Guidance for Industry 

 
M3(R2) Nonclinical Safety Studies for 
the Conduct of Human Clinical Trials 
and Marketing Authorization for 
Pharmaceuticals 
 
  
Questions and Answers(R2) 

 
 
 
 
 
 
 
 
 
 
 
 
 
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) 

 
February 2013 

ICH 

 
Revision 1 

  Contains Nonbinding Recommendations 
 
Guidance for Industry 

 
M3(R2) Nonclinical Safety Studies for 
the Conduct of Human Clinical Trials 
and Marketing Authorization for 
Pharmaceuticals 
  
Questions and Answers(R2) 

 
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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) 

 
February 2013 

ICH 

 
Revision 1 

   
 
TABLE OF CONTENTS 

 
 
I.   INTRODUCTION............................................................................................................. 1 
 
II.   QUESTIONS AND ANSWERS....................................................................................... 2 
 
A.   Limit Dose for Toxicity Studies (1)...............................................................................................2
  
B.   Metabolites (2)................................................................................................................................5
  
C.   Reversibility of Toxicity (3)...........................................................................................................8
  
D.   Combination Drug Toxicity Testing (4).....................................................................................10 
 
E.   Safety Pharmacology (5)..............................................................................................................15 
 
F.   Exploratory Clinical Trials (6) ...................................................................................................15
  
G.   Reproductive Toxicity (7)............................................................................................................20 
 
H.   Juvenile Animal Studies (8).........................................................................................................21 
 
i   
1
 
Guidance for Industry  
 
M3(R2) Nonclinical Safety Studies for the Conduct of Human 

Clinical Trials and Marketing Authorization for Pharmaceuticals 

 
Questions and Answers(R2) 

 
 
This guidance represents the Food and Drug Administration's (FDAs) current thinking on this topic.  It 
does not create or confer any rights for or on any  person and does not operate to bind FDA or the public.  
You can use an alternative approach if the approach satisfies the requirements of the applicable statutes 
and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for 
implementing this guidance.  If you cannot identify the appropriate FDA staff, call the appropriate 
number listed on the title page of this guidance.  
 
 
 
I.  INTRODUCTION2   
 
Although the ICH M3(R2) guidance is still in its early phases of the implementation, the 
complexity of the guidance, its broader scope, and numerous changes in recommendations from  
the M3(R1) guidance have generated questions that have an impact on its successful 
implementation. This question and answer (Q&A) document is intended to clarify the key issues.   
The Steering Committee has endorsed the establishment of an M3(R2) Implementation Working 
Group (IWG), which is currently working on the development of Q&As. 
 
This guidance is a revision of the ICH guidance of the same title dated February 2012 (M3(R2) 
Q&As). The February 2012 guidance addressed the first set of Q&As on Limit Dose for Toxicity 
Studies, Metabolites, and  Reversibility of Toxicity that was finalized under Step 4 in June 2011. 
 
In December 2011, a second set of Q&As addressing Combination Drug Toxicity Testing was 
developed and approved under Step 4 for integration in the M3(R2) Q&As.  In March 2012, an 
additional question on Limit Dose for Toxicity Studies and four additional sections addressing 
Safety Pharmacology, Exploratory Clinical Trials, Reproductive Toxicity, and Juvenile Animal 
                                                 
1  This guidance was developed within  the Expert Working Group (Multidisciplinary) of the International 
Conference on 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 Steering Committee at Step 4  of the ICH process, December 2011 and March 2012.  
At Step 4  of the process, the final draft is recommended for adoption to the regulatory bodies of the European 
Union, Japan, and the United States.  
2  Arabic  numbers reflect the organizational  breakdown in the document endorsed  by the ICH Steering Committee at 
Step 4 of  the ICH process,  June 2011.  
 
1 
  
Studies were approved under Step 4 for integration in the (M3(R2) Q&As.  This revised guidance 
incorporates the changes approved in December 2011 and March 2012. 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities. Instead, guidances describe the Agencys current thinking on a topic and should 
be viewed only as recommendations, unless specific regulatory or statutory requirements are 
cited. The use of the word should in Agency guidances means that something is suggested or 
recommended, but not required.  
 
 
II. 	 QUESTIONS AND ANSWERS 
 
A. 	 Limit Dose for Toxicity Studies (1) 
 
Q1: 	 Can you provide a definition of a 50-fold clinical exposure margin in terms of 
how it is calculated and whether it relates to the intended therapeutic clinical 
exposure or the maximum exposure achieved in phase 1 trials? 
 
A1: 	 Generally, the exposure margins should be calculated using the group/cohort 
mean area under the curve (AUC) values for animals at the highest dose tested 
and for humans at the anticipated therapeutic exposure.  In some special cases, 
based on prior knowledge of the compound class, exposure limits based on C  
max 
(maximum plasma concentration) might also be appropriate (e.g., if it is suspected 
that the drug could cause seizures). 
 
Using the 50-fold approach, the high dose in the toxicity studies should be 
selected to produce a 50-fold exposure margin over the anticipated clinical 
exposure at the highest dose proposed for phase 2 and 3 studies (see the exception 
for phase 3 trials in the United States (ICH M3(R2) guidance, section I.E High 
Dose Selection for General Toxicity Studies (1.5)), and the answers to Q2 and Q3 
of this section). For phase 1 clinical trials, it is recognized that the therapeutic 
exposure generally will be exceeded and smaller margins are appropriate (for 
example, see answers to Q2 and Q3). 
 
Q2: 	 When using the 50-fold exposure approach and there are no adverse findings in 
the rodent and nonrodent toxicity studies, if the clinical dose is escalated up to 
the agreed limit (1/50th of the exposure achieved at the top dose in animal 
studies) and there are no adverse findings in humans, is it possible to escalate 
the clinical dose further?  
 
A2: 	 In this situation, if the clinical dose is escalated to 1/50th of the maximum 
exposure in the animal studies and no treatment-related adverse effects are noted 
in volunteers/patients, for short-term clinical studies (e.g., 14 days duration) the 
2 
  
clinical dose could be cautiously further escalated up to 1/10th of the maximum 
exposure in the animal studies, or to a dose that produces adverse effects in 
humans, whichever occurs first.  This  is reasonable because exploratory trials 
Approach 4 (not intended to evaluate a maximum tolerated dose (MTD) supports 
dosing for 14 days up to 1/10th the NOAEL (no observed adverse event level) 
exposure with the same First-In-Human enabling toxicity studies. 
 
