve long-term exposure to a therapy because
they are generally of short-term duration (less than 6 months). This is especially true when the
trials are intended to determine pharmacokinetics rather than efficacy. As a result, long-term
exposure during postnatal developmental periods is not usually addressed in pediatric clinical
2 We update guidances periodically. To make sure you have the most recent version of a guidance, check the CDER
guidance Web page at http://www.fda.gov/cder/guidance/index.htm.
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trials. If the drug is indicated for chronic use then some assessment of the long-term
developmental effects of the drug in animals should be made before marketing. However, in
those cases when pediatric clinical studies do involve long-term exposure, we recommend
conducting juvenile animal studies before initiation of the long-term clinical studies. When
designing juvenile animal studies, the age of the pediatric population for which the drug is
intended is important. Neonates, infants, and older children are at very different developmental
stages, and appropriate nonclinical data should support the drug’s use in the intended pediatric
population.
2. 	Short-Term Exposure in Pediatric Subjects
Depending on the indication and use of the drug, safety concerns, and the number of subjects
exposed, there may be a need for juvenile animal studies in conjunction with clinical studies
even if the trials are designed for short-term exposure. Because juvenile animal studies may
identify potential hazards and these hazards may have relevance to human safety, it may be more
useful to complete juvenile animal studies before conducting clinical studies so that appropriate
monitoring can be incorporated into the clinical trial design to limit human risk.
3. 	Insufficient Clinical Data to Support Initiation of Pediatric Studies
Typically, pediatric subjects are included in clinical trials after there has been considerable
experience in the adult population. When there is insufficient clinical data or experience because
of minimal prior adult and pediatric experience, completed juvenile animal studies are needed
before initiation of pediatric clinical trials regardless of whether the clinical trials involve long-
term exposures. Similarly, when there have been reports of adverse effects with off-label use in
pediatric patients and there are inadequate data to evaluate the relationship between the drug and
the adverse effects, completed juvenile animal studies are needed before initiation of pediatric
clinical studies. The timing of juvenile animal studies relative to clinical testing of therapeutics
indicated for serious or life-threatening pediatric conditions will be considered on a case-by-case
basis by the review division.
C. 	Issues to Consider Regarding Juvenile Animal Studies
These considerations are important in determining the appropriateness and design of juvenile
animal studies: (1) the intended or likely use of the drug in children; (2) the timing of dosing in
relation to phases of growth and development in pediatric populations and juvenile animals; (3)
the potential differences in pharmacological and toxicological profiles between mature and
immature systems; and (4) any established temporal developmental differences in animals
relative to pediatric populations. We also recommend that endpoints relevant to identifying
target organ toxicity across species be included in the juvenile animal study design. Juveniles
generally undergo more dynamic development than is seen in the relatively stable adult.
Although the greatest concern is with chronic, long-term therapy, the duration of anticipated
treatment of the pediatric population should be considered in relation to the duration of
developmentally sensitive phases. For instance, a relatively short exposure time for neonates
may cover a period of more substantial development than would a longer exposure in
prepubescent children where development occurs over a much longer time frame. It is important
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for juvenile animal toxicology studies to be designed efficiently, using the least number of
animals to identify potential pediatric safety concerns. Whenever feasible, we recommend
designing an initial study to address endpoints of concern for multiple potential pediatric
populations. In all cases, studies using juvenile animals are appropriate when adequate
information cannot be generated using standard nonclinical studies or from clinical trials. The
following issues are specific to studies in juvenile animals for assessing toxicity.
1. 	Developmental Stage of Intended Population
Consideration should be given to the age of the intended population and thus the stage of
postnatal development. The condition to be treated may also influence the type, extent, and
timing of testing considered appropriate. Selection of appropriate endpoints in the nonclinical
studies to address concerns for the specific pediatric populations is important.
Recommendations regarding specific age ranges of pediatric subpopulations are discussed in the
ICH guidance for industry E11 Clinical Investigation of Medicinal Products in the Pediatric
Population.
2. 	Evaluating Data to Determine When Juvenile Animal Studies Should Be Used
Evaluation of the available data is important when considering the need for studies in juvenile
animals. Toxicity studies in juvenile animals may be appropriate when available nonclinical or
clinical data are insufficient to support reasonable safety of a therapeutic for pediatric patients.
