S11 N S T
  ONCLINICAL  AFETY  ESTING IN 
S D
UPPORT OF  EVELOPMENT OF 
P P
EDIATRIC  HARMACEUTICALS 
  Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
May 2021 
ICH 
 
     
S11 N S T
  ONCLINICAL  AFETY  ESTING IN 
S D
UPPORT OF  EVELOPMENT OF 
P P  
EDIATRIC  HARMACEUTICALS
 
Guidance for Industry 
 
 
 
 
 
 
 
 
Additional copies are available from: 
Office of Communications, Division of Drug Information  
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov  
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs 
and/or 
Office of Communication, Outreach and Development  
Center for Biologics Evaluation and Research 
 Food and Drug Administration 
10903 New Hampshire Ave., Bldg. 71, Room 3128 
Silver Spring, MD 20993-0002 
Phone: 800-835-4709 or 240-402-8010 
Email: ocod@fda.hhs.gov 
https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-guidances 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
May 2021 
ICH  
 
 
 
    Contains Nonbinding Recommendations 
 
FOREWORD 
 
The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for 
Human Use (ICH) has the mission of achieving greater regulatory harmonization worldwide to 
ensure that safe, effective, and high-quality medicines are developed, registered, and maintained 
in the most resource-efficient manner.  By harmonizing the regulatory expectations in regions 
around the world, ICH guidelines have substantially reduced duplicative clinical studies, 
prevented unnecessary animal studies, standardized safety reporting and marketing application 
submissions, and contributed to many other improvements in the quality of global drug 
development and manufacturing and the products available to patients.  
 
ICH is a consensus-driven process that involves technical experts from regulatory authorities and 
industry parties in detailed technical and science-based harmonization work that results in the 
development of ICH guidelines.  The commitment to consistent adoption of these consensus-
based guidelines by regulators around the globe is critical to realizing the benefits of safe, 
effective, and high-quality medicines for patients as well as for industry.  As a Founding 
Regulatory Member of ICH, the Food and Drug Administration (FDA) plays a major role in the 
development of each of the ICH guidelines, which FDA then adopts and issues as guidance to 
industry.  
     
 
TABLE OF CONTENTS 
I.  INTRODUCTION (1.)  ..............................................................1 
A.  Objectives of the Guidance (1.1) .................................................................................. 1 
B.  Background (1.2) .......................................................................................................... 1 
C.  Scope (1.3) .................................................................................................................... 1 
D.  General Principles (1.4) ................................................................................................ 2 
II.  CONSIDERATIONS FOR ADDITIONAL NONCLINICAL SAFETY 
INVESTIGATIONS (2)  ..............................................................3 
A.  Clinical Context (2.1) ................................................................................................... 3 
B.  Weight of Evidence Approach (2.2) ............................................................................. 3 
C.  Considerations To Inform the Weight of Evidence Evaluation (2.3) ........................... 4 
1.  Clinical Information (WoE Factors: Youngest Intended Patient Age; Amount/Type 
of   Existing Data; Clinical Treatment Duration) (2.3.1)  ............................4 
2.  Pharmacological Properties (WoE Factors: Effects on Developing Organ 
Systems; Pharmacological Target Has Role in Organ Development; Selectivity 
and Specificity of Pharmaceutical) (2.3.2)  .................................................5 
3.  Pharmacokinetic Data (WoE Factors: Amount/Type of Existing Data) (2.3.3).....6 
4.  Nonclinical Safety Data (WoE Factors: Effects on Developing Organ Systems; 
Amount/Type of Existing Data) (2.3.4)  .................................................6 
5.  Feasibility (2.3.5) ....................................................................................7 
D.  Application and Outcome of the Weight of Evidence Evaluation (2.4) ...................... 7 
III.  DESIGN OF NONCLINICAL JUVENILE ANIMAL STUDIES (3)............7 
A.  General Considerations/Study Objectives (3.1) ........................................................... 7 
B.  Dose Range-Finding Studies (3.2) ............................................................................... 7 
C.  Animal Test System Selection (3.3) ............................................................................. 8 
D.  Age of Animals, Dosing Period, and Dosing Regimen (3.4) ....................................... 9 
E.  Post-Treatment Period Assessments (3.5) .................................................................. 10 
F.  Route of Administration (3.6) .................................................................................... 11 
G.  Dose Selection (3.7) ................................................................................................... 11 
H.  Endpoints (3.8) ........................................................................................................... 12 
1.  Core Endpoints (3.8.1)  ...................................................................12 
a.  Mortality and Clinical Observations (3.8.1.1) ........................................12 
b.  Growth (3.8.1.2) ......................................................................................12 
c.  Food Consumption (3.8.1.3) ...................................................................12 
d.  Sexual Development (3.8.1.4) ................................................................12 
e.  Clinical Pathology (3.8.1.5) ....................................................................13 
f.  Anatomic Pathology (3.8.1.6) .................................................................13 
i  Contains Nonbinding Recommendations 
 
g.  Toxicokinetics (3.8.1.7) ..........................................................................13 
2.  Additional Endpoints To Address Identified Concerns (3.8.2)  .................13 
h.  Other Growth Endpoints (3.8.2.1) ..........................................................13 
i.  Bone Assessments 3.8.2.2) .....................................................................13 
j.  Clinical Pathology (3.8.2.3) ....................................................................14 
k.  Anatomic Pathology 3.8.2.4....................................................................14 
l.  Ophthalmologic Examinations (3.8.2.5) .................................................14 
f.   CNS Assessments (3.8.2.6) .........................................................14 
m.  Reproductive Assessments (3.8.2.7) .......................................................15 
n.  Immunologic Assessments (3.8.2.8) .......................................................16 
I.  Allocation of Animals to Study Groups and Endpoint Subsets (3.9) ..... ...............16 
1.  Preweaning Allocation (3.9.1)  .........................................................16 
2.  Postweaning Allocation (3.9.2)  .........................................................17 
J.    Animal Numbers and Sex (3.10)............................................................17 
IV.  CONSIDERATIONS FOR PEDIATRIC-FIRST/ONLY DEVELOPMENT 
(4)..........................................................................................................................18 
V.  Data Interpretation (5)  .............................................................19 
A.  Considerations for Endpoint Interpretation (5.1) ..........................................19 
B.  Overall Interpretation (5.2)..................................................................19 
VI.  OTHER CONSIDERATIONS (6)  ...................................................20 
A.  Excipients (6.1) . ...............................................................................20 
B.  Combination Pharmaceuticals (6.2) ...... ....................................................20 
VII.  Glossary  ...............................................................................21 
VIII.  References  ...............................................................................23 
IX.  Appendix A:  Overview of Age-Dependent Development of Organ Systems 
by Species...................................................................................24 
X.  Appendix B:  Case Studies Applying the Weight of Evidence Approach....38 
XI.  Appendix C:  Approaches To Preweaning Litter Allocation in the Rodent.42 
 
    Contains Nonbinding Recommendations 
 
S11 NONCLINICAL SAFETY TESTING IN SUPPORT OF DEVELOPMENT 
OF PEDIATRIC PHARMACEUTICALS1 
GUIDANCE FOR INDUSTRY 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page.   
 
 
I.  INTRODUCTION (1.)2 
 
A.  Objectives of the Guidance (1.1) 
 
The purpose of this document is to recommend international standards for, and promote 
harmonisation of, the nonclinical safety assessments to support the development of 
pharmaceuticals intended for pediatric use. Harmonisation of the guidance for nonclinical safety 
studies will define the current recommendations and reduce the likelihood that substantial 
differences will exist among regions. It should facilitate the timely conduct of pediatric clinical 
trials and reduce the use of animals in accordance with the 3R (replace/reduce/refine) principles. 
 
B.  Background (1.2) 
 
Guidances have previously been issued by various regulatory agencies and are not in complete 
agreement on whether a juvenile animal study (JAS) is advisable and its timing and design.  
 
This guidance is intended to complement and expand on existing ICH guideances (e.g., ICH E11, 
M3, S5 and S9) and reflects current thinking based on collations of examples by regulatory 
agencies, by industry surveys, and literature. 
 
C.  Scope (1.3) 
 
This guidance recommends an approach for the nonclinical safety evaluation of pharmaceuticals 
intended for development in pediatric populations. This can include products with prior adult 
use, as well as products being considered for initial human use in pediatrics (see section IV (4)).  
The ICH S9 guidance should be consulted for recommendations on whether to conduct a JAS for 
those pharmaceuticals included in the scope of the ICH S9 guidance, i.e., anticancer 
 
1 This guidance was developed within the Expert Working Group (Safety) of the International Council for  
Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and has been 
subject to consultation by the regulatory parties, in accordance with the ICH process. This document has been 
endorsed by the ICH Assembly at Step 4 of the ICH process, April 2020. At Step 4 of the process, the final draft is 
recommended for adoption to the regulatory bodies of the ICH regions. 
2 The numbers in parentheses reflect the organizational breakdown of the document endorsed by the ICH Assembly 
at Step 4 of the ICH process, April 2020. 
1  Contains Nonbinding Recommendations 
 
 
pharmaceuticals. The ICH S11 guidance should be consulted for study design in all cases, 
including oncology indications. 
 
Small molecule therapeutics and biotechnology-derived pharmaceuticals as defined in ICH S6 
are within the scope of this guidance. Tissue engineered products, gene and cellular therapies, 
and vaccines are excluded from the scope of this guidance because dedicated juvenile animal 
safety studies are generally not warranted for such products. However, some of the thinking 
outlined in this document about evaluating safety with existing information can apply. 
 
D.  General Principles (1.4) 
 
Pediatric patients who can receive pharmaceuticals during periods of rapid growth and/or 
postnatal development of several organ systems, represent a distinct population when compared 
to adults. Immaturity of organ systems and maturation of systems during drug treatment can 
affect drug pharmacokinetics (PK), pharmacodynamics (PD), and/or off target effects of 
pharmaceuticals, potentially leading to differences in safety and/or efficacy profiles between 
pediatric populations (as described in ICH E11) and/or when compared to adults. 
 
An understanding of the overall clinical development plan is needed to design an appropriate, 
efficient nonclinical plan. A weight of evidence (WoE; see section II (2)) based decision should 
be made to determine whether additional nonclinical investigations are warranted to support the 
pediatric population. As clinical development progresses, adjustments to the WoE can be made 
based on all the available data at that time. The outcome of a WoE assessment can be different 
for different applications of the same pharmaceutical depending on pediatric age, indication, and 
duration of treatment. Note that in accordance with ICH M3, juvenile animal toxicity studies are 
generally not considered important to support short-term PK studies in pediatric populations.  
 
An early consideration of nonclinical support for pediatric pharmaceutical development is 
recommended. In this respect, changing the design and/or timing of the traditional nonclinical 
program is one way to address potential safety concerns for the pediatric patient. For example, 
dosing can be initiated at a younger age in a repeated-dose toxicity study to support the 
corresponding developmental stages in pediatric patients. Another approach could be to conduct 
the Pre- and Postnatal Development (PPND) study earlier than the normal drug development 
paradigm (see ICH M3 and S6), with modifications such as toxicokinetics (TK) in offspring and 
additional endpoints. These changes can replace or refine the design of a JAS.  
 
The conduct of additional nonclinical investigations should be undertaken only when previous 
nonclinical and human data are judged to be insufficient to support pediatric studies. A JAS is 
designed to address safety concerns that cannot be adequately addressed in other nonclinical 
studies or pediatric clinical trials, including potential long-term safety effects. This guidance 
recommends a customised JAS that comprises core design endpoints and potential additional 
endpoints driven by specific concerns.  
Some regulatory agencies have procedures for defining pediatric development plans (see ICH 
E11). Early regulatory interaction, prior to initiating a JAS, is encouraged for efficient pediatric 
drug development. 
 
 
2  Contains Nonbinding Recommendations 
 
 
II.  CONSIDERATIONS FOR ADDITIONAL NONCLINICAL SAFETY 
INVESTIGATIONS (2) 
 
A.  Clinical Context (2.1) 
 
The pediatric clinical development plan for a pharmaceutical is discussed in the ICH E11 
guidance and needs to be understood before an appropriate nonclinical plan can be designed. The 
pediatric clinical plan includes the indication/condition, the intended pediatric age group(s), and 
the treatment regimen (particularly, the duration of dosing during the stages of development). 
The clinical development of a pharmaceutical for pediatric patients usually follows initial adult 
clinical studies but can occur in parallel or can be conducted without any adult clinical studies. 
Whether additional nonclinical investigations are advisable, and their design and timing, will 
depend on the identified safety concerns and the intended pediatric clinical use. 
 