Q3: 	 When toxicity study doses are selected by using the 50-fold exposure approach 
and there are adverse findings in at least one of the toxicity studies, but the 
findings are not dose-limiting, what is the limitation for clinical exposure? 
 
A3: 	 Doses might be escalated in the clinical studies based on the NOAEL for the 
adverse findings identified in the toxicity studies.  The clinical doses should not 
be limited by the 50-fold margin in this case but should be based on standard risk 
assessment approaches (e.g., whether the findings are reversible and/or 
monitorable, the severity of the indication, adverse effects in clinical studies). 
Note the exception for phase 3 trials in the United States (section I.E (1.5) of ICH 
M3(R2)). 
 
Q4: 	 Does the 50-fold exposure limit only apply to small molecules?  
 
A4: 	 Yes, the 50-fold margin of exposure limit dose applies to small molecules only.  
As stated in section I.C (1.3) of ICH M3(R2), the guidance only applies to 
biologics with regard to timing of nonclinical studies relative to clinical  
development.  High dose selection for nonclinical studies of biologics is different 
from that for small molecules (see ICH S6(R1)3). 
 
Q5: 	 When making a maximum feasible dose (MFD) argument, to what lengths 
should the sponsor go to justify the MFD? 
   
A5: 	 The MFD should be a dose that attempts to maximize exposure in toxicity studies, 
rather than maximize the administered dose.  However, formulation volumes that 
can be administered should be based on anatomical and physiological attributes of 
the test species and properties of the formulation, and can have an impact on the 
MFD. In addition, the chemical and physical stability of the formulation are 
important criteria for suitability for use in toxicity studies and could limit the 
selection of vehicles for determining the MFD. Solubility limits can restrict the 
dose for some routes, such as intravenous. Solubility limits are not usually 
considered sufficient to justify the MFD for some other routes of administration, 
such as inhalation or oral.  The characteristics of multiple formulations of the test 
                                                 
3 The ICH guidances referenced in this document are available on the FDA Drugs guidance Web page  at  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm. We update 
guidances  periodically.  To make sure you  have the most recent version of a guidance, check the Drugs  guidance  
Web page.   
3 
  
article, with a range of properties (e.g., aqueous and non-aqueous and various 
viscosities), should be investigated before dosing in animals. The most promising 
formulations (generally three) should be evaluated in animals to determine which 
formulation produces maximal exposure. The vehicles used should be well 
characterized in the scientific literature or selected based on experience (sponsor 
or regulatory agency information) to provide confidence that they will not cause 
significant toxicity under conditions of use. 
 
Q6: 	 What if dose-limiting toxicity is not identified in any species and there is only 
one nonclinical toxicity study in each species before the phase 3 study 
(regarding phase 3 recommendation for the United States)?  
   
A6: 	 The guidelines for high dose selection for general toxicity studies apply 
irrespective of the length or complexity of the drug development paradigm.  In 
accord with the recommendation to support phase 3 studies in the United States 
(see section I.E (1.5) of ICH M3(R2)), an assessment of doses up to an MTD, 
MFD or limit dose should be conducted in an attempt to identify toxicity. 
 
Q7: 	 Does the guidance on high dose selection and the 50-fold margin of clinical 
AUC apply to routes other than oral (e.g., dermal, inhalation)? 
 
A7: 	 For any drug intended to provide systemic exposure (including transdermal), the 
50-fold approach is considered appropriate.  For topical drugs intended to produce 
local effects, the high dose in topical toxicity studies should generally be based on 
the MFD or MTD and might not achieve high local concentrations or high 
systemic exposures compared to those achieved clinically. In this case, a 50-fold 
systemic margin is not relevant. 
 
For inhaled drugs with intended systemic action, the high dose in an inhalation 
toxicity study could be one that produces an AUC value of greater than or equal 
to 50-fold the clinical systemic exposure and a 10-fold margin over the calculated 
deposited lung dose. For inhaled drugs that are designed to work locally in the 
lungs, the high dose could be one that achieved a calculated deposited lung dose 
of 50 times the calculated clinical deposited lung dose and produced a 10-fold 
margin over the AUC achieved in humans at the clinical dose. 
 
Q8: 	 Does the 50-fold margin apply to juvenile animal studies?  Can the 50-fold 
margin be used to select the top dose for reproductive toxicity studies?   
 
A8: 	 Similar principles of reliance on exposure margins to limit the top dose should be 
applicable to some other types of toxicity testing, such as juvenile animal toxicity 
studies where toxicity is not anticipated.  Use of a 50-fold margin for top doses in 
reproductive toxicity studies has not been addressed; however, current ICH 
4 
  
guidance states that minimal toxicity is expected to be induced in the high dose 
dams although other factors can also limit the dose (see ICH S5(R2)). 
 
Q9: 	 What exposure limits should be applied for clinical development studies when 
the top dose for the nonclinical studies is the limit dose such as 1000 mg/kg or 
2000 mg/kg described in section 1.5 or an MFD and no toxicity is observed at 
this dose? 
 
A9: 	 The clinical dose could be conservatively escalated up to one that produced a 
plasma AUC exposure of 1/2 that seen in the animal species that gives the lowest 
exposure at the limit dose or MFD.  If there are no adverse effects in humans at 
this clinical exposure, further careful escalation might be justified based on 
risk/benefit considerations.  
 
B.  Metabolites  (2) 
 
Q1: 	 In the M3(R2) guidance, what does significantly greater mean in the 
following statement:  Nonclinical characterization of a human metabolite(s) is 
only warranted when that metabolite(s) is observed at exposures greater than 10 
percent of total drug-related exposure and at significantly greater levels in 
humans than the maximum exposure seen in the toxicity studies?  
 
A1: 	 The term  significantly greater is not meant to imply a statistically greater level. 
Differences of  2-fold in (mean) AUC are generally considered meaningful in 
toxicokinetic evaluations. Thus, characterization of metabolite toxicity would 
generally be considered adequate when animal exposure is at least 50 percent the 
exposure seen in humans.  In some cases, for example when a metabolite 
composes the majority of the total human exposure, it is appropriate for exposure 
to the metabolite in animals to exceed that in humans (see also Q12 of this 
section). In this latter case it is important to achieve a higher exposure to the 
metabolite in animals because this metabolite constitutes the bulk of human 
exposure. 
 
Q2: 	 What is the definition and calculation method of  10 percent?   
 
