Guidance for Industry 
 
M3(R2) Nonclinical Safety 
Studies for the Conduct of 
Human Clinical Trials and 
Marketing Authorization for 
Pharmaceuticals 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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) 
 
January 2010 
ICH   
 
Revision 1
   
 
 
Guidance for Industry 
 
M3(R2) Nonclinical Safety 
Studies for the Conduct of 
Human Clinical Trials and 
Marketing Authorization for 
Pharmaceuticals 
 
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http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.   
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Drug Evaluation and Research (CBER) 
  
January 2010 
ICH 
 
Revision 1 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
List of Abbreviations 
 
I.  INTRODUCTION (1).......................................................................................................1 
A.  Objectives of the Guidance (1.1)...................................................................................................1 
B.  Background (1.2)............................................................................................................................2 
C.  Scope of the Guidance (1.3)...........................................................................................................2 
D.  General Principles (1.4).................................................................................................................3 
E.  High Dose Selection for General Toxicity Studies (1.5)..............................................................3 
II.  PHARMACOLOGY STUDIES (2).................................................................................6 
III.  TOXICOKINETIC AND PHARMACOKINETIC STUDIES (3)...............................6 
IV.  ACUTE TOXICITY STUDIES (4).................................................................................6 
V.  REPEATED-DOSE TOXICITY STUDIES (5).............................................................7 
A.  Clinical Development Trials (5.1).................................................................................................7 
B.  Marketing Authorization (5.2)......................................................................................................9 
VI.  ESTIMATION OF THE FIRST DOSE IN HUMANS (6)............................................9 
VII.  EXPLORATORY CLINICAL TRIALS (7).................................................................10 
A.  Microdose Trials (7.1)..................................................................................................................10 
B.  Single-Dose Trials at Subtherapeutic Doses or Into the Anticipated Therapeutic Range (7.2)
  11 
C.  Multiple Dose Trials (7.3)............................................................................................................11 
VIII.  LOCAL TOLERANCE STUDIES (8)..........................................................................16 
IX.  GENOTOXICITY STUDIES (9)...................................................................................16 
X.  CARCINOGENICITY STUDIES (10).........................................................................16 
IX.  REPRODUCTION TOXICITY STUDIES (11)...........................................................17 
A.  Men (11.1).....................................................................................................................................17 
B.  Women Not of Childbearing Potential (11.2)............................................................................17 
C.  Women of Childbearing Potential (11.3)...................................................................................17 
D.  Pregnant Women (11.4)...............................................................................................................18 
XII.  CLINICAL TRIALS IN PEDIATRIC POPULATIONS (12)....................................19 
XIII.  IMMUNOTOXICITY (13).............................................................................................20 
XIV.  PHOTOSAFETY TESTING (14)..................................................................................20 
XV.  NONCLINICAL ABUSE LIABILITY (15)..................................................................20 
XVI.  OTHER TOXICITY STUDIES (16).............................................................................21 
  i Contains Nonbinding Recommendations 
 
XVII.     COMBINATION DRUG TOXICITY TESTING (17).............................................21 
XVIII.      CONTINUING EFFORTS TO IMPROVE HARMONIZATION (18)................23 
XIX.  ENDNOTES (19).............................................................................................................24 
XX.  REFERENCES (20)........................................................................................................25 
  ii Contains Nonbinding Recommendations 
 
LIST OF ABBREVIATIONS 
 
AUC  Area Under the Curve 
C   Maximum Plasma Concentration 
max
EU  European Union 
GLP  Good Laboratory Practices 
HCG  Human Chorionic Gonadotropin 
HIV  Human Immunodeficiency Virus 
ICH  International Conference on Harmonisation of Technical Requirements for 
Registration of Pharmaceuticals for Human Use 
i.v.  Intravenous 
MFD  Maximum Feasible Dose 
MTD  Maximum Tolerated Dose 
NOAEL  No Observed Adverse Effect Level  
PET  Positron Emission Tomography 
PD  Pharmacodynamics 
PK  Pharmacokinetics 
SAR  Structure-Activity Relationship   
siRNA  Small Interfering RNA  
WOCBP   Women of Childbearing Potential 
  i Contains Nonbinding Recommendations 
 
1
Guidance for Industry  
 
M3(R2) Nonclinical Safety Studies for the Conduct of Human 
Clinical Trials and Marketing Authorization for Pharmaceuticals 
 
 
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.  INTRODUCTION (1)2  
 
A.  Objectives of the Guidance (1.1) 
 
The purpose of this document is to recommend international standards for, and promote 
harmonization of, the nonclinical safety studies recommended to support human clinical trials 
of a given scope and duration as well as marketing authorization for pharmaceuticals. 
Harmonization of the guidance for nonclinical safety studies will help to define the current 
recommendations and reduce the likelihood that substantial differences will exist among 
regions. 
 