For severely debilitating or life-threatening diseases, or diseases with serious unmet medical 
need in a pediatric population, the sponsor and regulatory agencies should consider the benefit of 
producing data in addition to existing studies versus the potential delay in patient access to a 
pharmaceutical caused by additional nonclinical testing. The decision whether to perform 
nonclinical testing and its timing should be based upon a thorough risk-benefit evaluation. In 
these conditions, if a safety concern is identified as new information is generated, appropriate 
nonclinical studies should be considered, and could potentially be conducted in parallel with 
ongoing pediatric clinical investigations. 
 
B.  Weight of Evidence Approach (2.2) 
 
The nonclinical development plan for a pediatric pharmaceutical depends on an integrated 
assessment based on the totality of the evidence, including the clinical context together with the 
pharmacology, pharmacokinetic (ADME), and nonclinical in vitro and in vivo animal, and 
clinical safety data (adult and/or pediatric), i.e., a WoE approach. A WoE approach considers 
multiple factors evaluated together and, therefore, a single factor should not be considered in 
isolation. The importance of each factor should be considered such that the final decision 
concludes whether available data adequately address safety concerns in the proposed pediatric 
population or whether additional nonclinical studies would address those concerns. In addition, 
the translatability and biological relevance of the JAS data to humans should be considered.  
 
The WoE evaluation should be conducted when designing the initial pediatric development plan, 
but reassessed if there are new safety signals in nonclinical or clinical studies, changes in age 
ranges, route of administration, treatment duration, drug product formulation and/or indications. 
The WoE outcome can be different for each trial depending on the pediatric population and the 
disease to be treated. 
 
Figure 1 shows key factors that should be considered as part of the WoE evaluation. The most 
important factors (i.e., the most highly weighted) are the youngest intended patient age, and 
whether there are suspected adverse effects on developing organ systems of the patients. The 
other factors are not listed in order of importance in the figure. The list is not all inclusive for 
every situation, as there may be additional specific factors to consider (e.g., risk mitigation). The 
WoE factors are further described in the following sections.  
 
3  Contains Nonbinding Recommendations 
 
 
Figure 1: Key WoE Factors To Be Considered in Determining if Nonclinical Studies Are 
Warranted. The most important factors that should be highly weighted (listed first) are the 
youngest intended patient age and whether there are suspected adverse effects on developing 
organ systems of the patients during the conduct of the pediatric trial. The other factors are not 
listed in order of importance.  
WoE
Further  Further 
Factors
Studies  Studies 
Likely Unlikely
Youngest 
Intended
Patient Age
Neonates    Infant  Child  Adolescent 
(<1 Month) (123 Months)  (211 Years) (12 Years)
Effects on 
Developing Organ 
Systems Yes No
Amount/Type of
Existing Data
No Nonclinical        Adult Nonclinical  Adult  Pediatric 
or Clinical Data         Only Clinical Clinical
Pharmacological
Target has Role In Organ
Development
Yes  No
Selectivity and 
Specificity of 
Pharmaceutical
Low  High
Clinical Treatment 
Duration
Chronic Use  Acute Use
 
C.  Considerations To Inform the Weight of Evidence Evaluation (2.3) 
 
1.  Clinical Information (WoE Factors: Youngest Intended Patient Age; Amount/Type of 
Existing Data; Clinical Treatment Duration) (2.3.1) 
 
The youngest intended patient age is one of the most important factors to be considered. As 
stated in ICH E11, any classification of the pediatric population into age categories is to some 
extent arbitrary, but a classification such as the one in Figure 1 provides a basis for thinking 
about the nonclinical testing to support safety in such patients. Decisions on how to stratify by 
age should focus on developmental biology. Additional nonclinical studies are more likely to be 
warranted to support younger pediatric age ranges. 
 
The existing clinical data relevant for intended pediatric patients come from other pediatric 
subpopulations (if available) and adults exposed to the pharmaceutical. Therefore, this 
established clinical safety profile is usually one of the first points to consider when determining 
if additional nonclinical studies are warranted.  
 
4  Contains Nonbinding Recommendations 
 
 
The duration of clinical treatment is another factor in determining whether additional nonclinical 
studies are warranted. Longer durations of treatment (e.g., 3 month, 6 month, chronic 
intermittent) are more likely to expose a pediatric subject during a developmentally sensitive 
window and are, therefore, more likely to warrant further nonclinical studies than short-term 
treatments. Short-term exposure to a pharmaceutical is less likely to affect aspects of 
development such as growth. However, even a short-term exposure can have deleterious effects 
if it occurs at a vulnerable time of organ development.  
 
Additional nonclinical studies are not warranted when existing clinical safety data and risk 
mitigation strategies are considered sufficient to support pediatric use. In addition, a JAS is not 
warranted to confirm toxicity in target organs in which sensitivity to toxicity is not expected to 
differ between adults and pediatric patients. Differences in target or off-target tissue 
development are a concern that should be considered. 
 
If adult clinical data are available and exposure is not occurring at a vulnerable time of organ 
development, a JAS is not considered important to support initiation of short-term PK studies in 
pediatric patients (see ICH M3). 
 
2.  Pharmacological Properties (WoE Factors: Effects on Developing Organ Systems; 
Pharmacological Target Has Role in Organ Development; Selectivity and Specificity of 
Pharmaceutical) (2.3.2) 
 
Primary or secondary pharmacological properties of a pharmaceutical can be responsible for 
unwanted side effects. This can raise concerns for pediatric use if effects occur in systems/organs 
in development or if developing organs have a different sensitivity from mature organs. A review 
of the literature on the developmental expression and ontogeny of the pharmacological target(s) 
(e.g., receptors, enzymes, ion channels, proteins), or the known or potential role of the target 
during development is recommended. Existing data from genetically modified animals (e.g., the 
knock-out of a receptor) might also identify developmental effects of potential relevance for the 
pediatric population, which could be included in the WoE evaluation. 
 
If the known pharmacology of a pharmaceutical has the potential to impact development in the 
intended pediatric population, or the role of the pharmacology on development is not understood 
or not reasonably predictable, further nonclinical investigations should be considered. Potential 
adverse effects of pharmaceuticals with high selectivity and specificity for their target (e.g., 
monoclonal or bispecific antibodies) are more likely to be related to exaggerated pharmacology 
and therefore can be more predictable than effects of pharmaceuticals with lower selectivity or 
specificity for their pharmacological target. Pharmaceuticals with lower selectivity or specificity 
can have secondary pharmacodynamic effects and thus are more likely to warrant further 
nonclinical investigations. In vitro or ex vivo investigations using juvenile (i.e., animal) or 
pediatric (i.e., human) tissues or matrices (e.g., serum, urine) might be useful to determine 
potential age-related differences in sensitivity.  
 
Further nonclinical studies might not add value when the existing pharmacology information has 
already identified a particular hazard unless a more detailed understanding of the dose-response 
relationship or differences in sensitivity between adult and juvenile animals is warranted.  
5  Contains Nonbinding Recommendations 
 
 
 
3.  Pharmacokinetic Data (WoE Factors: Amount/Type of Existing Data) (2.3.3) 
 
Maturation of systems important for ADME such as the gastrointestinal, liver, and renal systems 
can result in rapidly changing systemic exposures in humans and in animals, leading to potential 
age-related differences in efficacy and toxicity. In humans, these differences are usually most 
prominent in neonates and infants. 
 
Clinical pharmacology and modelling and simulation tools are considered useful to contribute 
information on the pharmacokinetics, and also pharmacodynamics, efficacy, and safety of a drug 
in pediatric subjects (see ICH E11). In general, a JAS is not informative in predicting or 
recapitulating age-related differences in human ADME. 
 
4.  Nonclinical Safety Data (WoE Factors: Effects on Developing Organ Systems; 
Amount/Type of Existing Data) (2.3.4) 
 
Existing nonclinical data should be evaluated for findings that could indicate potential effects in 
organs undergoing development in pediatric subjects. Findings occurring in animals at similar 
exposures as those likely to be achieved in pediatric subjects are of increased concern, 
particularly if the findings occur in organs/tissues that undergo critical postnatal development at 
the intended pediatric age (see Appendix A). Safety signals in adult animals in more than one 
species are of increased concern. It can still be appropriate to evaluate the potential impact on 
pediatric subjects of a toxicity that occurred in adult animals that did not translate to adverse 
effects in adult humans if the target organ/system is undergoing development in the relevant 
pediatric population. Depending on the age of the animals at initiation of dosing and the 
endpoints included, some of these concerns may have been addressed in existing toxicity studies.  
 
Genotoxicity testing and safety pharmacology investigations are normally conducted to support 
adult clinical trials and, therefore, should be available before pediatric clinical trials commence. 
If a safety pharmacology study shows an effect in an organ system undergoing structural or 
functional development and maturation in the intended pediatric patient population, the possible 
impact of the effect should be considered. Additional genotoxicity and safety pharmacology 
assessments in juvenile animals are generally not appropriate to support pediatric indications. 
 
Reproductive and developmental toxicity study data may also be available and can be 
informative. If PPND/ePPND study data are available and have shown clinically relevant 
systemic exposures in offspring during the postnatal period, these data can contribute to the WoE 
evaluation (see section I.D (1.4)). For ePPND studies conducted in the non-human primate 
(NHP), the data from the offspring can characterize toxicity during early postnatal development, 
provided relevant exposure and/or PD effects are confirmed in the offspring.   
 
When available, PPND/ePPND data should be evaluated in combination with data from the 
general toxicity studies in assessing the potential added value of conducting additional 
nonclinical investigations. Maternal and fetal tolerance of the drug should be considered because 
they could influence interpretation of the findings in offspring. Observations of adverse effects in 
offspring would not, on their own, indicate that a JAS is recommended. However, if a safety 
concern was identified in the PPND/ePPND study, it should be considered in the WoE 
evaluation. In rodents, these data are primarily relevant to preterm and term neonates if exposure 
is demonstrated. However, the species-specific development of an organ system should be 
considered when determining human relevance. 
6  Contains Nonbinding Recommendations 
 
 
 
If data from a previously conducted JAS are available, they should be considered in the WoE. 
 
5.  Feasibility (2.3.5) 
 
The decision to conduct an additional animal study should also consider the technical and 
practical feasibility of the study design and endpoints. Some endpoints might not be practical in 
some species (see section III (3) for further discussion of this point). 
 
In addition, if a dose range finding (DRF) study in juvenile animals indicates that a definitive 
JAS cannot be conducted at appropriate systemic exposures or relevant ages in the range of those 
expected in pediatric patients, such a study might not be informative or warranted (see also 
section III.B (3.2) and III.F (3.6)). 
D.  Application and Outcome of the Weight of Evidence Evaluation (2.4) 
 
The WoE approach should be applied to determine whether additional nonclinical investigations 
are warranted, with emphasis on the factors considered most important to inform the clinical risk 
assessment. When a study is deemed warranted, the specifics of the identified safety concerns 
should define the aim of the nonclinical investigation; this could be a JAS or another study (e.g., 
in vitro or ex vivo investigations). For a JAS, the study objectives should be aligned with the 
WoE outcome and the intended pediatric use. This is essential to appropriately design and 
customize the JAS with regard to the treatment period and endpoints to be included. 
 
Examples of applying the WoE approach are in Appendix B.  
 
 
III.  DESIGN OF NONCLINICAL JUVENILE ANIMAL STUDIES (3) 
 
A.  General Considerations/Study Objectives (3.1) 
 
This section contains recommendations on study design, core endpoints, and additional 
endpoints that can be included to address specific concerns. A JAS design including all potential 
additional endpoints is not recommended without a rationale for each additional endpoint. A JAS 
should be conducted according to Good Laboratory Practices.  
 
The stage of maturation of human and animal organ systems can influence susceptibility to 
toxicity. Understanding the relative level of maturity and function across species during 
development is needed not only to design the appropriate JAS but also to aid the translation of 
nonclinical toxicity findings to human age categories. Comparison of development across species 
can be challenging and is not uniform across different organ systems. For example, the relative 
maturity at birth, rate of postnatal maturation, and/or regulation of maturation are quite different 
between humans and animals. While not comprehensive, Appendix A, Tables A1-A5, provide an 
overview of comparative development of organ systems by species. 
 