A2: 	 The 10 percent threshold refers to when a human metabolite comprises greater 
than 10 percent of the measured total exposure to drug and metabolites, usually 
based on group mean AUC (e.g., AUC  ). 
0-inf
  
Q3: 	 When characterization of metabolite toxicity is warranted, in what type(s) of in 
vivo nonclinical studies is it important that adequate systemic exposure to a 
metabolite be achieved? 
 
5 
  
A3: 	 It is important to have adequate exposure to the metabolite in one species used in 
the general toxicity evaluation, one species used in a carcinogenicity study when 
carcinogenicity evaluation is warranted (or one species used in an in vivo 
micronucleus study when carcinogenicity evaluation is not warranted), and one 
species used in an embryo-fetal development study. 
 
Q4: 	 Are in vitro genotoxicity studies recommended for metabolites? 
When genotoxicity assessment is warranted for a metabolite, is quantitative 
structure-activity relationship (QSAR) assessment sufficient or should 
genotoxicity studies be conducted? 
 
A4: 	   This topic is outside the scope of ICH M3(R2). 
 
Q5: 	 Is the metabolite exposure data provided from the single-dose radiolabeled 
human ADME (absorption, distribution, metabolism, and excretion)) study 
sufficient for comparison to the exposures observed in animal toxicity studies 
without evaluation of steady state levels, which cannot be done with radiolabel 
clinically? 
 
A5: 	   An evaluation of whether a metabolite is 10 percent of the total drug-related 
exposure can be based on single-dose data in humans.  It is not generally feasible 
to measure AUC of all metabolites by non-radiolabeled methods, particularly for 
those drugs that have many metabolites.  In these cases, a single-dose radiolabeled 
study provides a reasonable estimate of human total drug-related exposure and is 
an adequate basis for calculating whether a metabolite exceeds 10 percent. (A 
metabolite cannot be more than 10 percent of the total drug-related material if 
non-radiolabeled methods indicate that a metabolite is less than 10 percent of the 
parent or of any drug-related component(s).   For example, P+M +M +...M  = 
1 2 n
total; if M  is less than 10 percent of P or M  is less than 10 percent of any M, 
1 1
then M  is less than 10 percent of the total. In this case, no further assessment of  
1
that metabolite is warranted.) 
 
If during development exposure data normally collected from multiple-dose 
human studies indicate that steady state levels of a metabolite exceed 10 percent, 
then additional nonclinical evaluation of the metabolite should be considered. 
 
Generally, exposure data from nonclinical studies and single-dose clinical studies 
can be compared to determine whether further metabolite toxicity characterization 
is warranted.  For those metabolites that have been determined to exceed 10 
percent of drug-related material in humans only after repeated dosing, steady state 
levels (clinical and nonclinical) should be used to assess the adequacy of the 
exposure margins.  
  
6 
  
Q6: 	 The M3(R2) guidance says: Nonclinical characterization of a human 
metabolite(s) is only warranted when that metabolite(s) is observed at exposures 
greater than 10 percent of total drug-related exposure and at significantly 
greater levels in humans than the maximum exposure seen in the toxicity 
studies. 
 
When a human metabolite exposure is compared to the maximum exposure of 
that metabolite in toxicity studies, should it always be to the highest exposure 
achieved in the animal studies or is it more appropriate in some cases to use the 
exposure at the NOAEL, NOEL (no observed effect level), or MTD?  
 
A6:  	  Because the parent drug and metabolites contribute to the target organ toxicity 
profile observed in animals at the MTD, the exposure comparisons across species 
should be conducted at the MTD in the animal compared to the maximum 
exposure in humans at the therapeutic dose, assuming the toxicity of concern can 
be adequately monitored in humans and does not pose an unacceptable risk.  If the 
toxicity at the MTD is not monitorable in humans or poses an unacceptable risk, 
then the exposure comparison should be conducted at the NOAEL for the toxicity 
of concern. 
 
Q7: 	    When in development, should data on nonclinical metabolites be available? 
 
A7: 	 As described in  ICH M3(R2), section III Toxicokinetic and Pharmacokinetic 
Studies (3), paragraph 1, in vitro metabolism data for animals and humans should 
be evaluated before initiating human clinical trials.  Data on in vivo metabolism in 
test species and humans should be available before exposing large numbers of 
human subjects or treating for long duration (generally before phase 3).   
 
Q8: 	 Clarification is sought on metabolites that may not be of toxicological concern. 
In ICH M3(R2), what is meant by most in the phrase most glutathione 
conjugates? Would acyl glucuronides that can undergo chemical 
rearrangement be an example of a concern? What should we do about 
chemically reactive metabolites?  
 
A8: 	    Although there are relatively rare exceptions, most glutathione conjugates are 
formed by conjugation with reactive metabolites to form excretory metabolites 
that are not of toxicological concern.  Most glucuronides are not of concern, 
except those that undergo chemical rearrangement (e.g., reactive acyl 
glucuronides). Highly chemically reactive metabolites, although of toxicological 
concern, do not generally accumulate in plasma due to their short half-life. 
Generally, it is not feasible to test highly reactive metabolites independently 
because of their instability, but they are assumed to contribute to the overall 
nonclinical toxicity of the drug. 
 
7 
  
Q9: 	 Should  safety pharmacology studies be conducted for metabolites that warrant 
nonclinical characterization? 
 
A9: 	   Clinical studies assessing safety pharmacology endpoints are generally conducted 
during phase 1. These endpoints will have already been assessed in humans 
before a full characterization of the metabolites is conducted. Therefore,  
nonclinical safety pharmacology studies are generally not warranted for the 
characterization of metabolites.  However, if a safety pharmacology signal is seen 
in humans that was not predicted by nonclinical studies with the parent, then 
additional safety pharmacology studies of these human metabolites can be 
considered to better understand the mechanism (see ICH S7A and ICH S7B). 
 
Q10: 	   What does in vitro biochemical information mean in section III (3), 
paragraph 1, of ICH M3(R2)? 
 
A10: 	 In vitro biochemical information includes standard in vitro metabolic evaluation 
(e.g., cytochrome P450 (CYP) inhibition, pregnane X receptor (PXR) activation 
assays). It can include studies with hepatic microsomes/hepatocytes or studies on 
potential interactions via drug transporters. 
 
Q11: 	 What should be the design of nonclinical studies for metabolites (e.g., species, 
duration, study type)?  
 
A11: 	 This level of detail is generally out of scope for ICH M3(R2); study design should 
be considered on a case-by-case basis using scientific judgment in consultation 
with regulatory agencies. Also see answers to other questions in this section (e.g., 
Q3 and Q9). 
 