This is a revision of the ICH guidance M3 Nonclinical Safety Studies for the Conduct of 
Human Clinical Trials for Pharmaceuticals that published in 1997.  The revisions further 
harmonize recommendations in a number of areas and include a new section on exploratory 
clinical studies.  This revised guidance discusses other nonclinical studies that should be 
conducted on a case-by-case basis as appropriate, including phototoxicity studies, 
immunotoxicity studies, juvenile animal toxicity studies, and abuse potential studies.   
 
This guidance should facilitate the timely conduct of clinical trials, reduce the use of animals 
in accordance with the 3R (reduce/refine/replace) principles, and reduce the use of other drug 
                                                 
1 This guidance was developed within the Expert Working Group (Multidisciplenary) 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, June 2009.  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, [date].  
 
  1 Contains Nonbinding Recommendations 
 
development resources.  Although not discussed in this guidance, consideration should be 
given to use of new in vitro alternative methods for safety evaluation.  These methods, if 
validated and accepted by all ICH regulatory authorities, can be used to replace current 
standard methods. This guidance promotes safe, ethical development and availability of new 
pharmaceuticals. 
 
B.  Background (1.2) 
 
The recommendations of this revised guidance further harmonize the nonclinical safety 
studies to support the various stages of clinical development among the regions of the 
European Union (EU), Japan, and the United States.  The present guidance represents the 
consensus that exists regarding the type and duration of nonclinical safety studies and their 
timing to support the conduct of human clinical trials and marketing authorization for 
pharmaceuticals. 
 
C.  Scope of the Guidance (1.3) 
 
The nonclinical safety assessment for marketing approval of a pharmaceutical usually 
includes pharmacology studies, general toxicity studies, toxicokinetic and nonclinical 
pharmacokinetic studies, reproduction toxicity studies, genotoxicity studies and, for drugs that 
have special cause for concern or are intended for a long duration of use, an assessment of 
carcinogenic potential.  Other nonclinical studies to assess phototoxicity, immunotoxicity, 
juvenile animal toxicity and abuse liability should be conducted on a case-by-case basis.  The 
types of nonclinical safety studies and their relation to the conduct of human clinical trials is 
delineated in this guidance. 
 
This document applies to the situations usually encountered during the development of 
pharmaceuticals and should be viewed as general guidance for drug development.  
Nonclinical safety studies and human clinical trials should be planned and designed to 
represent an approach that is scientifically and ethically appropriate.  
 
For biotechnology-derived products (as defined in Ref. 1), appropriate nonclinical safety 
studies should be determined in accordance with ICH S6.3  For these products, ICH M3(R2) 
only provides guidance with regard to timing of nonclinical studies relative to clinical 
development.   
 
Pharmaceuticals under development for indications in life-threatening or serious diseases 
(e.g., advanced cancer, resistant human immunodeficiency virus (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.  In these cases and for products using innovative 
                                                 
3 The ICH guidances referenced in this document are available on the CDER guidance 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 CDER 
guidance page. 
 
  2 Contains Nonbinding Recommendations 
 
therapeutic modalities (e.g., small interfering RNA (siRNA)), as well as vaccine adjuvants, 
particular studies can be abbreviated, deferred, omitted, or added.  Where ICH guidances for 
specific product areas exist, they should be consulted. 
 
D.  General Principles (1.4) 
 
The development of a pharmaceutical is a stepwise process involving an evaluation of both 
animal and human efficacy and safety information.  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.  
This information is used to estimate an initial safe starting dose and dose range for the human 
trials and to identify parameters for clinical monitoring for potential adverse effects.  The 
nonclinical safety studies, although usually limited at the beginning of clinical development, 
should be adequate to characterize potential adverse effects that might occur under the 
conditions of the clinical trial to be supported. 
 
Human clinical trials are conducted to investigate the efficacy and safety of a pharmaceutical, 
starting with a relatively low systemic exposure in a small number of subjects.  This is 
followed by clinical trials in which exposure to the pharmaceutical usually increases by 
duration and/or size of the exposed patient population.  Clinical trials should be extended 
based on the demonstration of adequate safety in the previous clinical trial(s), as well as on 
additional nonclinical safety information that becomes available as clinical development 
proceeds. 
   