Gaps in the age ranges of rodent and nonrodent species used in standard toxicity testing are
widely acknowledged. These age gaps can affect assessment of nervous system toxicity
endpoints in particular because of the extended process of maturation. Standard toxicity studies
with adult animals cannot assess all of the relevant endpoints, especially growth present in the
immature animal. In other circumstances, however, juvenile animal studies would be neither
informative nor necessary. For example, juvenile animal studies might not be necessary when:
(1) data from similar therapeutics in a class have identified a particular hazard and additional
data are unlikely to change this perspective; (2) there are adequate clinical data and adverse
events of concern have not been observed during clinical use; (3) target organ toxicity would not
be expected to differ in sensitivity between adult and pediatric patients because the target organ
of toxicity is functionally mature in the intended pediatric population and younger children with
the functionally immature tissue are not expected to receive the drug.
Most drugs that are intended for use in pediatric patients have established efficacy and safety
profiles in adult humans. Some data may also be available from pediatric patients aged 12 years
or older. For some drugs a preponderance of clinical data will be obtained from children, as in
the case of inhaled corticosteroids (FDA Talk Paper, Class Labeling for Intranasal and Orally
Inhaled Corticosteroid Containing Drug Products Regarding the Potential for Growth
Suppression in Children, 1998). For approved drugs that have already undergone extensive
clinical testing, substantial nonclinical pharmacology and toxicology data will have already been
performed. The toxicology assessment generally includes studies of general toxicity,
reproductive toxicity, genetic toxicity, carcinogenicity, and special toxicities, as well as studies
in juvenile animals, if available. Target organs of toxicity of the drug both in humans and
animals should have been identified in these studies. A thorough evaluation of these data should
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enable scientists to: (1) judge the adequacy of the nonclinical information; (2) identify some of
the potential safety concerns for the intended population; and (3) identify any gaps in the data
that might be addressed by testing in juvenile animals.
3. 	Considering Developmental Windows When Determining Duration of Clinical
Use
Based on the observation that embryo-fetal development is especially sensitive to perturbation
during organogenesis, tissues that undergo significant postnatal development in pediatric patients
and juvenile animals may also have greater sensitivity to certain drug-induced toxicities than
mature tissues. Organ systems identified as undergoing considerable postnatal growth and
development include the nervous, reproductive, pulmonary, renal, skeletal, gastrointestinal,
hepatobiliary, and immune systems. Given the variable rate of postnatal development during
different periods of childhood, the definition of long-term treatment can vary by pediatric
population. Intended treatment of several weeks may not be considered long term in early
adolescence, but might involve considerable development for the neonate given the duration of
some developmental windows.
4. 	Timing of Exposure
The timing of the intended use of the drug as it relates to periods of rapid postnatal growth and
development is important. If the drug is intended for use in children undergoing phases of rapid
overall growth and development, it is important to evaluate an animal model undergoing a
corresponding growth phase. Organ systems mature at specific times in specific species.
Human-to-animal comparisons of developmental periods for the nervous, reproductive, skeletal,
pulmonary, immune, renal, cardiac, and metabolic systems are presented in Section VII at the
end of this guidance. These can be used as general guides to appropriate periods of treatment to
assess the development of specific systems in various animal models. Immature animals have
accelerated chronological development compared to humans, which can facilitate evaluation of
long-term effects following acute or chronic exposure using well-defined endpoints (e.g.,
assessment of reproductive or nervous function).
5. 	Selection of Study Models
In addition to consideration of models and endpoint assessments based on the intended pediatric
human use, target organs for toxicological and pharmacological activity identified in adults need
special consideration. It is important that organ systems identified as specific targets of drug
toxicity in adults and that undergo significant postnatal development be studied in juvenile
animals for those specific effects, even when the primary postnatal developmental period in
humans does not coincide with the intended treatment phase. This is based on the observation
that development is generally a continuous event. Additionally, a therapeutic target tissue may
be developmentally regulated by other tissues or organ systems. In such cases, it may be
advisable to examine the effects of the drug during the stages of development relevant to all of
those tissues/organs in a test species.