Q12: 	 Does the guidance on metabolites in  ICH M3(R2) apply to a prodrug (i.e., when 
a metabolite provides most of the pharmacologic activity)? 
 
A12: 	 The guidance does not specifically address prodrugs.  If the animal species 
converts the prodrug to the active metabolite similarly to humans, then a standard 
testing approach as recommended in ICH M3(R2) can be used.  If the active 
metabolite is not adequately produced in the animal species, then the target 
molecule for toxicological evaluation is the active metabolite and therefore 
additional testing beyond that recommended for metabolites can be appropriate. 
Timing of the nonclinical testing of the active metabolite in this case should 
follow the general timelines as outlined in ICH M3(R2) rather than the timing 
indicated for metabolite testing in section III (3) of M3(R2). 
 
C. 	 Reversibility of Toxicity (3) 
 
8 
  
Q1: 	 When is assessment of reversibility considered to be appropriate and is it 
important to demonstrate full reversibility or is it sufficient to demonstrate the 
potential for full reversibility? 
 
A1: 	 ICH M3(R2) states the following in section I.D General Principles (1.4):  The 
goals of the nonclinical safety evaluation generally include a characterization of 
toxic effects with respect to target organs, dose dependence, relationship to 
exposure, and, when appropriate, potential reversibility. 
 
Evaluation of the potential for reversibility of toxicity (i.e., return to the original 
or normal condition) should be provided when there is severe toxicity in a  
nonclinical study with potential adverse clinical impact.  The evaluation can be 
based on a study of reversibility or on a scientific assessment. 
 
The scientific assessment of reversibility can include the extent and severity of the 
pathologic lesion, the regenerative capacity of the organ system showing the 
effect and knowledge of other drugs causing the effect.  Thus, recovery arms or 
studies are not always critical to conclude whether an adverse effect is reversible.  
The demonstration of full reversibility is not considered essential.  A trend  
towards reversibility (decrease in incidence or severity), and scientific assessment 
that this trend would eventually progress to full reversibility, are generally 
sufficient.  If full reversibility is not anticipated, this should be considered in the 
clinical risk assessment. 
 
A toxicity study that includes a terminal non-dosing period is generally warranted 
if a scientific assessment cannot predict whether the toxicity will be reversible and 
if: 
1. 	  there is severe toxicity at clinically relevant exposures (e.g.,  10-fold the 
clinical exposure); or 
2. 	  the toxicity is only detectable at an advanced stage of the pathophysiology 
in humans and where significant reduction in organ function is expected. 
(The assessment of reversibility in this case should be considered even at 
> 10-fold exposure multiples.) 
 
A toxicity study that includes a terminal non-dosing period is generally not 
warranted when the toxicity: 
1. 	  can be readily monitored in humans at an early stage before the toxicity 
becomes severe; or  
2. 	  is known to be irrelevant to humans (e.g., rodent Harderian gland 
toxicity); or 
3. 	  is only observed at high exposures not considered clinically relevant (see 2 
above for exception); or 
4. 	  is similar to that induced by related agents, and the toxicity based on prior 
clinical experience with these related agents is considered a manageable 
risk. 
 
9 
  
If a study of reversibility is called for, it should be available to support clinical 
studies of a duration similar to those at which the adverse effects were seen 
nonclinically. However, a reversibility study is generally not warranted to 
support clinical trials of a duration equivalent to that at which the adverse effect 
was not observed nonclinically. 
 
If a particular lesion is demonstrated to be reversible in a short duration (e.g., 2
week or 1-month) study, and does not progress in severity in longer term studies, 
repeating the reversibility assessment in longer term toxicity studies is generally 
not warranted. 
 
If a reversibility study is warranted, it is efficient to conduct it as part of a chronic 
study so that all toxicities of concern can be assessed in a single study, provided 
that it is not critical to conduct it earlier to support a specific clinical trial. 
 
D. 	 Combination Drug Toxicity Testing (4) 
 
Q1: 	 If two (or more) late stage entities are combined but for one of them the human 
dosage/exposure will be higher than that already approved, is it important to 
conduct a combination toxicity study or are the existing nonclinical data and 
clinical experience with the lower dose considered adequate to address the 
nonclinical assessment?  
 
A1: 	 If there has been previous clinical experience with the two entities used together, 
a combination toxicity study would generally not be recommended for an increase 
in dose/exposure of one of the entities unless this gave cause for significant 
toxicological concern. The level of concern would depend on the new exposure 
margins, the established safety profile of the individual agents, the degree of 
experience with the co-administration, and the ability to monitor any potential 
adverse effects in humans.  If the increase in dose/exposure does cause concern 
and a study is conducted to address that concern, then it should generally be 
completed before carrying out clinical studies with the combination.  If there is no 
clinical experience with the entities used together, see paragraph 4 of section 
XVII (17) of ICH M3(R2). 
 
Q2: 	 Section XVII (17) of M3(R2) states, [i]f nonclinical embryo-fetal studies have 
indicated that neither agent poses a potential human developmental risk, 
combination studies are not recommended unless concerns exist, based on the 
properties of individual components, that their combination could give rise to a 
hazard for humans. Although this statement is in line with European 
Medicines Agency (EMA) guidance, it contradicts FDA guidance that states 
that embryo-fetal development studies of the combination should be conducted 
unless the marketed drug substance or the new molecular entity (NME) is 
already known to have significant risk for developmental toxicity (e.g., the 
10 
  
marketed drug has been assigned a pregnancy category D or X). Please 
provide clarity regarding the precedence of ICH guidance over regional  
guidances in those areas where such differences occur. 
 
A2: 	 Statements made in ICH guidances represent an agreed position across the 
participating bodies and reflect each regulatory bodys current recommendations 
on a given topic. 
 
Q3: 	 The current guidance states that for combinations of late stage products for 
which there is adequate clinical experience of co-administration, combination 
toxicity studies are generally not recommended unless there is a significant 
toxicological concern.  In this context, what is considered adequate clinical 
experience with co-administration?  Specifically, how do you get adequate 
clinical experience with the combination without having done combination 
toxicity testing? This guidance seems only to apply to marketed products that 
have been used together. Was that the intent? 
 
A3: 	 This section of the guidance was not intended to only apply to marketed products. 
Adequate clinical experience is defined in ICH M3(R2) as data from phase 3 
clinical studies and/or postmarketing use.  Adequate clinical experience can be the 
result of common clinical practice with drug combinations. 
 
Co-administration of two or more late stage entities is a common practice in many 
therapeutic areas of clinical development where add-on therapy to the standard of 
care or combination therapy is common, such as with hypertension, diabetes, 
human immunodeficiency virus (HIV), hepatitis C, and cancer.  
  