B.  Dose Range-Finding Studies (3.2) 
 
A DRF study with small group sizes of juvenile animals is recommended to assess tolerability in 
relation to exposure and age. This is particularly valuable to design a definitive JAS when dosing 
starts prior to weaning to avoid unexpected mortality or excessive toxicity, often due to 
irrelevant exposures. Dosing should include the youngest planned starting age of the animals in 
7  Contains Nonbinding Recommendations 
 
 
the definitive JAS to evaluate the most critical period for tolerability and exposure differences. 
DRF studies typically are of short duration, have limited endpoints and are not expected to 
include all core endpoints (e.g., pathology). DRF studies can also be used to explore particular 
endpoints and thus refine the study design of the definitive JAS. DRF studies are not necessarily 
conducted according to Good Laboratory Practices.  
 
A DRF study can reveal important information for pediatric development. Differences in 
exposure between age ranges can be identified in a DRF. This might necessitate an adjustment to 
the dosing regimen in the definitive JAS (see sections III.D (3.4) and III.G (3.7)).  
 
Alternatively, lack of tolerability in a DRF at anticipated pediatric clinical exposure might 
indicate a significant concern for the corresponding clinical age range (i.e., the juvenile animals 
are unexpectedly sensitive, potentially related to their immaturity, which can have clinical 
pediatric relevance). When the reason for greater sensitivity or significant differences in toxicity 
is not understood, additional investigations guided by review of available ADME, safety and 
developmental biology knowledge can be useful for the interpretation of these differences. This 
situation might warrant a customized investigative JAS to further define the sensitive age 
window and/or understand the possible mechanism of toxicity. Results could have safety 
implications for a specific pediatric age which might alter the intended pediatric clinical age 
range, and thus the WoE should be revisited (see section II.B (2.2)). 
 
C.  Animal Test System Selection (3.3) 
 
When a JAS is warranted, in most cases a single species is considered sufficient. In principle, the 
same species as used in adult repeated-dose studies should initially be considered as the species 
for a JAS, preferably a rodent. In all cases, the selected species should be justified, as nonclinical 
studies in a pharmacologically non-relevant species can give rise to misinterpretation and are not 
recommended. 
 
The following factors should be considered when selecting a relevant species: 
 
  An understanding of the ontogeny of the pharmacological or toxicological target (e.g., the 
receptor) in animals in comparison to that in the intended pediatric population  
 
  Preference for a species and strain for which adult repeated-dose toxicity data are available to 
facilitate a comparison of the toxicity and systemic exposure profiles between juvenile and 
adult animals 
 
  Toxicological target organs  
 
o  The relative stage of organ/system development in the juvenile animal as compared to the 
intended pediatric population (see also section III.D (3.4))  
 
o  The ability of the animal model to detect toxicity endpoints of concern 
 
  Similarity to human ADME characteristics 
 
  The technical/practical feasibility to conduct the study in the selected species  
8  Contains Nonbinding Recommendations 
 
 
Advantages and disadvantages of using different rodent (rat, mouse) or non-rodent (rabbit, dog, 
minipig, NHP) species are outlined in Appendix A, Table A6.  
 
While NHPs are pharmacologically relevant in many cases for biopharmaceuticals, the conduct 
of a JAS in NHPs prior to weaning can be challenging for both scientific and practical reasons 
(e.g., breeding/transport and management of dam/infant pairs). There is limited added value of 
performing a JAS in postweaning NHP. In postweaning NHPs, organ system maturity is 
generally beyond that relevant for many pediatric ages (see Appendix A). Only in rare cases is 
the value of a JAS conducted in preweaning NHP justifiable (e.g., intended neonatal pediatric 
use and inadequate exposure from an ePPND study). Therefore, alternative approaches are 
encouraged (also see section IV (4)).  
 
Use of an available homologous protein, as discussed in ICH S6, can be considered for the 
purposes of hazard identification in the juvenile rodent or non-rodent species. 
 
JAS in two species can be warranted in a pediatric-first situation (see section IV (4)) or where 
there are multiple specific concerns for postnatal development and one species alone is not able 
to address them. Biopharmaceuticals should only be assessed in pharmacologically relevant 
species, consistent with ICH S6. The conduct of a JAS in a second species to confirm findings in 
the first species is generally not warranted. 
 
If an animal model of a pediatric disease exists (e.g., for enzyme replacement therapy) and is 
being used to support pharmaceutical development, appropriate safety endpoints (e.g., 
histopathology, clinical pathology) can be incorporated in these studies. This information could 
contribute to the WoE evaluation, potentially providing sufficient information without 
conducting a dedicated JAS.  
 
D.  Age of Animals, Dosing Period, and Dosing Regimen (3.4) 
 
The age of animals at dosing initiation should developmentally correspond to the youngest 
intended patient age and will depend on a human-to-animal comparison of developmental 
periods of organ system(s) of toxicological concern. As comparative organ system correlations 
are not aligned for each organ across species, priority should be given to any target organ/system 
of potential concern or to particularly vulnerable developing systems in the intended patient 
population. The animal age at dosing initiation should be justified using relevant information 
(such as provided in Appendix A). 
 
In contrast to nonclinical studies for adult populations (see ICH M3), the recommended dosing 
period is not always directly related to the clinical treatment duration for the pediatric 
population. When determining the duration of administration in a JAS, it is important to consider 
the pediatric age range and the shorter developmental period of animals compared to humans, the 
safety concern for the intended pediatric population, and the relevant period of organ 
development for the target organ of concern. Dosing in a JAS should usually occur during the 
critical and active periods of growth and development identified in the tables in Appendix A for 
a system of concern. 
 
Thus, for example, a pharmaceutical with only the kidney as target organ of concern, could be 
supported with a rat JAS of limited duration focusing on the relevant period of renal 
development irrespective of the clinical treatment duration. Alternatively, even if the clinical 
9  Contains Nonbinding Recommendations 
 
 
treatment duration is short, a longer dosing period in animals can be appropriate to address 
concerns of organ systems that develop at different times or over an extended time. For example, 
a pharmaceutical with a CNS concern with a 10-day clinical treatment duration intended for 
patients more than 2 years of age could be supported with a rat JAS with animals dosed from 
weaning to maturity (see Appendix A). 
 
If clinical treatment is intended through adolescence, dosing to maturity is typically conducted in 
rodents. The interval between birth and maturity for NHPs is several years, making dosing to 
maturity impractical. Furthermore, NHPs show considerable inter-individual variation in the age 
of onset of puberty and maturity. Dosing up to maturity can, however, be possible in other 
typical non-rodent species when these species mature over a period of a few to several months 
and with relative consistency (e.g., minipig, see Appendix A). 
 
The dosing regimen should be designed to achieve and maintain relevant exposures during 
developmental periods of concern. Therefore, dosing regimens in a JAS may not be exactly the 
same as in the clinic. For example, even though a clinical regimen is once a week, more frequent 
dosing in juvenile animals might be more appropriate. If drug accumulation is a concern in 
juvenile animals, dosing could be less frequent than in adult toxicity studies (e.g., every other 
day vs. daily). 
 
When data demonstrate that animals at different ages have different tolerability or exposures to 
the drug, dose adjustment over time can be considered to provide information with clinically 
relevant exposures at the appropriate developmental stages.  
 
If the duration of dosing is not expected to be tolerable in a JAS, it may be possible to achieve 
the clinically relevant exposure by separating the dosing period into different subgroups (e.g., a 
6-week JAS dosing period is split into two subgroups of 3 weeks dosing, each starting at 
different ages). If subgroups with different dosing periods are used, all subgroups may need to be 
followed through to maturity to detect late effects. The benefits of this approach should be 
considered along with the drawbacks, such as substantially increasing the number of animals and 
difficulties interpreting data at different ages. 
 
E.  Post-Treatment Period Assessments (3.5) 
 
Inclusion of an evaluation period after treatment has stopped in a JAS is generally recommended 
to help address two issues: (1) whether any effects observed during treatment are reversible, 
persistent, or progressive and; (2) whether any effects emerge later in development as a result of 
early life exposure (i.e., delayed onset). 
 
Whether a post-treatment period is advisable is dependent on the outcome of the WoE 
assessment and the endpoints to be evaluated in the study. The principles of evaluation of the 
potential for reversibility in ICH M3 apply. The duration of the post-treatment period should be 
sufficient to allow the potential recovery of the effect and should take into account the 
elimination of the pharmaceutical. However, the demonstration of full reversibility is not 
considered essential. A trend towards reversibility (e.g., decreased incidence and/or severity) and 
a scientific assessment that this would eventually progress to full reversibility could be sufficient. 
Likewise, if irreversibility of a specific effect is well characterised in adult animals, it is 
generally not necessary to confirm this in a JAS. There are endpoints in a JAS that are not 
amenable to the classic approach of reversibility assessment, such as the timing of onset of 
10  Contains Nonbinding Recommendations 
 
 
puberty. Additionally, the timing of the post-treatment period in relation to the life stage of the 
animals should be considered. If the post-treatment period begins prior to maturity, the capacity 
for recovery can be influenced by the continued growth and development of some organ systems 
and should be carefully interpreted. 
 
Some alterations can only be identified following an appropriate post-treatment period to allow 
maturation of an organ system and expression of the alteration. Therefore, some assessments can 
only be meaningfully performed after a certain level of maturity is reached (e.g., learning and 
memory, immunological function). These assessments can be conducted in post-treatment 
periods after exposure has covered all developmental windows relevant to the clinical use. This 
is especially relevant in cases when the JAS dosing duration would cease at an immature age and 
the animals will continue to mature during the post-treatment period to an age at which an 
appropriate assessment can be conducted.  
 
Conducting assessments in the post-treatment period can also address delayed onset changes that 
can be a result of early life exposure, especially in cases when the JAS dosing stops at an 
immature age. 
 
In non-rodents, depending on the species, the addition of post-treatment groups for a JAS is 
generally less useful due to the more protracted development period, high inter-individual 
variability, and fewer and less well-established assessments available to identify delayed or 
altered development (e.g., learning and memory tests).  
 
F.  Route of Administration (3.6) 
 
The intended clinical route of administration should be used when feasible, but obtaining 
adequate systemic exposure is paramount (see section III.G (3.7)).  
 
Alternative administration routes should be considered in cases of practical difficulties (e.g., use 
of oral route in preweaning rats for a dermal product); changing routes during the course of the 
study can also be considered (e.g., subcutaneous injection until intravenous is feasible). The 
validity of using an alternative dosing route should be justified (e.g., supported by exposure data 
in representative juvenile animals).  
 
If the pharmaceutical is intended for use by two or more clinical routes of administration, a JAS 
with a single route of administration is sufficient but should provide adequate exposure in 
juvenile animals for all intended clinical routes of administration. 
 
G.  Dose Selection (3.7) 
 
It is desirable to establish a dose-response relationship for adverse effects and to determine a no-
observed adverse effect level (NOAEL) in juvenile animals. Dose levels should be selected to 
achieve some overlap in the range of exposure in adult animals to enable comparison of effects 
between young and adult animals. However, the high dose should not result in marked toxicity 
that can confound the growth and development endpoints and complicate the assessment. Body 
weight loss or lack of weight gain during rapid growth periods has the potential to confound 
results, and is therefore not desirable in a JAS. At least one dose should result in exposure levels 
similar to the anticipated exposure in the intended clinical population if tolerable. For small 
molecules, selection of the high dose in accordance with ICH M3 applies. For biotechnology-
derived products, the principles for dose selection described in ICH S6 apply. 
11  Contains Nonbinding Recommendations 
 
 
Dose adjustment (increase or decrease) during the course of a JAS should be considered 
in cases of substantial changes in systemic exposure due to maturation of the ADME systems. 
Adjusting doses is intended to keep the exposures somewhat consistent and clinically relevant. 
Generally, more than one dose adjustment during a JAS would not be expected. 
 
H.  Endpoints (3.8) 
 
Generally, each JAS should include the core endpoints defined in section III.H.1 (3.8.1) below. 
Each additional endpoint (see section III.H.2 (3.8.2)) should be considered and justified to 
address an identified safety concern. In some cases, such as a follow-up investigational JAS, all 
core endpoints might not be included when justified. 
 