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V. 	GENERAL CONSIDERATIONS IN DESIGNING TOXICITY STUDIES IN
JUVENILE ANIMALS
A. 	Types of Studies
Testing approaches can use generalized screening tests to provide hazard identification or can be
designed to specifically address identified concerns. We recommend the selection of an
appropriate, scientifically justified study design. The effects of dosing and handling on
immature animals can be systematically assessed. Studies conducted in juvenile animals to
support the safety of pediatric therapeutics may either be protocols designed to address a specific
safety concern, or modified peri- and postnatal developmental study protocols. Dedicated
juvenile animal protocols can be designed to address specific concerns based on known
properties of the drug, product class, or other information. Modified repeat-dose toxicity studies
can provide a more general screen for potential hazards in some instances. However, we
recommend that such studies modify the animal age at study initiation, duration of treatment, and
endpoints assessed to address the specific concerns. Modification of standard ICH studies
designed to address developmental stages C-F3 would include ensuring adequate exposure in
juvenile animals during the postnatal period and assessment of developmental endpoints
appropriate for the intended pediatric population. Assessment of developmental endpoints not
usually included in standard repeat-dose toxicity studies also may be appropriate. In addition to
ensuring adequate exposure to the drug, histopathologic examinations and effects on specific
growth parameters and functionally immature tissues in the juvenile animal would be important.
In these modified designs dosing can be initiated with animals younger than usual and extended
until the developmental period for the intended pediatric population has been completed in the
animal species in accordance with the age of the pediatric patients who would use the drug.
Information from such studies can be compared with the findings from treated adults of the same
species to evaluate whether the effects are specific to juvenile animals.
B. 	Animals
1. 	Species
The species of the juvenile animal tested should be appropriate for evaluating toxicity endpoints
important for the intended pediatric population. Traditionally, rats and dogs have been the
rodent and nonrodent species of choice. In some circumstances, however, other species may be
more appropriate. For example, when drug metabolism in a particular species differs
significantly from humans, an alternative species (e.g., minipigs, pigs, monkeys) may be more
appropriate for testing. When determining an appropriate species, sponsors are encouraged to
consider certain factors, such as the following:
• 	Pharmacology, pharmacokinetics, and toxicology of the therapeutic agent
• 	Comparative developmental status of the major organs of concern between juvenile
animals and pediatric patients
• 	Sensitivity of the selected species to a particular toxicity
3 ICH guidance for industry S5A Detection of Toxicity to Reproduction for Medicinal Products
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A study in juveniles from one animal species may be sufficient to evaluate toxicity endpoints for
therapeutics that are well characterized in both adult humans and animals. It is anticipated that
this evaluation often can be accomplished in the rodent using modified perinatal and postnatal
developmental studies, although other approaches can be used.
2. 	Age
The age of the animals at initiation of dosing should be determined by the postnatal development
parameters of interest. It is important that the stage of development in the animals being studied
be comparable to that in the intended pediatric population.
3. 	Sex and Sample Size
We recommend including both male and female animals in these studies. It is important that
adequate numbers of animals are used to demonstrate the presence or absence of effects of the
test substance. When determining the sample size, consideration of the magnitude of the
biologic effect that is of concern is also important. The particular study design used (e.g., a
screening study or one designed to address an identified concern, modification of a standard
design, composite or split litter design) will influence the number of animals it takes for an
adequate evaluation.
C. 	Exposure
1. 	Route of Administration and Dosage Formulation
When performing nonclinical studies, the intended clinical route of administration and dosage
formulation4 should be used unless an alternate route of administration and dosage formulation
provides greater exposure or is less invasive with adequate exposure. Assessment of toxic
effects by more than one route of administration can be appropriate if the drug is intended for
clinical use by more than one route of administration. When different routes are expected to
result in differences in systemic and local exposure of such magnitude that occurrence of
postnatal toxicity would be expected, sponsors should consider testing by multiple routes. When
the intended clinical administration is intravenous, this route should be sufficient. Since the
primary purpose of these studies is to identify potential hazards, small changes in
exposure/distribution by route generally would not be considered important.
Since adverse effects can sometimes be related to metabolic differences between adult and
juvenile animals, toxicokinetic studies can provide useful information for assisting in study
interpretation. Assessment of developmental differences in parent drug disposition and profiles
of significant metabolites in juvenile animals should be made according to established guidelines
(see the ICH guideline for industry S3A Toxicokinetics: Assessment of Systemic Exposure in
Toxicity Studies).