Q4: 	 For non-fixed-dose combinations, if one of the agents is a member of a class 
containing multiple approved products, should each member of the class be 
tested in a combination toxicity study? 
 
A4: 	 Generally, combination toxicity studies are recommended when there is an intent  
to combine (co-package or administer in a single dosage form) specific drugs, or 
when the product information of one drug recommends co-use with another 
specified drug. There is no recommendation for combination toxicity testing in 
the guidance for the situation described in this question.  When there is a specific 
cause for concern with an agent, combination toxicity testing should be done with 
the agent.  When there is a class-related cause for concern, a combination toxicity 
study with a representative agent in the class could be informative (see also 
Q&A3 in this section). A rationale should be provided for the agent selected for 
testing. 
 
Q5:   How are dosage, duration, and endpoint of a combination toxicity study 
selected? 
11 
  
 
A5: 	 ICH M3(R2) is intended primarily to address the timing and duration of 
nonclinical studies relative to clinical development.  Provided a nonclinical 
combination toxicity study is warranted to support the combination clinical trial, 
the duration of the study should be equivalent to that of the clinical trial it is 
intended to support, up to a maximum of 90 days (which would also support 
marketing).  A combination study of shorter duration can be used to support 
marketing, depending on the duration of clinical use.  A combination toxicity 
study intended to address a particular cause for toxicological concern, based on 
the experience with the individual agents, should be of a duration that is 
appropriate to address the concern. 
  
The combination toxicity study should incorporate endpoints to evaluate additive 
and synergistic effects for known toxicities that might be predicted from what is 
known of the pharmacological, toxicological, and pharmacokinetic (PK) profiles 
of the individual entities, as well as the available clinical data, and standard 
endpoints typically used in a general toxicity study. Detailed discussion of 
experimental design (e.g., choice of species, dose and dosing frequency 
justifications)  is outside the scope of this guidance.  However, dosages should be 
appropriate to address any identified cause for concern or to provide exposure 
margins that are clinically relevant (e.g., when conducting a study with two early 
stage agents). 
 
Q6: 	 When there is a cause for concern for multiple entities being used together (e.g., 
more than two), how should the multiple entity combinations be assessed in the 
toxicity studies? 
 
A6: 	 Because of the potential complexity of performing and interpreting a combination 
toxicity study with more than two entities, it is generally more practical for initial 
studies to evaluate combinations of no more than two entities. Additional testing 
would then depend on the outcome of these studies and should be considered on a 
case-by-case basis, and in consultation with appropriate regulatory authorities. 
 
Q7: 	 If a compound is being developed that aims to reduce another compounds side 
effect, such combination effects would be evaluated in clinical or nonclinical 
pharmacology studies.   Do the pharmacology studies replace the combination 
toxicity study? 
  
A7: 	 When combination toxicity studies are warranted, they generally cannot be 
replaced by combination pharmacology studies, except for anticancer 
pharmaceuticals (see ICH S9).  The purpose of a combination toxicity study is to 
evaluate toxicity endpoints that could give rise to an unanticipated hazard for 
humans.  These toxicity endpoints are not usually adequately evaluated in the 
pharmacology studies.  Situations where combination toxicity studies are not 
warranted are described in section XVII (17) of the M3(R2) guidance. 
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Q8: 	 Section XVII (17) of M3(R2) indicates that if there is a concern for a potential 
human developmental risk of a combination and a combination embryo-fetal 
development study is warranted, such a study should be available to support the 
marketing application.  Please clarify whether such a study should be 
performed before the commencement of a clinical trial that includes women of 
childbearing potential (WOCBP). 
 
A8: 	 As described in the M3(R2) guidance, the combination embryo-fetal toxicity 
study should be provided to support marketing.  Any trial enrolling WOCBP 
before completing a combination embryo-fetal development study should include 
appropriate precautions, including informed consent, to minimize the risk of 
unintentional exposure of the embryo or fetus as outlined in section XI.C (11.3). 
 
Q9: 	 In ICH M3(R2), section I.C Scope of the Guidance (1.3) states that 
[p]harmaceuticals under development for indications in life-threatening or 
serious diseases (e.g., advanced cancer, resistant HIV infection, and congenital 
enzyme deficiency diseases) without current effective therapy also warrant a 
case-by-case approach to both the toxicological evaluation and clinical 
development in order to optimize and expedite drug development.  Although 
not specifically stated in the combination section of M3(R2) (see section XVII 
(17)), it is generally accepted that combination toxicity studies on advanced 
cancer and HIV products are not warranted unless there is a specific cause for 
concern. Can this be confirmed? Would this also extend to hepatitis C virus 
(HCV) products, as discussed in the recently issued FDA draft  guidance for 
industry on Chronic Hepatitis C Virus Infection:  Developing Direct-Acting 
Antifiral Agents for Treatment (September 2010),4 and to other therapeutic 
areas where cocktails of drugs are standard clinical practice? 
 
A9: 	 It is accepted that combination toxicity studies on advanced cancer, tuberculosis, 
and HIV products are generally not warranted unless there is a specific cause for 
concern under clinically relevant conditions.  Combination toxicity studies are 
also not generally warranted for antiviral agents for treatment of hepatitis C.  
There are other situations where combinations of drugs are standard clinical 
practice for serious or life-threatening conditions without current effective 
therapies, and a similar approach might also apply. 
 
Q10: 	 In case of combinations with at least one biotechnology-derived product, does 
section XVII Combination Drug Toxicity Testing (17) apply as such, or only 
with regard to timing as suggested in  section I.C (1.3) on the scope of the 
M3(R2) guidance? And in case of the latter, which guidance would (still) apply 
in deciding whether and which types of studies would be recommended? 
 
                                                 
4  When final, this guidance will represent FDAs current thinking on this topic. 
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A10:  	For biotechnology-derived products, appropriate nonclinical safety studies should 
be determined in accordance with ICH S6(R1).  However, the topic of 
combination toxicity studies is not addressed by that guidance.  When the 
combination consists of a biotechnology-derived component and a non
biotechnology-derived component, the design and feasibility of any nonclinical 
combination study are complex and should be considered on a case-by-case basis. 
The rationale for such a study should be clearly scientifically justified, using the 
principles of ICH S6(R1) and ICH M3(R2). 
  