The justification for including an endpoint should consider that invasive or prolonged procedures 
should be limited as much as possible during preweaning and at the time of weaning as they can 
contribute to mortality. 
 
1.  Core Endpoints (3.8.1) 
 
a.  Mortality and Clinical Observations (3.8.1.1) 
 
Mortality should be evaluated throughout the experimental period. Clinical observations, 
including physical examinations, should be conducted both during treatment and post-treatment 
as they can identify overt behavioral effects. 
 
Clinical observations of the maternal animals should include assessment of nursing behavior and 
maternal care of offspring when treatment of juvenile animals is initiated prior to weaning. 
Clinical observations can be different between suckling animals and adults and have different 
implications on the overall health status of the animal (e.g., hydration status, which reflects 
nutritional status in pups). Therefore, clinical observations of offspring should also capture 
observations specific to suckling animals. After weaning, clinical observations should be 
recorded as is appropriate for adult animals. 
 
b.  Growth (3.8.1.2) 
 
Growth should be assessed by body weights in conjunction with long bone length. As body 
weight increases dramatically during the early postnatal period, individual weight measurements 
should be assessed at intervals appropriate for informing dose calculations. Generally, one long 
bone (e.g., femur) measured for length at necropsy is sufficient (see section V (5)). 
 
c.  Food Consumption (3.8.1.3) 
 
Food consumption during the postweaning period should be assessed as appropriate for the 
species and housing conditions. 
 
d.  Sexual Development (3.8.1.4) 
 
The physical indicators of onset of puberty (e.g., for rodents, the age of vaginal opening in 
females and balanopreputial separation in males) are recommended when the study design 
encompasses the relevant developmental window. 
12  Contains Nonbinding Recommendations 
 
 
 
e.  Clinical Pathology (3.8.1.5) 
 
Standard clinical pathology examinations (clinical chemistry and haematology) should be 
assessed as a terminal endpoint at necropsy if evaluation is planned at an age at which clinical 
pathology ranges are known and can support interpretation of histopathology findings. 
 
f.  Anatomic Pathology (3.8.1.6) 
 
At the end of the treatment and/or post-treatment periods, gross pathology, organ weights, and 
comprehensive collection and preservation of tissues should be conducted for animals allocated 
to necropsy. Microscopic evaluation should be performed on major organs (e.g., bone/marrow, 
brain, gastrointestinal tract, heart, kidney, liver, lung, ovary, testis with qualitative evaluation of 
spermatogenic progression in mature males), those with macroscopic lesions and previously 
identified target organs. 
 
If a JAS is supporting first-in-pediatric trials (see section IV (4)) then a standard set of tissues as 
used in adult toxicity studies is recommended for histopathology. 
 
g.  Toxicokinetics (3.8.1.7) 
 
When designing the TK component of a JAS, the use of microsampling and sparse sampling (see 
ICH S3A) is strongly encouraged. 
 
Toxicokinetic sampling should be conducted near the beginning and end of the dosing period. If 
dosing is started preweaning, interim TK assessment(s) should be considered. If dose levels are 
adjusted during the study, additional sampling for TK is recommended. A DRF JAS with TK 
assessment (see section III.B (3.2)) will inform on the sampling day and the timepoints of sample 
collection. The TK assessment should consider both active pharmaceutical ingredient and 
relevant major human metabolites. 
 
For biopharmaceuticals, samples for anti-drug antibodies should be collected, and evaluated if 
appropriate (see ICH S6). 
 
2.  Additional Endpoints To Address Identified Concerns (3.8.2) 
 
The decision to include additional endpoints should be based on the type and strength of the 
concern identified in the WoE evaluation. 
 
a.  Other Growth Endpoints (3.8.2.1) 
 
As appropriate for the species, crown rump length, body length (e.g., nose/tail), and/or withers 
height can be used as an indicator of growth. Serial non-invasive measurements of long bone 
length using suitable imaging techniques (e.g., X-ray) can be useful in non-rodents in addition to 
a direct measurement at necropsy. 
 
b.  Bone Assessments 3.8.2.2) 
 
13  Contains Nonbinding Recommendations 
 
 
When there is an identified concern specific to bone metabolism or structure, additional 
endpoints should be considered. Examples include assessments of bone mass and geometry using 
densitometric techniques, serum and urinary biomarkers of bone formation and resorption, and 
bone histomorphometry.   
 
c.  Clinical Pathology (3.8.2.3) 
 
Additional haematology, serum chemistry, and/or biomarkers can be considered to further 
characterise identified target organ/tissue concerns. Other parameters such as urinalysis or 
coagulation assessments can be added when warranted and feasible. 
Due to the limitation in obtaining adequate sample volumes from juvenile animals (especially 
rodents), additional samples can require additional animals and, therefore, are only 
recommended when critical to address a concern. When sample volume constraints exist, the 
parameters to be measured should be selected according to a priority based on the identified 
concern(s).  
 
d.  Anatomic Pathology 3.8.2.4 
 
Additional tissues/organs can be evaluated to address specific concerns. Immunohistochemical 
or other special staining methods for tissue sections, electron microscopy, histomorphometry, or 
other imaging techniques can be used for further characterisation when warranted.  
 
e.  Ophthalmologic Examinations (3.8.2.5) 
 
Standard ophthalmologic examinations (e.g., palpebral reflex, ophthalmoscopy) are not routinely 
included in a JAS, because structural development of the eye is largely completed during the 
prenatal period in humans. However, when there is concern for ocular toxicity, assessment of 
ocular endpoints should be considered. 
 
f.  CNS Assessments (3.8.2.6) 
 
There are different categories of CNS assessments, such as:  
  Detailed clinical observations  
  Behavioral tests  
  Learning and memory tests, and  
  Expanded neurohistopathology evaluations. 
The selection of any additional CNS assessments should be based on concerns identified in the 
WoE evaluation. The timing of any such additional assessments within the JAS should take into 
consideration whether the results will be used to investigate adverse effects that are due to 
exaggerated pharmacology, developmental neurotoxicity (i.e., effects that are still present or 
emerge after the cessation of treatment), or both. 
 
If a compound has a target in the CNS, the extent of distribution across the blood-brain barrier 
and which region of the brain is potentially affected (e.g., by target distribution and related 
functional pathways) should be considered. Such information, if known, can help inform 
selection of the appropriate additional CNS endpoints (e.g., when determining whether 
assessments of learning and memory or other endpoints are warranted). 
14  Contains Nonbinding Recommendations 
 
 
 
Detailed clinical observations are a key component of CNS assessment and, therefore, should be 
assessed as appropriate throughout the study during the treatment and post-treatment periods. 
These observations should document the severity, the time of onset and the duration of the 
clinical signs relative to the time of dosing to determine whether or not effects are temporally 
associated with exposure. 
 
There are many different behavioral tests, including assessment of locomotor activity, evaluation 
of coordination and reflexes, and/or acoustic startle response (e.g., habituation or prepulse 
inhibition). The functional observational battery (FOB) or modified Irwin test are considered to 
have relatively low sensitivity in juvenile rodents and are of limited utility. The selected test(s) 
should be appropriate for the species being tested and the timing of these assessments should 
consider the level of maturity in the test species at the age of the assessment. Before deciding 
whether behavioral testing is performed during the treatment period, the potential for 
confounding pharmacological effects (e.g., sedation, decreased motor coordination) should be 
considered. 
 
When specific aspects of learning and memory have been identified as areas of concern in the 
WoE evaluation, then an appropriate complex learning task capable of assessing such aspects 
should be selected. For evaluation of persistent or delayed effects on learning and memory, these 
assessments should be conducted in the post-treatment period. 
 
Postnatal CNS assessments are most commonly conducted and characterised in the rodent. For 
those pharmaceuticals where the rodent is an inappropriate species, some behavioural tests are 
also available in other species (e.g., dogs, minipigs). In NHPs, behavioral observations in a JAS 
or ePPND study can provide an assessment of potential CNS effects. Learning assessments 
similar to those used in pediatric subjects have also been developed for NHPs, but these are 
infrequently conducted because of study design complexities and substantial interindividual 
variability. 
 
Lastly, any CNS regions or components (e.g., hippocampus, myelin) identified in the WoE 
evaluation as potentially affected should be assessed with expanded neurohistopathological 
evaluations as appropriate (e.g., additional sections examined, immunohistochemistry, special 
stains). These assessments are typically performed at the scheduled necropsy at the end of 
treatment and following a post-treatment period. Imaging technologies can also be useful in 
specific circumstances. 
 
g.  Reproductive Assessments (3.8.2.7) 
 
If there is an identified concern for effects on female and/or male reproductive organs or 
function, histopathology examinations and organ weights can be expanded to include 
reproductive and/or endocrine tissues in addition to the gonads. It is not important to confirm in a 
JAS reproductive system effects that were identified as irreversible in adult animals.  
 
In rodents, for concerns relevant to females, assessment of estrous cyclicity is recommended for 
assessment of reproductive and endocrine function. For concerns relevant to male rodents, sperm 
analysis (e.g., counts, motility, morphology) and/or testicular immunohistochemistry (e.g., 
apoptosis) can be considered if of value to further characterise effects. 
 
15  Contains Nonbinding Recommendations 
 
 
The timing of the treatment and assessments in relation to that of sexual maturation in the species 
tested is critical. The timing of folliculogenesis and spermatogenesis should be considered in the 
study design and timing of reproductive assessments. Assessment of reproductive organs or 
function (e.g., estrous cyclicity, sperm count, or qualitative histologic assessment of 
spermatogenesis) can only be conducted in sexually mature animals. If the clinical age range 
includes prepubertal stages, there can be a concern whether the pharmaceutical could cause any 
delayed effect on sexual maturation or reproductive function in adulthood. If the clinical 
treatment is only during prepubertal stages, a JAS should be designed to treat only during 
immaturity, and then allow the animal to mature without further treatment and conduct 
assessments after maturation is reached. 
 
Mating assessments are not generally recommended in a JAS. Most effects on reproductive 
organs related to male fertility are detectable by histopathology. In female rodents, assessment of 
estrous cyclicity and ovarian histology can identify many developmental reproductive hazards. In 
dogs and NHPs, mating assessments are difficult due to the protracted duration of development 
and high degree of individual variability.  
 
Hormonal assessments are generally not recommended in a JAS as there is considerable 
variability in hormone levels, especially during puberty. Therefore, any hormonal assessment 
should be justified, and the timing and specific hormones assessed should be well characterised 
for the age at which the assessment is conducted. 
 
The feasibility of reproductive assessments is such that the large majority are conducted in 
rodents, although they can be considered for those non-rodent species that achieve maturity 
during the conduct of a JAS. 
 
h.  Immunologic Assessments (3.8.2.8) 
 
If the pharmacological class or data in animals or humans gives cause for concern for the 
development of the immune system, assessments for immunotoxicity should be considered as 
outlined in ICH S8. Such concerns, when considered developmentally important, can include a 
transient, prolonged, or permanent decrease or increase in the number or function of a 
lymphocyte subtype, or a sustained increase or decrease in immunoglobulin class. Functional 
assays should be performed at appropriate stages of development, e.g., for T-Cell-Dependent 
Antibody Response (TDAR) after PND 45 for the rat. Confirmation of immunotoxicity is 
generally not warranted in a JAS if the toxicity is already well characterised. 
 
I.  Allocation of Animals to Study Groups and Endpoint Subsets (3.9) 
 
1.   Preweaning Allocation (3.9.1) 
 
A definitive JAS can become large and complex; therefore it is important that the study design 
balances scientific rigor against animal use. Before designing the study, investigators should 
know all the planned endpoints (core and additional). Efficiency in study design is critical to 
reduce animal use as per the 3R principles and should be measured by the total number of 
maternal animals (and litters) needed to supply the study, including pups that are not used in 
standardized litters and pups that are not assigned to specific endpoints.  
 
16  Contains Nonbinding Recommendations 
 
 
There are different allocation methods for litter management of multiparous non-rodent animals. 
For species with single offspring or small and variable litter sizes (e.g., NHP, dogs), group 
allocation design can be modelled following the principles applied in general toxicity studies. 
 