4 We recommend safety evaluations of inactive ingredients be conducted to determine potential adverse effects in
pediatric subjects. The type of testing is dependent on the extent to which this information is already well
understood.
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2. 	Frequency and Duration of Exposure
The frequency of administration should be relevant to the intended clinical use of the drug. In
some cases, however, the use of dosing frequencies similar to those anticipated for clinical
administration are not feasible because of technical considerations for the animal models used.
Changes in frequency can be made when variables such as metabolic and kinetic differences are
considered.
The duration of treatment in animals should include at least the significant periods of relevant
postnatal development for the selected species. When the aim of the study is to evaluate
potential long-term effects, dosing duration should be increased relative to the intended
therapeutic use. One approach to consider is establishing exposure and initial tolerability in a
dose-range finding study followed by a definitive study powered to assess specific concerns.
Treatment-free periods designed to assess reversibility of possible adverse effects should also be
considered. Inclusion of recovery periods in studies can be valuable in distinguishing acute to
intermediate pharmacodynamic effects from frank developmental toxicity, and this information
could influence the evaluation of potential human risk. Depending on the concern being
addressed, it may be sufficient to assess delayed toxicity through organ maturity or it may be
necessary to continue until the juvenile animal reaches adulthood.
3. 	Dose Selection
It is important to establish a clear dose-response relationship for adverse effects in juvenile
animals, when possible. The high dose should produce identifiable toxicity (either
developmental or general). The intermediate dose should produce some toxicity so that a dose-
response relationship can be demonstrated if one exists. The low dose should produce little or no
toxicity, and a NOAEL should be identified, if possible. We recommend evaluating and
potentially modifying intermediate and low doses in relation to those that produce the desired
pharmacodynamic effect in the test species.
D. 	Toxicological Endpoints and Timing of Monitoring
The selection of toxicological endpoints to be monitored in a juvenile animal study is critical for
assessing the effects of a drug on development and growth. Designing studies to determine drug
effects on overall postnatal growth as well as postnatal development of specific organ systems
(e.g., skeletal, renal, lung, nervous, immunologic, cardiovascular, and reproductive) is
appropriate. It is important that studies include measurement of overall growth (e.g., body
weight, growth velocity per unit time, tibial length), clinical observations, measurement of organ
weights, gross and microscopic examinations, assessment of sexual maturation (mating, fertility),
and neurobehavioral testing. More specific measurements can be reserved for case-specific
evaluations based on the knowledge of the pharmacologic or toxicologic target. Clinical
pathology determinations can also be useful but can be limited by the technical feasibility of
obtaining adequate samples for analysis, particularly in the case of rodents. For developmental
neurotoxicity assessments, well-established methods should be used to monitor key central
nervous system (CNS) functions, including assessments of reflex ontogeny, sensorimotor
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function, locomotor activity, reactivity, learning, and memory. Modifications of existing toxicity
designs or de novo juvenile studies should be used depending on the concerns to be addressed.
It can be helpful to determine the relationship between toxicologic endpoints and drug exposure
(e.g., predosing, immediately postdosing, time of peak plasma concentration). To differentiate
long-term effects on development from acute effects, it might be appropriate to measure certain
endpoints immediately before daily administration of the drug. Also, adding recovery group
animals is helpful in determining whether the drug-induced effects are reversible. The more
specific the concern, the more directed the study design approach can be. A more generalized
screening approach may be useful if little information is available.
VI. 	GENERAL CONSIDERATIONS FOR APPLICATION OF JUVENILE ANIMAL
DATA IN RISK MANAGEMENT
A. 	Use in Clinical Trials
It is important that nonclinical toxicology studies designed to support the safety of clinical trials
in pediatric subjects identify hazards specific to the treated population. These studies can
provide information useful in limiting the risk of experiencing adverse events and identify
appropriate clinical monitoring. When adverse effects are observed in nonclinical toxicology
studies, there are a number of possible uses of these findings. Biomarkers of adverse effects
could be identified in nonclinical studies that would be useful in monitoring subjects in clinical
trials. In cases where biomarkers cannot be identified or safely used in clinical studies,
nonclinical pharmacokinetic data could be useful because a given adverse effect would be
associated with a particular level of systemic exposure which might be extrapolated to clinical
use. Blood level monitoring could then be used in clinical trials to minimize the probability of
such an adverse effect occurring. If toxicities identified in juvenile animal studies are likely to
occur in pediatric patients, cannot be monitored clinically, and would not be considered an
acceptable potential consequence of exposure, it may not be possible to safely conduct pediatric
clinical trials. Consideration of the risk-benefit analysis of a given drug therapy is important.