Q11: 	 In the discussion of inclusion of WOCBP in combination drug development, 
M3(R2) states, where . . . individual agent(s) have shown findings indicative of 
embryo-fetal risk, combination studies are not recommended as a potential 
human developmental hazard has already been identified.  What is meant by 
the phrase have shown findings indicative of embryo-fetal risk?  FDA's 
guidance for industry on Nonclinical Safety Evaluation of Drug or Biologic 
Combinations references Pregnancy Category D and X only as yielding this 
exclusion. Is this the intent for the ICH as well?  
 
A11: 	 A finding indicative of embryo-fetal risk includes any observations for 
reproductive hazard at relevant exposure multiples (within approximately an order 
of magnitude of the clinical exposure) or directly related to the 
pharmacodynamics of the drug.  In these cases, recommendations about patient 
actions to minimize the identified hazard would likely be unchanged even if data 
from a combination study showed an enhanced effect, because a significant risk to 
patients has already been identified.  Therefore, combination reproductive toxicity 
studies are not recommended when a finding with one of the individual agents 
indicates embryo-fetal risk; that information would be made available to patients 
and physicians as part of the risk communication, irrespective of pregnancy 
category. For example, if studies with one of the agents showed fetal death or 
terata at approximately 10-fold the clinical exposure, even if observed in only one 
species, a combination study would not be warranted, provided that this 
information was present in the single agent product labeling. 
 
Q12: 	 There is no mention about pharmacology studies, and pharmacodynamic or 
pharmacokinetic drug-drug interaction studies in section XVII (17).  Please 
indicate whether and when these studies should be conducted. 
 
A12: 	 Presumably, the pharmacodynamic activities and pharmacokinetic profile, 
including the effects on the CYP450s of the individual drugs, are known before 
the drugs are combined.  Because potential pharmacodynamic interactions are 
anticipated based on the nonclinical and clinical experiences with the individual 
entities or their combination, no nonclinical pharmacodynamic interaction studies 
are warranted.    If the pharmacology information indicates potential interactions 
that could lead to toxicity, then combination nonclinical toxicity studies might be 
warranted. 
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Concerns regarding pharmacokinetic interactions can often be addressed by 
lowering the initial doses administered below those that might be appropriate for 
the individual drugs or by conducting a clinical pharmacokinetic drug-drug 
interaction study. 
 
E. 	 Safety Pharmacology (5) 
 
Q1: 	 ICH M3(R2) states that including the in vivo safety pharmacology evaluations 
in toxicity studies to the extent feasible should be considered.  Does this mean 
that the safety pharmacology assessment conducted as part of general toxicity 
studies can be less thorough than that obtained in stand-alone safety 
pharmacology studies? 
 
A1: 	 No. Assessment of safety pharmacology as part of the general toxicity studies 
should provide rigor similar to that in stand-alone safety pharmacology studies. 
This can be achieved with current technology, provided the methods have been 
adequately assessed. 
 
F. 	 Exploratory Clinical Trials (6) 
 
Q1: 	 To support exploratory clinical trials, why should the extended single-dose 
studies be done in both sexes when the clinical exploratory studies are likely to 
be done in one sex? 
 
A1: 	 Exploratory clinical studies do not represent a commitment to full development.  
Therefore, when intent is to conduct the exploratory clinical study in one sex 
only, the single-dose toxicity studies can be restricted to that sex.  However, in 
such cases, animal group sizes for the Day 2 termination should be increased, as it 
is normal to combine effects from both sexes with respect to identifying and 
characterizing toxicities that are not sex-specific.  For extended single-dose 
toxicity studies using a single sex, the usual animal numbers should be 15/group 
(rodents) or 5/group (nonrodents) for the Day 2 termination, and 7/group (rodents) 
or 3/group (nonrodents) for the Day 14 termination. 
 
Q2a: 	 Please clarify the differences between Approaches 3, 4, and 5. 
 
A2a: 	 Approach 3 involves just a single dose in humans supported by extended single-
dose toxicity studies in rodents and nonrodents conducted up to the animal 
maximum tolerated dose (MTD), maximum feasible dose (MFD), or limit dose. 
 
Approach 4 involves multi-dose clinical trials (up to 14 days) supported by 14
day toxicity studies (in rodents and nonrodents) in which dose selection for 
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animals is based on multiples of proposed human exposure in the exploratory 
clinical trial.  If no toxicity is observed in either species, it is recommended that 
the maximum clinical dose not exceed 1/10th the lower exposure (AUC) in either 
species at the highest dose tested in the animals.  If toxicity is observed, see 
answer A2b below. 
  
Approach 5 involves multi-dose clinical trials (up to 14 days) supported by a 14
day study in rodents up to the MTD, MFD, or limit dose and a nonrodent 
confirmatory study (at least equivalent to the duration of the exploratory clinical 
trial) that indicates that the nonrodent is not more sensitive than the rodent.  In 
this case, the highest exposure appropriate in the exploratory clinical trial should 
be determined by the findings in the toxicity studies. 
  
Thus, the differences between Approach 4 and Approach 5 include how the 
standard nonclinical toxicity study recommendations are modified, and how the 
clinical exposure limit is established.   Approach 5 probably uses less drug than 
Approach 4, but relies heavily on the rodent for identifying safety risks.  
Approach 4 gives equal weight to the rodent and nonrodent, but might not identify 
target organ toxicity in either species.  In this case, clinical progression is 
supported by the knowledge that a reasonable safety margin exists. 
 
The series of examples are intended to provide sponsors flexibility in exploratory 
clinical trial approaches so that they can do what best fits their purpose.  The 
approaches given are only examples, and sponsors can propose alternatives that 
do not fit neatly into one of the described approaches. 
 
Q2b: 	 Why does Approach 4 have a more stringent maximum clinical dose than 
Approaches 3 and 5? 
 
A2b: 	 Approach 4 is the only one of these approaches that does not rely on the standard 
high-dose criteria described in section I.E (1.5) (MTD, MFD, 50X exposure 
multiple, or limit dose) in at least one species. 
 
In Approach 4, the high dose in both the rodent and nonrodent studies is based on 
multiples of the proposed human exposure, and thus the high-dose selection 
recommendations described in section I.E (1.5) are not applied to either species.  
This is in contrast to Approach 3, in which the standard high-dose criteria should 
be met in both species, and in contrast to Approach 5, in which the standard high-
dose criteria should be met in rodents.  In Approach 3 and Approach 5, the use of 
standard high-dose selection criteria reduces the uncertainty around potential 
unidentified toxicities that might be relevant to humans. 
 