In most species, initiation of a JAS during the preweaning phase presents a unique situation for 
dosing offspring within a litter. Although the maternal animal is a critical component of the study 
providing nutrition and care, only the offspring are the test system. The study should be designed 
to reduce potential confounders. For JAS, maternal care and litter size are generally considered 
more important confounders than genetics. Reduction of confounding can be achieved by the 
way the litters are constructed and standardized in combination with how they are assigned to 
dose groups and how the individual pups are assigned to endpoints.  
 
When constructing study litters, select dams demonstrating good maternal care and pups in 
apparent good health. It is advisable that study litters be standardized with respect to number of 
offspring and sex ratio (i.e., 4 to 5 pups/sex/litter in rats) at the same postnatal age. This can be 
accomplished by either fully fostering (arbitrarily mixing up all birth litters) or minimally 
fostering (keeping the birth litter as intact as possible and fostering only as necessary to obtain 
study litter size and sex ratio requirements). When feasible, standardize study litters at a time that 
allows some acclimation to the new litters prior to the first dose. As a change in litter size can 
alter pup growth rate, maintaining a consistent litter size across and within dose groups is 
recommended during the preweaning phase. 
 
For assigning study animals to dose groups, it is preferred that each standardized litter be 
assigned to a single dose group to minimize the risk for cross-contamination and to avoid treated 
and control offspring competing for suckling position and time.  
 
When assigning individual animals to endpoints in definitive studies, it is recommended that 
litter mates of the same sex not be assigned to the same endpoint to avoid maternal care biases 
(see Appendix C).  
 
The fostering approach, litter standardisation, dose group, and endpoint allocation methods 
should be clearly described in the study plan/protocol and report. Appendix C provides a case 
study using one allocation approach for a rat JAS that minimizes potential for genetic, maternal 
care, and litter size confounders. There are other ways to successfully allocate litters, depending 
on the study objectives and endpoints, but other approaches should also consider and avoid these 
biases. 
 
2.   Postweaning Allocation (3.9.2) 
 
In multiparous animal species, it is recommended to design the study with consideration 
of potential confounders when possible. In particular, when dosing starts in the early 
postweaning phase, and, when offspring are supplied from a limited number of natural mothers, 
the study should be designed in consideration of the potential confounders similar to those at 
preweaning allocation. 
 
J.  Animal Numbers and Sex (3.10) 
 
A JAS should use a group size that is considered generally appropriate in a definitive toxicity 
study based on the selected endpoints (e.g., approximately 10 animals/sex for end-of-treatment 
necropsy endpoints similar to repeated-dose toxicity study for rats). To reduce the number of 
17  Contains Nonbinding Recommendations 
 
 
animals, combining assessment of endpoints in the same subset of animals can be effective (See 
Appendix C). It is recommended that a JAS generally be performed in both female and male 
animals.  
 
 
IV.  CONSIDERATIONS FOR PEDIATRIC-FIRST/ONLY DEVELOPMENT (4) 
 
A common clinical approach for the development of a pediatric-first/only pharmaceutical starts 
with a First in Human (FIH) study in healthy adult volunteers prior to any pediatric trial. As per 
ICH M3, this approach generally includes repeated-dose toxicity studies in rodent and non-
rodent animals, as well as safety pharmacology and genetic toxicology studies. The principles of 
ICH S6 can also apply. The toxicity studies to support the FIH adult study could be performed as 
standard repeated-dose toxicity studies in two species. Alternatively, one or both of these studies 
could be initiated in juvenile animals, and treatment can be continued into maturity in some 
species (see section III.D (3.4)). Studies in juvenile animals should include additional relevant 
endpoints (see section III.H (3.8)). The approach that includes juvenile animals can be more 
efficient, as it could support initiation of clinical trials in pediatric patients shortly after the adult 
FIH study. 
 
There are cases, however, where pediatric patients are treated without any prior adult patient or 
healthy volunteer data (e.g., for a life-threatening or debilitating disease that only exists in 
children or when the pharmaceutical cannot be given safely to adult volunteers). In these cases, 
the FIH trial would be in pediatric patients and the nonclinical program would generally include 
one JAS in a rodent and one JAS in a non-rodent species. Safety pharmacology and genotoxicity 
testing would be conducted as appropriate for adult use, although these studies need not be 
conducted in juvenile animals (see section II.C 4 (2.3.4)). 
 
If the pharmaceutical is intended to treat a chronic pediatric disease, chronic toxicity studies 
should be conducted in one rodent and one non-rodent species. In at least one of these studies, 
dosing should start at an age developmentally matched to the lowest age of the intended patient 
population. In principle, chronic studies that start dosing from ages that developmentally 
correlate to the youngest pediatric patient age can be sufficient to cover all ages and durations of 
pediatric use. These can replace adult animal chronic studies and a separate JAS. Further 
nonclinical assessments of reproductive toxicity and carcinogenic potential can be warranted. 
 
When studies in juvenile animals are warranted for biopharmaceuticals, these should be limited 
to relevant species, as per ICH S6. Non-invasive safety pharmacology endpoints can be included 
in the juvenile or standard NHP repeated-dose studies. Genotoxic and carcinogenic potential 
should be addressed as outlined in ICH S6. 
 
A JAS in postweaning NHP is typically conducted in animals starting at 10 to 12 months of age, 
thus limiting the coverage of the lowest pediatric age ranges. A JAS in preweaning NHP should 
only be conducted in the situation of pharmaceuticals with first and primarily neonatal clinical 
use, and where alternative approaches to nonclinical safety assessment are not feasible. Studies 
with direct dosing of NHPs prior to weaning can require large numbers of mature dams to 
populate even a relatively small JAS. Therefore, the design and endpoints should be clearly 
justified based on the clinical concern. Design expectations should also be flexible because, for 
example, variability in sex distribution and starting weights of offspring is expected. In cases 
where a JAS is not feasible to support the youngest pediatric age, alternative approaches (e.g., in 
18  Contains Nonbinding Recommendations 
 
 
vitro assays, genetically-modified animal models, surrogate molecules) should be considered if 
available and relevant. 
 
Section III (3) should be consulted to determine study designs. 
 
 
V.  DATA INTERPRETATION (5) 
 
A.  Considerations for Endpoint Interpretation (5.1) 
 
Many observations in a JAS (e.g., body weight, clinical pathology) are age, sex, and 
species/strain dependent. Age-matched concurrent control data are, therefore, critical for 
interpretation. If available, appropriate historical control data or reference materials (e.g., tissue 
database or atlas) can also be helpful to interpret results, especially in cases of low incidence 
findings or unscheduled early deaths (i.e., when control data are often lacking or insufficient).  
 
Interpretation of clinical observations can differ between juvenile and adult animals and 
assessments in preweanlings should also consider maternal care and the overall health status of 
the litter. 
 
An assessment on growth should typically be considered a key objective of a definitive JAS and 
should preferably be conducted at necropsy by an evaluation of long bone length in conjunction 
with body weight, and food intake if available. The latter parameters are needed to differentiate 
between potential direct effects on skeletal development or indirect effects (secondary to toxicity 
causing malnutrition and body weight loss/decrease) on long bone length. An effect solely on 
decreased body weight gain is not necessarily an effect on growth. Frequent assessments of long 
bone length for transient effects on growth are challenging to interpret and offer limited value 
because of the inter-individual variability in growth rate during development. 
 
Assessment of organ weight data and the onset of sexual development should be performed in 
the context of growth. Organ weight changes are not always proportional to body weight changes 
because some organs grow at different rates throughout development (i.e., allometric versus 
isometric growth). Additionally, organs have different sensitivity to growth effects (e.g., the 
brain can be less affected than other organs). Thus, interpretation of absolute and relative organ 
weights should consider these aspects. 
 
For statistical analysis, data collected from offspring while part of a study litter should not be 
considered an independent variable (see ICH S5). 
 
B.  Overall Interpretation (5.2) 
 
An integrated assessment should be made across all appropriate studies, comparing available 
findings in juvenile and adult animals, and evaluating clinical relevance. Relevant findings in 
juvenile animals not observed in adults should be discussed, as well as any marked differences in 
sensitivity compared to adults. The overall interpretation of relevant findings should consider the 
type, severity, and recovery (if known) of the effects; the age of the animals; and the exposures 
and/or doses at which any effects were observed, and relate these to the intended pediatric use.  
 
19  Contains Nonbinding Recommendations 
 
 
 
VI.  OTHER CONSIDERATIONS (6) 
 
A.  Excipients (6.1) 
 
Pharmaceutical formulations occasionally contain excipients for which only limited experience 
exists in pediatric populations. To assess the safety of the excipients in a pediatric clinical 
formulation, available information on the excipients should be evaluated and a WoE approach 
should be followed (see section II (2)). If there are insufficient data to support the use of the 
excipient in the intended pediatric population, further safety evaluation can be warranted, for 
example, an additional group evaluating the excipient alone in a JAS. 
 
B.  Combination Pharmaceuticals (6.2) 
 
The development of combination pharmaceuticals for pediatric use should have a nonclinical 
evaluation consistent with the principles outlined in ICH M3 for combination products in 
general, together with the WoE principles outlined in this guidance. Consequently, a JAS of a 
combination pharmaceutical should be considered only if previous human and animal data are 
determined to be insufficient to support pediatric development, and the WoE evaluation suggests 
that a JAS would address identified concerns. If additional nonclinical information is warranted, 
the study design should consider which endpoints are appropriate to address any concerns of 
administering the particular combination. If a JAS is considered appropriate, assessment of the 
combination as it is to be used clinically might be sufficient and testing of the individual active 
ingredients might not be critical. Alternatively, an extra group with the combination could be 
added to a JAS that is already being conducted with one of the single entities. This could provide 
information that would otherwise be obtained in a separate study with the combination product. 
   
20  Contains Nonbinding Recommendations 
 
 
GLOSSARY 
Allometric and isometric growth 
Isometric growth occurs when proportional relationships are preserved as size changes during 
growth. Allometric growth is any deviation from isometric growth. With allometric growth, 
properties such as bone length, organ weight and body surface area can change according to an 
exponential function of body mass. 
 
Endpoint Subset:  
A set of individual animals within a dose group that are assigned to the same endpoint. 
 
Enhanced Pre- and Postnatal Development Study (ePPND):  
This study design is based on biopharmaceutical experience, often in NHP, and is a PPND study 
which includes elements of the embryofetal development (EFD) study in newborns and infants 
instead of the fetus.  
 
Fostering: 
The act of nurturing or offering parental care to offspring that are not genetically related. The 
fully fostering technique arbitrarily mixes up litters with the intent not to have dams with their 
genetic pups. The minimally fostering technique retains the natural litter as intact as possible, 
fostering only as necessary to achieve desired litter size and sex ratio. 
 
Juvenile Animal: 
An animal in any postnatal stage not fully matured in terms of organ or system morphology and 
function. 
 
Juvenile Animal Study (JAS)  A nonclinical safety study typically conducted with the 
objective to provide an assessment of the toxicity profile of a pharmaceutical in juvenile animals. 
 
Pediatric-First Development: 
Pediatric-first development describes development for treatment of pediatric patients before any 
development for an adult indication. 
 
Pediatric-Only Development: 
Pediatric-only development describes development for treatment exclusively in pediatric ages 
(e.g., neonatal respiratory distress syndrome). 
 
Weight of Evidence:  
An approach that evaluates information from several sources to decide if there is sufficient 
evidence to support the development of pharmaceuticals for pediatric use or whether additional 
nonclinical testing is warranted to address potential safety concerns. 
 
