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 
1
Guidance for Industry  
Nonclinical Safety Evaluation of Pediatric Drug Products
 
 
 
 
 
This guidance represents the Food and Drug Administrations (FDAs) 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.   
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the Agencys 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.   
  1 Contains Nonbinding Recommendations 
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 Physicians 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 
  2 Contains Nonbinding Recommendations 
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 (t  = 26 h) compared to adults (t  = 4 h) 
12 12
(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 Reyes 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) 
  3 Contains Nonbinding Recommendations 
  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). 
 
 
  4 Contains Nonbinding Recommendations 
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 drugs 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. 
  5 Contains Nonbinding Recommendations 
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 drugs 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 
  6 Contains Nonbinding Recommendations 
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 
  7 Contains Nonbinding Recommendations 
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.  
 
 
  8 Contains Nonbinding Recommendations 
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 
  9 Contains Nonbinding Recommendations 
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. 
  10 Contains Nonbinding Recommendations 
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 
  11 Contains Nonbinding Recommendations 
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 
  12 Contains Nonbinding Recommendations 
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  Postnatal Developmental Period 
Event 
  Human  Primate  Dog  Rat 
(Years)  (Weeks)  (Weeks)  (Days) 
Glutamate receptors1  1-2 Cortex      28 
(Maximal binding)  Decline to  Decline to 
adult 2-16  adult >28 
Monoamine system2  2-4      21-30 
Maximum  Adult 
receptor  levels 
density 
Ocular dominance3  0-3      21-35 
Cerebellum  0.6-2      0-21 
persistent external 
germinal layer3 
Rapid phase of  2      25-30 
myelination ends4 
Cognitive  1-2  9-36  12-16  10-35 
development 
Delayed response 
learning5 
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 
  13 Contains Nonbinding Recommendations 
 
 
Table 2:  Reproductive System 
Developmental  Postnatal Developmental Period 
Event  Human  Rhesus  Dog  Mouse Rat 
(Years)  Monkey  (Days) (Days)  (Days) 
(Years) 
Puberty1  11-12  2.5-3  180- 35-45  40-60 
240 
1 DeSesso and Harris 1995; Marty et al. 2003; Beckman and Feuston 2003; Lewis et al. 
2002 
 
 
Table 3:  Skeletal System 
Developmental  Postnatal Developmental Period 
Event 
Fusion of 2o  Human  Monkey Dog  Rabbit  Rat  Mouse 
Ossification  (Years)  (Years)  (Years) (Weeks) (Weeks)  (Weeks)
Centers1 
Femur Distal  14-19  3-6  0.7-0.9  32  15-162  12-13 
Epiphysis 
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 
 
 
  14 Contains Nonbinding Recommendations 
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  0  14 
response1  41-56 Adult level 
T-independent Antibody  45-90  0 
response1  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  45-180  15-21 
secretion1,2 
1 Snodgrass 1992 
2 Travis 1991 
 
 
Table 6b:  Renal  Anatomical 
Developmental  Postnatal Developmental Period 
Event  (Weeks) 
  Human Dog  Rabbit  Rat  Mouse  Pig 
       
Completion of  Prenatal 2  2-3  4-6  Prenatal  3 
Nephrogenesis1  Week 
35 
1 Zoetis 2003 
 
 
  15 Contains Nonbinding Recommendations 
Table 7:  Metabolism 
Developmental Modulation of Phase I/II Metabolism 
  Maturation of Enzyme Activity 
Enzyme  Human  Rat  Rabbit 
(Years)  (Days)  (Days) 
CYP2D61,2  0-3  NA*  NA* 
CYP2E12,3,4  0-1  4-17  14-35 
 Post weaning  2X adult 
male>female  @35 
CYP1A21,5,6,7  0.5  7-100 Low  21-60 
1(> adult)  levels 
CYP2C81,2  <1  NA*  NA* 
CYP2C91,2  <0.5  NA*  NA* 
0.5 (> 
adult) 
CYP3A42  0-2  NA*  NA* 
Acetylation1,2  1  NA*  NA* 
(35% 
adult) 
Methylation1,2  <1  NA*  NA* 
(50% 
adult) 
Glucuronidation1,2  0 (>adult)    NA* 
12 
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 
 
 
  16 Contains Nonbinding Recommendations 
Table 8:  Cardiac1 
Cardiac  Postnatal Developmental Period 
Parameter  (Maturation Level Similar to Adult) 
Human  Dog  Rat 
(Years)    (Weeks) 
Electrophysiology  5-7 years  NA*  3-8 
(ECG) 
Cardiac Output  Birth 138 bpm;  Increase in BP and  Early increase in HR 
(CO) and  Adults 85 bpm.  decrease in HR from 1  then constant into 
Hemodynamics  <2 yrs:  Smaller  week to 0.5 years  adulthood 
ventricular vol.,   
stroke index,  High CO and low 
ejection fraction vs.  PVR 
adult   
Birth BP 62/40; 2  Neonate-puberty 
months 85/47; 0.5-8  systolic BP doubles 
yrs. Diastolic 58-62  reaches maturity by 
10 weeks 
Myocytes  Diploid at birth  NA*  Primarily diploid in 
compared to 60% in  infant and adult 
adults (40% 
polyploidy) 
Coronary  Diameter of arteries  Capillary  Capillary 
Vasculature  doubled at 1 yr. max  angiogenesis occurs  angiogenesis occurs 
at 30 yrs. Capillary  postnatally and  postnatally arterial 
angiogenesis occurs  density decreases with  maturation by 1 
postnatally and  age  month 
density decreases 
with age 
Cardiac  Neuron number  Continued  Adrenergic pattern 
innervation  increase and reach  development during  mature by 3 weeks 
adult pattern/density  2-4 months  and nerve density 
in childhood  mature by 5 weeks.  
Cholinergic matures 
postnatally 
* NA = not available 
1 Hew and Keller 2003 
 
 
  17 Contains Nonbinding Recommendations 
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  22