Because Approach 4 uses exposure multiples for the high-dose selection in both 
species, it is possible that potential toxicity might not be identified in either 
species. In this case, more conservative limits on clinical exposure (e.g., 1/10th  
the exposure obtained using the lower exposure of the two species) are 
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recommended because the dose-limiting toxicities of potential concern for clinical 
monitoring have not been identified. If toxicity is identified in one species, then 
the limit on clinical exposure is based on the No Observed Adverse Effect Level 
(NOAEL) exposure in the species with toxicity or 12 the NOAEL exposure in the 
species without toxicity, whichever is lower.  This  can yield a higher limit in 
Approach 4 than in the case where toxicity in neither species has been observed. 
The limit on clinical exposure for Approach 4 when based on toxicity can be 
comparable to the limit on clinical exposure in Approach 5.  If dose-limiting 
toxicity is identified in both species using Approach 4, then the high-dose 
recommendations of section I.E (1.5) have been met or exceeded in both species 
and a maximum clinical dose can be based on standard risk assessment used for 
phase 1 trials and a clinical MTD can be explored. 
   
Q2c: 	 In cases where toxicity is demonstrated (e.g., Approaches 3 and 5) why is the 
maximum allowable human dose (equal to or 1/2 the NOAEL) different from 
usual practice ( i.e., (1) where toxicity is nonserious and/or monitorable, human 
doses above the NOAEL would normally be allowed, and (2) where toxicity is 
serious and non-monitorable the maximum human dose would usually be 
limited to 1/10th the NOAEL). 
 
A2c: 	 The more stringent limits on maximum exposure in exploratory clinical trials 
compared to standard phase 1 trials are consistent with the more limited 
nonclinical recommendations compared with the standard toxicity study 
recommendations described in section I.E (1.5) and section V (5) in M3(R2).  For 
example, in Approach 3, extended single-dose studies are recommended rather 
than the typical recommendation of a study of at least 2 weeks duration (see 
Table 1 and Approach 5); the nonrodent study is only confirmatory in nature and 
can be limited to 3 animals at a single-dose level targeted to be a NOAEL.  The 
recommendation that the maximum human exposure allowed could be up to 1/2  
the NOAEL exposure assumes that the toxicity defining the NOAEL is not severe 
and is monitorable.  If this is not the case, it might be appropriate to adjust the 
exposure margin based on the nature of the dose-limiting toxicity. 
 
Q3: 	 Why is an MFD treated like an MTD in Approaches 3 and 5 when considering 
the maximum clinical exposure in the exploratory clinical trial?  If no toxicity 
is observed in either species when using an MFD, shouldnt this be treated 
similarly to the case in Approach 4 when there is no toxicity in either species 
(i.e., limit the clinical exposure to 1/10th rather than 1/2 the exposure at the 
highest dose tested)? 
 
A3: 	 In any situation in which the MFD is used as the top dose for a toxicity study, it is 
simply not possible to test a higher dose/exposure.  If the top dose used is the 
MFD and no toxicity is observed, this situation is similar to that of the limit dose 
when toxicity has not been identified (i.e., the limit dose is the NOAEL) where 
clinical exposures up to 1/2 the AUC at the NOAEL can be used (see section I.E 
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(1.5) of the M3(R2) guidance, and section II.A (1) of this Q&A guidance, Limit 
Dose for Toxicity Studies, Q&A9). The 1/10th exposure limit is not applied when 
the high dose is limited by an MFD, because this could prevent adequate clinical 
testing of a drug under the exploratory clinical trial concept.  When no toxicity is 
identified using Approach 4, a more stringent safety limit has been recommended 
because it would have been possible to test higher doses in animals to characterize 
the toxicity profile of the drug. 
 
Q4: 	 The M3(R2) guidance provides advice on establishing the maximum dose 
(exposure) permitted in exploratory clinical trials but provides minimal 
guidance for establishing the maximum dose in standard phase 1 or clinical 
development trials.  Can the maximum dose in standard phase 1 trials be based 
on the principles described for exploratory clinical trials (Table 3 of M3(R2))? 
 
A4: 	 When the package of nonclinical studies meets the general recommendations of 
section V.A (5.1) of ICH M3(R2), then the maximum clinical dose for a clinical 
development phase 1 study can be based on standard risk assessments (e.g., 
whether the findings are reversible and/or monitorable, the severity of the 
indication, adverse effects in clinical studies; also see section VI (6) of ICH 
M3(R2) and regional guidances). This would normally support a higher clinical 
dose than that recommended for exploratory clinical trials.  However, a sponsor 
has the option to set a lower maximum clinical dose for a phase 1 study (e.g., 
based on the principles described for exploratory approaches). 
 
Q5: 	 What are reasonable strategies for exploratory clinical trials with 
biotechnology-derived products? 
  
A5: 	 Exploratory clinical trial approaches can be applicable to biotechnology-derived 
products. Biotechnology-derived products include a wide variety of molecular 
structures and targets (e.g., peptides, polypeptides, therapeutic proteins, and 
monoclonal antibodies). The designs of the exploratory clinical trial and 
supporting toxicity studies for biotechnology-derived products should reflect their 
special features as described in ICH S6(R1).  This includes the duration of 
exposure, the potential for immunogenicity in animals or humans, and the 
possibility that dose-limiting toxicity might be due to on-target, 
pharmacodynamic-related mechanisms.  ICH S6(R1) recommends that 
exploratory clinical trial approaches be discussed with the appropriate regulatory 
authorities. 
 
Note that some biotechnology-derived products, for example monoclonal 
antibodies, are not active in rodents, and in such cases a nonhuman primate can be 
used as a single relevant species for toxicity testing.  In such cases, an approach 
analogous to Approach 5 would not be applicable because it relies on a rodent 
toxicity study and confirmatory nonrodent study.  Also, for standard toxicity 
studies of biotechnology-derived products, the high dose is routinely based on 
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exposure multiples (i.e., 10X the maximum clinical exposure) rather than on an 
MTD, an MFD (unless these are lower), or a limit dose.  Thus, the high dose 
recommendation in Approach 4 is not substantially different from the standard 
recommendations for biotechnology-derived products. 
   
Q6: 	 In exploratory Approaches 1 and 2 that use doses of < 100 micrograms (g), 
why is the cross-species exposure conversion for intravenous administration 
based on milligram (mg)/kilogram (kg) and not mg/square meter (m2) as it is for 
oral administration? 
 