The weight given to the available evidence depends on factors such as the quality of the data, 
consistency of results, nature and severity of effects, and relevance of the information. The 
weight of evidence approach requires use of scientific judgment and, therefore, should consider 
the robustness and reliability of the different data sources.   
21  Contains Nonbinding Recommendations 
 
 
LIST OF ABBREVIATIONS 
 
ADME  Absorption, Distribution, Metabolism, and Excretion 
CNS  Central Nervous System 
CYP  Cytochromes P450 
DRF  Dose Range-Finding 
ePPND  Enhanced Pre- and Postnatal Development  
FIH  First in Human 
FOB  Functional Observational Battery 
GABA  Gamma Aminobutyric Acid  
GFR  Glomerular Filtration Rate 
GI  Gastrointestinal 
HPG  Human Pituitary Gonadotropin 
ICH  International Council for Harmonisation  
JAS  Juvenile Animal Study 
NHP  Non-Human Primate 
NOAEL  No Observed Adverse Effect Level 
PND  Postnatal Day 
PPND  Pre- and Postnatal Development  
PK  Pharmacokinetics  
PD  Pharmacodynamics  
TDAR  T-Cell-Dependent Antibody Response 
TK  Toxicokinetic 
WoE    Weight of Evidence 
   
22  Contains Nonbinding Recommendations 
 
 
 
REFERENCES 
1.  ICH guidance for industry E11 Clinical Investigation of Medicinal Products in the 
Pediatric Population (August 2017) 
 
2.  ICH guidance for industry M3 Guidance on Nonclinical Safety Studies for the Conduct of 
Human Clinical Trials and Marketing Authorization for Pharmaceuticals (June 2009)  
 
3.  ICH guidance for industry S3A Note for Guidance on Toxicokinetics: The Assessment of 
Systemic Exposure in Toxicity Studies (October 1994) 
 
4.  ICH guidance for industry S5 Detection of Toxicity to Reproduction for Medicinal 
Products and Toxicity to Male Fertility (November 2000) 
 
5.  ICH guidance for industry S6 Preclinical Safety Evaluation of Biotechnology-Derived 
Pharmaceuticals (June 2011) 
 
6.  ICH guidance for industry S8 Immunotoxicity Studies for Human Pharmaceuticals 
(September 2005) 
 
7.  ICH guidance for industry S9 Nonclinical Evaluation for Anticancer Pharmaceuticals 
(October 2009)
23  Contains Nonbinding Recommendations 
 
 
APPENDIX A:  OVERVIEW OF AGE-DEPENDENT DEVELOPMENT OF ORGAN SYSTEMS BY 
SPECIES 
 
These tables reflect a high-level overview of organ system development by species to illustrate 
similarities and differences between the commonly used toxicology species, as compared to 
humans, for the timing and relative duration of development.  
 
The tables are intended to aid in the assessment of the relevance of existing nonclinical data, as 
well as the selection of species, starting age, and dosing duration for a JAS. They are based on a 
review of current knowledge, but are not comprehensive. Species-specific and/or organ system 
reviews in the literature can provide additional detail and should be consulted for each specific 
situation. Factors such as strain, breeding, and animal supplier can impact age-dependent 
development and should also be considered. 
 
The following legend applies to Tables A1-A5 and Figure A1: 
 
  Critical period of structural and 
functional growth and 
development 
  Active period of growth and/or 
functional maturation 
  Slow continued growth and/or 
refinement of function 
  Structurally and functionally fully 
mature 
 
The human age categories are aligned with those described in the ICH E11 guidance, with 
approximate milestones (e.g. birth, introduction of solid foods, weaning, puberty, and adulthood) 
corresponding to development in nonclinical species. The tables only provide an approximation 
of the ages when these milestones occur. For example, the active developmental period of 
several organ systems can last until 12 or 18 months of age in humans, which coincides with the 
regionally and culturally diverse milestones of introduction of solid food and weaning in 
humans. 
 
Developmental toxicity is of particular concern during critical and active periods of functional 
and structural growth and less so during periods of slow growth or refinement of function. In 
some organ systems, such as the immune system, the tissues are largely competent to respond at 
birth, but then undergo substantial expansion in the immediate postnatal period in parallel with 
environmental stimulation. Likewise, some organ systems such as the CNS are highly complex, 
with different timelines for establishment of reflexes, pain pathways, sleep patterns, myelination, 
coordination, and cognitive function. This complexity can influence periods of susceptibility 
during development, so the milestones listed provide only a general guide. Functional maturation 
occurs into adulthood in humans for some aspects of CNS development, and cannot be fully 
modelled by animal test systems. Puberty is a period of intense endocrine activity with important 
structural and functional maturation of the reproductive system, but also with effects on 
musculoskeletal growth and CNS development in humans. The timing of puberty can be variable 
across all species, particularly in primates, where onset and progression are typically monitored 
by external sex characteristics, as in Tanner staging in humans. Females typically achieve full 
reproductive functionality prior to males across species. Finally, slow continued growth with 
24  Contains Nonbinding Recommendations 
 
 
refinement of function occurs into adulthood for several organ systems, as is indicated in the 
tables.
25  Contains Nonbinding Recommendations 
 
 
Table A1:  Age-dependent Development of Human Organ Systems
Birth 1stSolid Food Weaning Puberty
Milestones:
Age Categories: Premature Neonate  Infant/Toddler Child Adolescent Adult
through full term  (Term Birth-27 days) (28 days-23 months) (2-11 years) (12-18 years) (> 18 years)
System: birth
Cardiovascular Critical neonatal physiologic transitions (pulmonary  Progressive increase in ion  Adaptive myocardial and vascular changes
and vascular resistance,  closure of fetal shunts) channels/conductance
Endocrine Fetal hormone production begins second trimester & structurally well developed at term  Adrenarche in late childhood Pubertal sex hormone 
Endocrine functions critical for growth/development surge and maturation
Eye O-sensitive retinal  Morphologically well  Development of retina (fovea), lens,  Training of vision important between 1-4 years (focus and track at  Star sutures of lens form continuously during adolescence and into 
an2giogenesis developed at term birth iris pigmentation complete by 1 year 1-4 months; color vision starts at 3 months) adulthood
Gastrointestinal Variable  preterm suck/swallow reflex  Increased digestive functionality,  Progressive adaptations in digestive function to 
Critical functionality present in term neonates and absorptive capacity with growth accommodate shift in diet/complexity
Acidification, transit, microbiome
Hepatobiliary Structurally well developed at term Important increases in  Continued refinement of metabolic and elimination function and capacity
Important neonatal transitions in bile production and  metabolic and elimination capacity
elimination
Immune Neonatal structural expansion of primary and  Progressive population of immune tissues and development of memory as a function of time and environment
secondary immune tissues 
Integument Cornification &  Critical neonatal function (barrier, water, thermoregulation, sensation) Progressive surface acidification, local microbiome and immune function
vernix High surface area relative to weight as compared to adults  Pubertal hair growth, oil production
Nervous Neuronal subsets  Maximum neuron count and brain:bodyweight at term birth Refinement of sensorimotor control and expanded capability for complex learning and  CNS development 
defined and active  Myelin and glia present at term birth with continued refinement  memory function continues into 
excitatory signaling  Postnatal neuronal apoptosis, synapse pruning, migration and circuit integration adulthood
initial myelination Metabolic functions, neurotransmitter and conduction systems mature at variable rates
Pulmonary Critical liquid air transition at birth  Alveolizationprogresses  Increased alveolar surface area with growth to maximum aerobic function in adolescence
Canalicularsaccularalveolarstructure
Surfactant made preterm, secreted at term
Renal Nephrogenesis not complete until term  Increases in renal function  Tubular growth and refinement of function including 
GFR precedes concentration erythropoietin and angiotensin axis 
Reproductive Oocyte meiosis &  Testes descended Quiescent period Sertoli cell expansion 
testicular descent Postnatal HPG surge of sex hormones  Breast development
(mini-puberty) Spermarche/Menarche
Skeletal Growth plate  Growth plates present at term birth Slower growth Pubertal growth Growth plate closure
development Critical period of rapid growth driven by growth hormone and thyroid hormone
 
    Contains Nonbinding Recommendations 
 
 
Table A2:  Age-dependent Development of Rat Organ Systems
Puberty
Neonate 1stSolid Food Weaning Adulthood
System General Considerations (M ~ PND 42,
(~ PND 1-10) (~ PND 15) (~ PND 21-25) (~ PND 70)
F ~ PND 35)
Cardiovascular   Critical neonatal physiologic transitions (pulmonary and systemic vascular resistance)
 Adaptive myocardial and vascular changes
 Progressive increase in cardiomyocytesand ion channels to PND 21
Endocrine  Most glands are well developed at birth and critical for growth
Eye  Morphological development of retina, lens, iris, cornea and adnexa ongoing until PND14
 Eyelids open on PND14
 Eyes morphologically fully developed by weaning; continued growth and refinement of vision through puberty
Gastrointestinal  Immature at birth; lack gastric acid and poor pancreatic enzyme production until PND 14
 Highly permeable proximal small intestine initially allows absorption of intact proteins 
 Adaptations in 3rd week of age to accommodate shift in diet
Hepatobiliary   Structurally immature at birth
 Progressive development of organized hepatic cords and plates, with increase in metabolic functionality, over first 4 weeks 
of age 
Immune  Progressive population of secondary immune tissues and development of memory as a function of time and environment
 TDAR typically assessed after PND 45 
Integument  Critical neonatal function (barrier, water and thermoregulation, conductance, sensation); thicker epidermis first 2 weeks of 
age
 Adnexa and hair develop postnatally, structurally resembles adult by PND 21
 Sexual dimorphism by PND 35 to 42 
Nervous  Structural maturation of olfactory bulbs, cerebellum, hippocampus, and cerebral cortex occurs over first 3 weeks of age
 Maximum neuron count and brain:bodyweight at PND7, with extensive postnatal apoptosis, pruning and migration
 Postnatal myelination of spinal cord caudal to cranial; brain for reflexes, sensorimotor, then learning and memory 
 Conduction systems, opiate receptors/metabolism, GABA, serotonin & noradrenalin pathways mature at different rates
Pulmonary  Saccular at birth
 Alveolizationoccurs over first 2 to 3 weeks of age
Renal  Nephrogenesisincomplete at birth
 Progressive increase in GFR and renal function over first 3 to 5 weeks of age
Reproductive  Period of decreased androgen production by Leydig cells during 3rd week of age necessary for expansion of Sertoli and 
germ cells
 Remaining reproductive changes and appearance of sexual dimorphism occur at onset of puberty (5 to 7 weeks of age)
Skeletal  Rapid postnatal growth through adulthood
 Long bone growth plate structure not evident until PND 14 to 21, and remain open into adulthood
 
    Contains Nonbinding Recommendations 
 
 
Table A3:  Age-dependent Development of Beagle Dog Organ Systems
Puberty
Neonate Weaning (M ~ 5-8  Adulthood
1stSolid Food
System General Considerations (< 3  (~ 8  months,  (> ~ 12 
(~ 3 weeks)
weeks) weeks) F ~ 6-12  months)
months)
Cardiovascular   Critical neonatal physiologic transitions (pulmonary and systemic vascular resistance) 
 Adaptive myocardial and vascular changes 
 Significant increase in blood pressure and decrease in heart rate from week 1 to 6 months of age
Endocrine  Endocrine tissue development and initial hormone production occurs in utero
 Endocrine functions critical for growth/development and progression through puberty
Eye  Morphological development almost complete at birth; eyelids first open at 10-14 days of age
 Mature retina by 7 weeks; corneal transparency and iris pigmentation complete by ~ 8 weeks
 Vision fully mature by 12-14 weeks
Gastrointestinal  At birth gastrointestinal tract is fully formed; suck/swallow and rooting reflexes fully functional within 1-2 days postnatal ?
 Colostrum stimulates intestinal maturation and growth
 Metabolic functional development primarily between birth and weaning
Hepatobiliary   Hepatobiliary structural maturation by 1 week of age 
 Bile secretory function matures more slowly (30 to 70 % of adult at 4 to 6 weeks of age)
Immune  Agammaglobulinemic at birth; need IgG transfer from dam via colostrum within 12-24 hours postnatal 
 Immunologic tissues are largely structurally mature and functional at or shortly after birth 
 Rapid postnatal growth of thymus to maximum size at weaning, with involution at puberty
Integument  Critical neonatal functions (barrier, hydration, conductance, sensation) develop in initial postnatal phase (~2 weeks)
 Initial haircoat and adnexa at birth; planum and pads thicken through weaning, and transition to adult coat
Nervous  Hypertonic flexion at birth, then extension develops first week; neonatal (primitive) reflexes disappear at ~ PND 28 
 Functional locomotor development occurs postnatally with rapid progression through 8 weeks
 Spinal cord reaches structural maturity by 6 weeks postnatal, but nerve conduction speed increases over 6-12 months
 Rapid cognitive development with critical developmental period for learning at approximately PND 18 to 28
Pulmonary  Lung maturation continues postnatally, and is generally comparable to a term human neonate by 1-2 weeks postnatal
 Regular daily rhythm of respiratory rate (RR) by 8 weeks; RR decreases with growth through puberty
 Continued development through 8 weeks and growth to maximum alveolar capacity by ~1 year of age  
Renal  Kidney is structurally and functionally immature at birth, with completion of nephrogenesis at approx. 2 weeks of age 
 Tubular growth and corpuscle maturation continues through 3 weeks postnatal, reaching maturity by weaning
 Concentrating ability develops prenatally, but acid-base homeostasis develops postnatally 
Reproductive  Testes descend postnatally at 5 to 6 weeks of age 
 Males reach sexual maturity at ~ 5 to 8 months of age; Females at  ~ 6 to 12 months of age
Skeletal  Appearance of long bone ossification centersbetween 1 to 10 weeks of age 
 Limbs cannot support weight until 2 weeks of age
 Most rapid long bone growth is complete by 5 months of age, with slower continued growth until ~ 18 months of age
 