B. 	Use in Product Approval
Nonclinical toxicology studies in juvenile animal models could demonstrate adverse effects that
should be considered in seeking postmarketing commitments by the sponsor, in labeling a
product for pediatric use, or in determining the approvability of a drug for pediatric use. Delayed
or irreversible adverse effects might be identified in animal studies but not in clinical trials
because the pediatric clinical trial might have been of insufficient duration to demonstrate the
adverse effect. It is possible that biomarkers of adverse effects could be identified in nonclinical
studies that were not seen in clinical trials, but might nevertheless be important to include in the
product label. Depending on the nature and severity of these adverse effects and the risk-benefit
relationship of the intended use, the sponsor might conduct long-term follow-up human safety
studies as a postmarketing commitment. The sponsor might conduct long-term follow-up studies
even following acute drug exposure if these effects were found to be delayed or irreversible. Use
of the drug could be restricted to serious indications based on nonclinical findings even if the
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adverse effects were not demonstrated in clinical trials. In this case, the product label would
include information on the relevant adverse effects observed in nonclinical studies. Adverse
effects associated with chronic drug exposure in nonclinical studies might not have been
observed in clinical trials of comparable length. In such a case, the label might be written to
reflect these findings. Juvenile animal studies might also be useful in identifying specific age
groups in which the drug should not be used or in determining unsafe parameters of exposure.
Finally, it is possible that nonclinical findings could result in a product label that specifically
warns against use in pediatric patients based on a risk-benefit analysis.
VII. 	HUMAN-TO-ANIMAL COMPARISONS OF DEVELOPMENTAL PERIODS
The information on comparative developmental timing, shown in Tables 1 – 8, was considered
current at the time this guidance was developed. These comparisons should be considered with
new information as it becomes available in deciding how best to design appropriate juvenile
animal studies to address risks to the pediatric population. Neither the human nor the animal
data represent a precise determination of the timelines of development due to the inherent
variability and different endpoints examined. Because of the nature of science, these tables
should only serve as a general starting point.
Table 1: Nervous System
Developmental
Event
Postnatal Developmental Period
Human
(Years)
Primate
(Weeks)
Dog
(Weeks)
Rat
(Days)
Glutamate receptors1
(Maximal binding)
1-2 Cortex
Decline to
adult 2-16
28
Decline to
adult >28
Monoamine system2 2-4
Maximum
receptor
density
21-30
Adult
levels
Ocular dominance3 0-3 	21-35
Cerebellum
persistent external
germinal layer3
0.6-2 	0-21
Rapid phase of
myelination ends4
2 	25-30
Cognitive
development
Delayed response
learning5
1-2 	9-36 	12-16 	10-35
1 Ikonomidou et al. 1999
2 Rice and Barone 2000
3 Sidhu et al. 1997; Kimmel and Buelke-Sam 1994
4 Radde 1985
5 Wood et al. 2004
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Table 2: Reproductive System
Postnatal Developmental Period	Developmental
Event 	Human
(Years)
Rhesus
Monkey
(Years)
Dog
(Days)
Mouse
(Days)
Rat
(Days)
Puberty1 11-12 	2.5-3 	180-
240
35-45 	40-60
1 DeSesso and Harris 1995; Marty et al. 2003; Beckman and Feuston 2003; Lewis et al.
2002
Table 3: Skeletal System
Developmental
Event
Postnatal Developmental Period
Fusion of 2o
Ossification
Centers1
Human
(Years)
Monkey
(Years)
Dog
(Years)
Rabbit
(Weeks)
Rat
(Weeks)
Mouse
(Weeks)
Femur Distal
Epiphysis
14-19 	3-6 	0.7-0.9 	32 	15-162 	12-13
1 Zoetis 2003
Table 4: Pulmonary System1
Developmental Event 	Postnatal Developmental Period
(Days)
Alveoli Formation2,3,4 Human 	Rat 	Mouse
Onset 	Prenatal 	1-4 	1-2
Completion 	730 	28 	28
1 The stages of lung development (glandular, canalicular, saccular, alveolar) at birth varies
with the species. Human lungs have few alveoli and are considered in the alveolar stage at
birth. Rodent lungs are less developed and considered in the saccular stage without
alveoli at birth (Zoetis and Hurtt 2003).