A6: 	 The intravenous (i.v.) approach of using mg/kg and permitting dosing with 1/100th  
of the NOAEL reflects a conservative risk mitigation strategy, considering the 
low levels of drug being administered.  The use of mg/kg for i.v. and mg/m2 for 
oral administration when determining dose multiples for microdose studies 
reflects the thinking that it is appropriate to use a more conservative scaling factor 
for oral versus i.v. administration.  With oral administration, there is the 
additional complexity of potential differences in absorption between species and, 
therefore, the more conservative mg/m2 basis was used rather than the mg/kg 
basis used for i.v. administration. 
   
Q7: 	 For Approach 1, the M3(R2) guidance says: 
(a) Total dose  100 g (no inter-dose interval limitations) AND Total dose   
1/100th NOAEL and  1/100th pharmacologically active dose (scaled on 
mg/kg for i.v. and mg/m2 for oral). 
But it also says: 
(b) Extended single-dose toxicity study (see footnotes c and d) in one species, 
usually rodent, by intended route of administration with toxicokinetic data, 
or via the i.v. route. A maximum dose of 1000-fold the clinical dose on a 
mg/kg basis for i.v. and mg/m2 for oral administration can be used. 
It is unclear whether the margin of exposure should be 100-fold the NOAEL or 
1000-fold. 
 
A7:  	The 1/100th the NOAEL in the animals is one of the criteria that could limit the 
clinical dose. Statement Q7(b) above refers to defining a limit dose for testing in 
animals for the microdose approaches rather than a clinical margin based on dose. 
  
Q8: 	 For positron emission tomography (PET) tracers, please confirm that for 
Approaches 1 and 2, toxicokinetics (TK) is not needed for either oral or i.v. 
administration. 
 
A8: 	 A nonclinical toxicity study conducted to support a clinical microdose trial should 
include TK assessment unless the study is conducted by the intravenous route.  
This is to demonstrate that systemic exposure has occurred.  However, it is 
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recognized that for some PET tracers, the clinical microdose can be very low and 
in such cases it might not be possible to characterize a full TK profile. 
 
Q9: 	 What chemistry, manufacturing, and control (CMC) information should be 
available for an exploratory clinical trial? 
 
A9: 	 CMC information for exploratory clinical trials was not addressed in ICH 
M3(R2). Consult appropriate regulatory authorities and regional guidances.  
 
Q10: 	 Does evaluation of potential mutagenic impurities (e.g., structure-activity 
relationship (SAR) or testing) apply to exploratory clinical trial support? 
  
A10: 	 The drug substance should be considered appropriate from a CMC perspective. 
For approaches 1 and 2 (microdose studies), SAR or genotoxicity testing is not 
recommended for the parent drug or for the impurities.  For other exploratory 
clinical trial approaches where higher doses and longer treatments are used, 
available guidance on mutagenic impurities should be followed. 
 
G. 	 Reproductive Toxicity (7) 
 
Q1a: 	 Endnote 4: In the preliminary embryo-fetal developmental study, what is the 
definition of adequate dose levels? Does this mean maternal toxicity at least 
one dose level? If only one or two dose levels have surviving fetuses, would that 
be adequate?   
 
A1a: 	 The same dose selection criteria used for a definitive embryo-fetal development 
study should be used for the preliminary study (see ICH S5).  
 
Q1b: 	 Endnote 4: The text specifies a minimum of six dams per group.  Does this 
mean a minimum of six litters per group should be evaluated? 
 
A1b: 	 No. Sometimes pregnant females have total loss of litters.  Dosing should be 
initiated with a minimum of  six presumed pregnant females per group, with all 
surviving litters evaluated. 
 
Q1a:  Are embryo-fetal development studies or the demonstration that the drug and/or 
metabolites do not partition into semen important for male-only products? 
  
A1a: 	 The ICH M3(R2) guidance does not address recommendations for embryo-fetal 
development studies in products intended for use only in males.  Embryo-fetal 
development studies for a male-only drug should be considered on a case-by-case 
basis. 
20 
  
 
Q2b:  	 Should contraception be used in male-only studies until reproductive risks have 
been evaluated?  
 
A2b: 	 It is general practice to use contraception in males until the potential for 
reproductive and developmental risk has been addressed.  
 
H. 	 Juvenile Animal Studies (8) 
 
Q1: 	 What is the appropriate duration of treatment for a toxicity study using juvenile 
animals to address a specific issue of concern? 
 
A1: 	 Specific aspects of the design of juvenile toxicity studies are outside the scope of 
ICH M3(R2). However, in general, the duration of such a study will depend on 
the toxicity to be addressed, the organ system involved, and the information 
available from previous studies. The design and duration of the study should 
address the concerns for the products potential to affect the developing organ 
systems of the intended clinical population.   
 
To reduce animal use, the specific issue of concern can sometimes be evaluated 
by incorporating developmental endpoints into a general repeated-dose toxicity 
study or into a pre/postnatal toxicity study in which the pups were adequately 
exposed to the drug. 
 
Q2: 	 Clarify when a second species might be important. 
The guidance states that when a juvenile animal toxicity study is warranted, one 
relevant species (preferably rodents) is generally considered adequate.  It may 
be difficult to prospectively describe the majority of instances in which a second 
study in another species is scientifically justified, but can parameters be 
described that are not reasonable justifications?   
 
A2: 	 There are few circumstances for which juvenile animal studies in two species 
would be recommended besides (1) an absence of adult human data (i.e., a 
pediatric-only indication), or (2) there are multiple specific issues of  
developmental concern and no one species is able to address them adequately. 
Some situations for which a juvenile study in a second species is not warranted 
include the following:  solely because a therapeutic is first-in-class, when 
verifying adverse findings in a juvenile study in one species, or when further 
examining behavioral effects of agents for which such effects are known or can be 
expected. 
 
Q3: 	 Please clarify what is important for pediatric-only indications.  Should a 
juvenile animal study be conducted to support a pharmacokinetic (PK) study in 
21 
  
pediatric populations if you dont have any adult data?  Should a second species 
be studied? 
 
A3: 	 Generally, data from adult human volunteers and the supporting nonclinical data 
(in two species) should be available before pediatric clinical trials are initiated, 
even when the product is not intended for development in adults.  Section XII 
(12) of ICH M3(R2), Clinical Trials in Pediatric Populations, generally provides 
recommendations for the situation in which adult clinical trials should precede 
pediatric trials and indicates that juvenile animal toxicity studies are not 
considered important to support short-term PK trials in pediatric populations.  
However, if data from adult humans are not available and the drug will be  
developed only for pediatric subjects, then this is a case where juvenile animal 
studies in two species would be appropriate to support pediatric PK trials. 
 
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