    Contains Nonbinding Recommendations 
 
 
Table A4:  Age-dependent Development of Gottingen Minipig Organ Systems
Neonate Weaning Adulthood
1stSolid Food Puberty
System General Considerations (< 2  (~ 4-6  (> ~ 6 
(~ 2-3 weeks) (~ 4-6 months) 
weeks) weeks) months)
Cardiovascular   Critical neonatal physiologic transitions (pulmonary and systemic vascular resistance)
 Adaptive myocardial and vascular changes and growth
Endocrine  Endocrine tissue development and initial hormone production occurs in utero
 Endocrine functions are critical for growth/development and progression through puberty
Eye  Eyes morphologically well-developed at birth; eyes open within 1st3 days postnatal
 Full eyelid retraction by 3 weeks allowing ophthalmic examination
Gastrointestinal  Highly comparable to human postnatal GI development; critical neonatal absorption capacity
 Postnatal acidification, microbiome, motility; adaptive digestive/transport functions achieved by weaning
Hepatobiliary   Structurally and functionally immature at birth; structurally similar to adult by 4 weeks
 Progressive increase in metabolic functionality, especially over first 3 to 4 months
Immune  Minimal innate and passive immunity at birth; piglets require colostrum within ~ 2-4 hours postnatal
 Anatomically fully developed by ~ 4 weeks of age
 Progressive population of secondary immune tissues and development of memory as a function of time and 
environment
Integument  Critical neonatal function (barrier, conductance, sensation)
 Poor thermoregulation from birth through 2-3 weeks
Nervous  Sexually dimorphic postnatal brain growth rapid through weaning and continuing until puberty
 Myelin, glia and most cortical neurons present at birth 
 Neuromuscular system is more functionally mature at birth than in human
Pulmonary  Lungs are well developed at birth, with completion of alveolizationover first 2 weeks postnatal
 Continued growth and refinement of function through puberty
Renal  Nephron formation up to ~ 3 weeks after birth
 Functionally mature at approximately 3 months of age
Reproductive  Structurally well developed and testes descended at birth
 Sexual maturity in males by 4-5 months of age and in females by 5 to 6 months of age
Skeletal  Able to stand at birth with increased mobility in first few days
 Rapid postnatal growth with closure of the epiphyseal growth plates at 18 months of age
 
    Contains Nonbinding Recommendations 
 
 
Table A5:  Age-dependent Development of Cynomolgus Monkey Organ Systems
Neonate Weaning
1stSolid Food Puberty Adulthood
System General Considerations (< 1  (~ 6 
(~ 3 months) (~ 3-4 years) (~ 4 years)
month) months)
Cardiovascular   Critical neonatal physiologic transitions (pulmonary and systemic vascular resistance)
 Adaptive myocardial and vascular changes
 Myocardiocyteexpansion through 3 months of age, then progressive growth
Endocrine  Most glands are well developed at birth and critical for growth
 Zona reticularisof adrenal cortex expands at 3 to 6 months of age (adrenarche)
 Endocrine function of gonads expands at puberty
Eye  Structurally well-developed and eyes open at birth
 Postnatal maturation of fovea and lens
Gastrointestinal  Functional at birth and comparable to human postnatal GI development
 Postnatal acidification, microbiome, motility
 Adaptive digestive/transport functions achieved by weaning to accommodate shift in diet/complexity and expand 
microbiome
Hepatobiliary   Structurally well developed at birth
 Progressive increase in metabolic functionality, especially over first 3 to 6 months
Immune  Neonatal structural expansion of immune tissues 
 Progressive population of secondary immune tissues and development of memory as a function of time and environment 
Integument  Functional (barrier, water and thermoregulation, conductance, sensation) with hair and adnexa present at birth
Nervous  Defined sequential and progressive development into adulthood
 Postnatal apoptosis, pruning and migration most prominent before weaning
 Myelin and glia present at birth, with further expansion postnatally 
 Neurotransmitter and conduction systems mature at variable rates (i.e.: GABA, serotonin, dopamine and noradrenalin all 
differ)
Pulmonary  Structurally mature at birth with progressive growth
Renal  Nephrogenesiscomplete at term birth
 Progressive increase in GFR and renal function over first 6 months of age
Reproductive  Testes descended at birth, populated by germ cells, Sertolicells and Leydigcells
 Follicular development and atresia begins at 3 to 6 months
 Subsequent reproductive changes (menarche and spermarche) occur at onset of puberty and continue until adulthood
Skeletal  Infant able to cling to dam from birth 
 Growth plates present at birth
 Most rapid postnatal growth occurs prior to weaning, followed by slower growth until growth plates close during adulthood
 
    Contains Nonbinding Recommendations 
 
 
Figure A1: Comparison of Rat and Human Organ System Development
RAT
PULMONARY
GIHEPATOBILIARY
IMMUNE
RENAL
CENTRAL NERVOUS SYSTEM
SKELETAL
REPRODUCTIVE
Sertoli cells Hormonal signaling
Birth 1                  2              W    e3a  n                4                 5                  6                  7            8                  9
Postnatal Weeks
Infant Child Adolescent
Neonate  
< 1 month 1 23 months 2 11 years 12 18 years
Birth
* REPRODUCTIVE 
SKELETAL
CENTRAL NERVOUS SYSTEM
RENAL
IMMUNE
GIHEPATOBILIARY
PULMONARY
HUMAN
* Human neonatal mini puberty, (See Table A1), also note leading dot with line to reproductive box denotes early pubertal hormonal signaling 
  Contains Nonbinding Recommendations 
 
 
Table A6.  Principal Advantages and Disadvantages of Various Mammalian Species for Use in 
Juvenile Animal Studies 
 
Species      Advantages       Disadvantages  
Rat    Well-studied species in juvenile animal    Small body size, high metabolic rate and 
studies with extensive historical control  rapid growth can lead to fast decline in 
data  general condition and death 
  Several consistent developmental    Several organ systems are less developed at 
milestones (general growth, preputial  birth relative to man (particularly CNS, 
separation/vaginal opening, puberty)  lung, kidney, GI tract and immune system; 
  Often used for (adult) general and  eyes do not open until PND 12-14) 
reproductive toxicology    ADME characteristics of oral 
  Body size allows most  pharmaceuticals given in the preweaning 
manipulations/administrations starting  phase often translate poorly to humans due 
early preweaning  to immaturity of the GI tract 
  Litter size allows balanced sex distribution    Compressed development can make it 
and allocation of pups to different  difficult to identify distinct windows of 
endpoints and dedicated cohorts of pups  vulnerability 
  Compressed development (~10 weeks)    Conventional blood samples are often 
allows for inclusion of wide range of  terminal collections, particularly 
endpoints   preweaning 
  Compressed development allows for    Can easily become very large studies as 
inclusion of additional endpoints which are  most endpoints or collections require 
difficult to perform using large animals  dedicated cohorts of pups 
(such as developmental neurotoxicity,    Less sensitive than humans to fertility 
immunotoxicity, fertility/breeding)   perturbations 
  Small body weight requiring low amount    Can have more limited application for 
of test material  foreign proteins 
  Relatively easy transportation, housing,    Often not pharmacologically relevant 
and management  for highly targeted therapies 
  Pups and dams are amenable to fostering     Potential impact of immunogenicity 
 
32  Contains Nonbinding Recommendations 
 
 
Species      Advantages       Disadvantages  
  Easy to obtain many pups with the same   
postnatal stage   
  Passive immunity present at birth   
Mouse    Advantages are generally similar to those  Similar to rat, additionally: 
of the rat, but postnatal development    Small pup size allows fewer manipulations 
occurs slightly faster  /administrations than rat from early on 
  Broad CYP enzymes; metabolism can be    Requires dedicated cohorts of pups for each 
more relevant than rat for some  endpoint or collection and can require 
compounds  sample pooling  
  Mice have a gall bladder (unlike rats)    Less historical background information than 
  Detailed literature available, especially for  the rat 
development and characterization of the   
CNS and immune system 
  Many genetic modification models 
available including some models that 
increase pharmacological relevance for 
highly targeted therapies   
33  Contains Nonbinding Recommendations 
 
 
Species      Advantages       Disadvantages  
Dog    Often used in general (adult) toxicology     Protracted development (~5-12 months to 
  Relatively large at birth  sexual maturity, 12-18 months to skeletal 
  Relatively easy to handle  maturity) with interindividual variability in 
  Litter size allows allocation of pups to  growth and developmental milestones 
different endpoints    Altricial at birth (i.e., eyes do not open and 
  Puppies can be separated from dams for a  cannot bear weight until ~ 2 weeks 
few hours   postnatally) 
  Breeding can be planned in advance    Require colostrum for passive transfer of 
  Postnatal development of several organ  maternal Ig in immediate perinatal period 
systems reasonably comparable to that of    Variable litter sizes and sex distribution can 
human infants (cardiovascular, pulmonary,  make it difficult to populate study with 
immune system)  minimal bias (genetic/litter, sex 
  CNS maturation relatively well  distribution) across groups 
characterized, with defined critical window    Limited historical background data, 
for learning/cognitive development  especially for nonstandard endpoints  
    Seasonal breeder (supply & study start over 
weeks or months) 
  Not amenable to fostering  
  Large body size requires comparably large 
amounts of test compound compared to 
rodents 
Minipig/   Many similar developmental milestones as    Less well-established historical control data 
Pig  humans  than dog or NHP toxicology species 
  Relatively large at birth    Require colostrum for passive transfer of 
  Relatively easy to handle  maternal Ig in immediate perinatal period 
  Breeding can be planned in advance     Some organ systems relatively mature at 
  Litter size allows allocation of piglets to  birth compared to human infants (e.g., 
different endpoints  lungs, musculoskeletal)   
  Amenable to fostering     Large body size requires comparably large 
  Relatively large litters usually allow  amounts of test compound compared to 
balanced sex distribution  rodents 
34  Contains Nonbinding Recommendations 
 
 
Species      Advantages       Disadvantages  
  Neonatal GI tract similar to human for    IV and gavage administration can be 
orally administered drugs  challenging in very young piglets 
  All routes of administration feasible   
(except inhalation); best model for dermal 
studies (low density of adnexa and hair 
follicles, similar thickness of epidermis) 
  Short development (~6-9 months), 
relatively easy transport and housing 
compared to other large non-rodents 
NHP    Usually cynomolgus but rhesus and    Protracted development (~3-6 years for 
marmosets also feasible  sexual maturity, ~5-8 years for skeletal 
  Many similar developmental milestones as  maturity in macaques) makes an extensive 
humans  juvenile study to cover all developmental 
  Neonates/infants similar to human for GI  phases impractical 
tract, immune system, cardiovascular,    Single offspring for macaques with high 
renal, and special sense (eye, ear)  inter-individual variability in growth and 
development  development  
  Macaque infants are relatively large at    Marmosets typically have twins and require 
birth  both maternal and paternal care in 
  Extensive reference and historical  preweaning phase; offspring are relatively 
background data from birth available  small  
  Often used for (adult) general and    Offspring highly dependent on maternal 
reproductive toxicology (e.g., ePPND),  care over first month (minimal procedural 
especially for biopharmaceuticals  intervention recommended; preweaning 
  Maternal transfer of immunoglobulin is  manipulation & dosing feasible with risk of 
similar to humans, so infants are born with  maternal rejection), and are cohoused with 
passive immunity (serum IgG)   dam for first 3-6 months; with shipping and 
  Often the most pharmacologically relevant  quarantine requirements it is rarely feasible 
animal model for highly targeted therapies  to initiate studies in juvenile monkeys < 9 
  months of age 
 
35  Contains Nonbinding Recommendations 
 
 
Species      Advantages       Disadvantages  
  Neonatal NHP are precocious relative to 
human neonates in terms of 
musculoskeletal, CNS, endocrine and 
respiratory system  
  Cannot synchronize breeding (supply & 
study start over weeks or months for 
seasonal breeders such as rhesus) 
  Ethical reservations (strong rationale to 
justify use of juvenile NHP for toxicity 
testing) 
 
Rabbit    Compressed development (~5-6 months)    Developmental milestones less well 
and small body size requiring comparably  established than other non-rodent species  
low amount of test material    Not routinely used/well accepted in (adult) 
  Relatively easy to handle  general toxicology  
  Often used for reproductive toxicology;    Less well-established historical control data 
can also be used for ocular administration,  than dog or NHP toxicology species 
evaluation of bone growth     Handling young offspring can provoke 
  Litter size allows balanced sex distribution  cannibalism or rejection by the mother 
and allocation of kits to different endpoints    Limitations similar to rat for foreign 
  Relatively easy transport and housing  proteins, and immunogenicity can lead to 
  Passive immunity present at birth  acute hypersensitivity reactions 
  Sensitive to gastrointestinal disturbances 
  General physical condition difficult to 
monitor using clinical signs 
 
36  Contains Nonbinding Recommendations 
 
 
Species      Advantages       Disadvantages  
Other    Other species could be considered for cause when pharmacologically and toxicologically 
Species  relevant. Examples of alternative mammalian test systems include the hamster, guinea pig, tree 
shrew, ferret, cat, sheep, and goat.  
  Advantages tend to be species and program specific, but often reflect use of that species in 
genetic or disease models, or when there are data supporting interpretation and translatability 
of specific endpoints. 
 