2 Burri 1997
3 Merkus et al. 1996
4 Tschanz and Burri 1997
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Table 5: Immune System
Developmental Event 	Postnatal Developmental Period
(Days)
Human 	Mice
B-cell Development1 Prenatal 	Prenatal
T-cell Development1 Prenatal 	Prenatal
NK-cell Development1 Prenatal 	21
T-dependent Antibody
response1
0 	14
41-56 Adult level
T-independent Antibody
response1
45-90 	0
14-21 Adult level
Adult level IgG1 1825 	42-56
1 Holladay and Smialowicz 2000
Table 6a: Renal — Functional
Developmental Event 	Postnatal Developmental Period
(Days)
Human 	Rat
Glomerulo-/Nephrogenesis1,2 Prenatal 	8-14
Adult GFR and tubular
secretion1,2
45-180 	15-21
1 Snodgrass 1992
2 Travis 1991
Table 6b: Renal — Anatomical
Developmental
Event
Postnatal Developmental Period
(Weeks)
Human 	Dog 	Rabbit 	Rat 	Mouse 	Pig
Completion of
Nephrogenesis1
Prenatal
Week
35
2 	2-3 	4-6 	Prenatal 	3
1 Zoetis 2003
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Table 7: Metabolism
Developmental Modulation of Phase I/II Metabolism
Maturation of Enzyme Activity
Enzyme 	Human
(Years)
Rat
(Days)
Rabbit
(Days)
CYP2D61,2 0-3 	NA* 	NA*
CYP2E12,3,4 0-1 	4-17
↓ Post weaning
male>female
14-35
2X adult
@35
CYP1A21,5,6,7 0.5
1(> adult)
7-100 Low
levels
21-60
CYP2C81,2 <1 	NA* 	NA*
CYP2C91,2 <0.5
0.5 (>
adult)
NA* 	NA*
CYP3A42 0-2 	NA* 	NA*
Acetylation1,2 1
(35%
adult)
NA* 	NA*
Methylation1,2 <1
(50%
adult)
NA* 	NA*
Glucuronidation1,2 0 (>adult)
12
NA*
Sulfation1,2 0 	NA* 	NA*
* NA = not available
1 Kearns and Reed 1989
2 Leeder and Kerns 1997
3 Waxman, Morrissey, Le Balnc 1989
4 Peng, Porter, Ding, Coon 1991
5 Ding, Peng, Coon 1992
6 Imaoka, Fujita, Funai 1991
7 Pineau, Daujat, Pichard, Girard, Angevain 1991
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Table 8: Cardiac1
Postnatal Developmental Period
(Maturation Level Similar to Adult)
Cardiac
Parameter
Human
(Years)
Dog 	Rat
(Weeks)
Electrophysiology
(ECG)
5-7 years 	NA* 	3-8
Cardiac Output
(CO) and
Hemodynamics
Birth 138 bpm;
Adults 85 bpm.
<2 yrs: Smaller
ventricular vol.,
stroke index,
ejection fraction vs.
adult
Birth BP 62/40; 2
months 85/47; 0.5-8
yrs. Diastolic 58-62
Increase in BP and
decrease in HR from 1
week to 0.5 years
Early increase in HR
then constant into
adulthood
High CO and low
PVR
Neonate-puberty
systolic BP doubles
reaches maturity by
10 weeks
Myocytes 	Diploid at birth
compared to 60% in
adults (40%
polyploidy)
NA* 	Primarily diploid in
infant and adult
Coronary
Vasculature
Diameter of arteries
doubled at 1 yr. max
at 30 yrs. Capillary
angiogenesis occurs
postnatally and
density decreases
with age
Capillary
angiogenesis occurs
postnatally and
density decreases with
age
Capillary
angiogenesis occurs
postnatally arterial
maturation by 1
month
Cardiac
innervation
Neuron number
increase and reach
adult pattern/density
in childhood
Continued
development during
2-4 months
Adrenergic pattern
mature by 3 weeks
and nerve density
mature by 5 weeks.
Cholinergic matures
postnatally
* NA = not available
1 Hew and Keller 2003
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22
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