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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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 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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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 (t1⁄2 = 26 h) compared to adults (t1⁄2 = 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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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). 

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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 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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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 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 longterm 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. 

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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

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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. 

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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 doseresponse 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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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 

Event 

Postnatal Developmental Period 

Human 

(Years) 

Primate 

(Weeks) 

Dog 

(Weeks) 

Rat 

(Days) 

Glutamate receptors

1

(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 layer

3

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 

Developmental Postnatal Developmental Period 

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

16
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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

17
Contains Nonbinding Recommendations 

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22

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