Disadvantages include: 
  Developmental milestones less well established than in rat, mouse, dog, minipig/pig and NHP  
  Not routinely used / well accepted in (adult) general toxicology 
  Limited historical control toxicology data 
  Limited use (model in special indications such as heart failure) 
  Many require colostrum for passive transfer of maternal Ig in perinatal period 
  Limited availability of purpose-bred animals and suitable laboratory housing 
   
37  Contains Nonbinding Recommendations 
 
 
APPENDIX B:  CASE STUDIES APPLYING THE WEIGHT OF EVIDENCE APPROACH  
A.  A small molecule with known pharmacology has available adult clinical and nonclinical 
data including repeated-dose toxicity data. None of these data suggest a safety concern in 
a developing organ for the intended pediatric population of adolescents (12 years and 
above) for a one month duration of clinical treatment. The WoE analysis indicates that 
additional nonclinical investigations will not contribute useful information. 
 
 
WoE
Further  Further 
Factors
Studies  Studies 
Likely Unlikely
Youngest 
Intended
Patient Age Neonates    Infant  Child  Adolescent 
(<1 Month) (123 Months)  (211 Years) (12 Years)
Effects on 
Developing Organ 
Systems Yes No
Amount/Type of
Existing Data
No Nonclinical        Adult Nonclinical  Adult  Pediatric 
or Clinical Data         Only Clinical Clinical
Pharmacological
Target has Role In Organ
Development
Yes  No
Selectivity and 
Specificity of 
Pharmaceutical Low  High
Clinical Treatment 
Duration
Chronic Use  Acute Use
 
   
38  Contains Nonbinding Recommendations 
 
 
B.  A small molecule with a novel mode of action intended for chronic use starting in 
neonates or infants has limited Phase 1 clinical and nonclinical safety data with no 
significant safety concerns identified. There are potential effects on developing organ 
systems based on the pharmacology. The WoE analysis indicates further nonclinical 
investigation, such as a JAS with additional endpoints based on the targeted developing 
organ systems, would be useful. 
 
WoE
Further  Further 
Factors
Studies  Studies 
Likely Unlikely
Youngest 
Intended
Patient Age Neonates    Infant  Child  Adolescent 
(<1 Month) (123 Months)  (211 Years) (12 Years)
Effects on 
Developing Organ 
Systems Yes No
Amount/Type of
Existing Data
No Nonclinical        Adult Nonclinical  Adult  Pediatric 
or Clinical Data         Only Clinical Clinical
Pharmacological
Target has Role In Organ
Development
Yes  No
Selectivity and 
Specificity of 
Pharmaceutical Low  High
Clinical Treatment 
Duration
Chronic Use  Acute Use
 
   
39  Contains Nonbinding Recommendations 
 
 
C.  A small molecule with a pharmacological target that has an established critical role in 
CNS development is intended for chronic use in children (6 years and above). Nonclinical 
and adult clinical data are available. The concern for a potential effect on the developing 
CNS cannot be addressed clinically by monitoring and management. Existing data 
adequately address other developing systems. The WoE analysis warrants a postweaning 
JAS design that includes core endpoints and additional CNS endpoints to address the 
specific concern.  
 
WoE
Further  Further 
Factors
Studies  Studies 
Likely Unlikely
Youngest 
Intended
Patient Age Neonates    Infant  Child  Adolescent 
(<1 Month) (123 Months)  (211 Years) (12 Years)
Effects on 
Developing Organ 
Systems Yes No
Amount/Type of
Existing Data
No Nonclinical        Adult Nonclinical  Adult  Pediatric 
or Clinical Data         Only Clinical Clinical
Pharmacological
Target has Role In Organ
Development
Yes  No
Selectivity and 
Specificity of 
Pharmaceutical Low  High
Clinical Treatment 
Duration
Chronic Use  Acute Use
 
   
40  Contains Nonbinding Recommendations 
 
 
D.  A monoclonal antibody targets a soluble cytokine and is intended for chronic pediatric 
use in rheumatologic and allergic diseases (>2 years old). The only findings are reversible 
decreased serum Ig and occasional injection site reactions (in both animals and adult 
patients). In a monkey ePPND study, offspring exposure was comparable to dams through 
PND 28 and decreased thereafter. T-cell-dependent antibody response (TDAR) results 
were similar to controls (between 3-6 months postnatally). The WoE analysis does not 
warrant a JAS. 
 
WoE
Further  Further 
Factors
Studies  Studies 
Likely Unlikely
Youngest 
Intended
Patient Age Neonates    Infant  Child  Adolescent 
(<1 Month) (123 Months)  (211 Years) (12 Years)
Effects on 
Developing Organ 
Systems Yes No
Amount/Type of
Existing Data
No Nonclinical        Adult Nonclinical  Adult  Pediatric 
or Clinical Data         Only Clinical Clinical
Pharmacological
Target has Role In Organ
Development
Yes  No
Selectivity and 
Specificity of 
Pharmaceutical Low  High
Clinical Treatment 
Duration
Chronic Use  Acute Use
 
 
41  Contains Nonbinding Recommendations 
 
 
APPENDIX C:  APPROACHES TO PREWEANING LITTER ALLOCATION IN THE RODENT 
Initiation of dosing rat pups during the preweaning phase of a JAS presents a unique situation in 
which the study should be designed to reduce potential confounders related to maternal care, 
litter size and other factors such as genetics. This can be achieved by how the study litters are 
constructed and standardized in combination with how the study litters are assigned to dose 
groups and how the individual pups are assigned to endpoint subsets. See also section III.I.1 
(3.9.1). 
 
One approach to achieve the goal of an even distribution of potential confounders is to  
  standardize the litter using a minimal fostering technique in which the majority of pups 
remain with their natural mother (i.e., those pups in good health) and, when necessary, a 
small number of pups are fostered into the litter to achieve the desired litter size and sex 
ratio 
  assign each standardized whole litter to a single dose group 
  assign individual pups within the study litter to endpoint subsets 
 
In the following example, the definitive rat JAS design has a dosing interval from PND 14 
through 63 and includes assessments of both core endpoints (with TK assessments on PND 14, 
22, and 63) and study-specific additional endpoints (post-treatment necropsy and CNS 
assessments). Each dose group contains 10 minimally fostered rat litters (identified as A, B, C for 
each dam with a litter, etc.). The litters are standardized to 10 pups per litter (5 male and 5 female 
pups when possible) with each colored line in the figure representing an individual pup. The 
genetic offspring are assigned arbitrarily (F1, F2, etc. for the first and second female, 
respectively). Pups that were fostered are re-assigned the last positions in the pup identification 
scheme (e.g., F5 for the fifth female). The dam identifiers for both their genetic (if available) and 
foster mothers are recorded in the study data. The whole litter is then assigned to the same dose 
group so all 10 pups in a litter will be treated with the same dose. Generally, the number of litters 
will depend on the total number of pups for the selected endpoints. Microsampling can minimize 
the number of animals for TK assessment, and thus is always encouraged. 
 
In this example, the pups are assigned to core and study-specific additional endpoints across the 
10 litters in an inter-litter fashion (i.e., one or two males and/or females from each litter to the 
specific endpoint subset). The end-of-treatment necropsy and other key endpoints are assigned to 
pups with low identification numbers (i.e., M1 or M2) so that fostered pups are more likely 
assigned to endpoints less affected by confounders. Depending on the specific study endpoints, 
there are different ways that the subsets can be allocated to accomplish the same objectives.  
 
42  Contains Nonbinding Recommendations 
 
 
Figure Represents One Dose Group of 10 litters with 5 pups/sex
Males Females
Litter
M1  M2  M3  M4  M5               F1    F2   F3   F4   F5
A
B
C EndofTreatment Necropsy (10/sex)
PostTreatment Necropsy & CNS Behavior (10/sex)
D CNS Behavioral Assessment (+10/sex) = 20/sex for CNS
TK PND 63 (3/sex/timepoint; 5 timepoints, nonterminal)
E TK PND 22 (2/sex/timepoint; 5 timepoints, terminal)
F
G
H
I
J
 
In this example, one pup/sex/litter is allocated for the end-of-treatment necropsy (M1/F1 blue 
lines; for a total of 10 pups/sex/dose group). The 10 pups/sex/dose group of the second set 
(M2/F2 half yellow and green lines) are assigned to two assessments, CNS behavioral 
assessments and post-treatment necropsy. These 10 pups/sex/dose group along with the 
10 pups/sex/dose group of the third set (M3/F3 green lines) are combined for a total subset of 
20 pups/sex/dose group for the CNS behavior assessment. This CNS assessment includes 
detailed clinical observations, behavior tests, and learning and memory tests, which can be 
conducted during the post-treatment period with clinical observations also conducted during 
treatment. Both of the necropsy subsets (end-of-treatment and post-treatment) include an 
expanded neuropathology assessment. The fourth set (M4/F4 red lines) is assigned to the PND 63 
TK subset (serial sampling; non-terminal serial sampling is possible in post-weaning rats of this 
age, therefore only a portion of this subset is needed for TK and could be used for other 
assessments). Because maternal and litter confounders would not be relevant for a single dose 
TK assessment, the TK blood samples after the first dose on PND 14 are collected from spare 
litters (not shown) that would not continue on study after the blood collection (approximately 3-
4 pups/timepoint/dose group). Lastly, 1 pup/sex/litter (M5/F5 black lines) is assigned to the 
PND 22 TK subset (an age that typically requires terminal blood collection unless microsampling 
is available).  
 
The pups in the end-of-treatment necropsy (M1/F1) and the post-treatment necropsy subsets 
(M2/F2) also have core assessments for post-weaning food consumption, sexual development, 
clinical pathology, and long bone length. The core endpoints of mortality, clinical observations, 
and body weights are assessed for all pups.  
 
43  Contains Nonbinding Recommendations 
 
 
In addition to the example above, there are other options that can decrease the number of pups 
assigned to the study. For instance, one set of 10 pups/sex/dose group could be split to serial 
sampling TK (4 pups/sex/dose group) and post-treatment necropsy (6 pups/sex/dose group). 
Alternatively, in the example above, the fourth set of 10 pups/sex/dose group (M4/F4) could be 
split into 4 pups/sex/dose group for the PND 63 TK assessment (an age when non-terminal serial 
blood collections are generally feasible) and 6 pups/sex/dose group for the CNS assessment. The 
third set of 10 pups/sex/dose group (M3/F3 green lines) could also be assigned to the CNS subset 
to achieve a total of 16 pups/sex/dose group. Both of these options obviate the need for extra 
animals for the CNS assessments. Thoughtful study designs which include appropriate endpoints 
and animal allocation can minimize the number of litters and total animals used. 
44