Serious adverse clinical or nonclinical findings can influence the continuation of clinical 
trials. Within the overall clinical context, these findings should be evaluated to determine the 
appropriateness and design of additional nonclinical and/or clinical studies. 
   
Clinical trials are conducted in phases for which different terminology has been utilized in the 
various regions.  This M3(R2) document generally uses the terminology as defined in the ICH 
E8 guidance (Ref. 2).  However, as there is a growing trend to merge phases of clinical 
development, in some cases this document also relates the nonclinical studies to the duration 
and size of clinical trials and the characteristics of the subjects included. 
 
E.  High Dose Selection for General Toxicity Studies (1.5) 
 
Generally, in toxicity studies, effects that are potentially clinically relevant can be adequately 
characterized using doses up to the maximum tolerated dose (MTD). It is not essential to 
demonstrate the MTD in every study. Other equally appropriate limiting doses include those 
that achieve large exposure multiples or saturation of exposure or use the maximum feasible 
dose (MFD).  These limit doses (see additional details below and Figure 1) prevent the use of 
doses in animals that would not add value to predicting clinical safety. These 
recommendations are consistent with those for reproduction and carcinogenicity study designs 
that already have defined limit doses and/or exposures (Refs. 3 and 4).   
 
Limit doses for acute, subchronic, and chronic toxicity studies of 1000 milligrams 
(mg)/kilogram (kg)/day for rodents and nonrodents are considered appropriate in all cases 
  3 Contains Nonbinding Recommendations 
 
except those discussed below. In the few situations where a dose of 1000 mg/kg/day does not 
result in a mean exposure margin of 10-fold to the clinical exposure and the clinical dose 
exceeds 1 gram (g) per day, then the doses in the toxicity studies should be limited by a 10-
fold exposure margin or a dose of 2000 mg/kg/day or the MFD, whichever is lower. In those 
rare situations in which the dose of 2000 mg/kg/day results in an exposure that is less than the 
clinical exposure, a higher dose up to the MFD can be considered.  
 
Doses providing a 50-fold margin of exposure (usually based on group mean area under the 
curve (AUC) values (see Note 1) of the parent drug or the pharmacologically active molecule 
of a pro-drug) to the clinical systemic exposure generally are also considered acceptable as 
the maximum dose for acute and repeated-dose toxicity studies in any species.  
 
To support phase 3 clinical trials for the United States, dose-limiting toxicity generally should 
be identified in at least one species when using the 50-fold margin of exposure as the limit 
dose.  If this is not the case, a study of 1 month or longer duration in one species that is 
conducted at the 1000 mg/kg limit dose, MFD or MTD, whichever is lowest, is 
recommended. However, on a case-by-case basis this study might not be warranted if a study 
of a shorter duration identifies dose-limiting toxicity at doses higher than those resulting in a 
50-fold exposure margin. 
 
If genotoxicity endpoints are to be incorporated into a general toxicity study, then an 
appropriate maximum dose should be selected based on a MFD, MTD, or limit dose of 1000 
mg/kg/day. 
  4 Contains Nonbinding Recommendations 
 
Figure 1. Recommended High Dose Selection for General Toxicity Studies  
 
 
  5 Contains Nonbinding Recommendations 
 
II.  PHARMACOLOGY STUDIES (2) 
 
Safety pharmacology and pharmacodynamic (PD) studies are defined in ICH S7A (Ref. 5).  
The core battery of safety pharmacology studies includes the assessment of effects on 
cardiovascular, central nervous, and respiratory systems, and should generally be conducted 
before human exposure, in accordance with ICH S7A and S7B (Refs. 5 and 6).  When 
warranted, supplemental and follow-up safety pharmacology studies can be conducted during 
later clinical development.  Consideration should be given to inclusion of any in vivo 
evaluations as additions to general toxicity studies, to the extent feasible, in order to reduce 
animal use. 
 
In addition, primary PD studies (in vivo and/or in vitro) are intended to investigate the mode 
of action and/or effects of a substance in relation to its desired therapeutic target. Such studies 
are generally conducted during the discovery phase of pharmaceutical development and as 
such, are not generally conducted in accordance with Good Laboratory Practices (GLP). 
These studies can contribute to dose selection for both nonclinical and clinical studies. 
 
III.  TOXICOKINETIC AND PHARMACOKINETIC STUDIES (3) 
 
In vitro metabolic and plasma protein binding data for animals and humans and systemic 
exposure data (ICH S3A, Ref. 7) in the species used for repeated-dose toxicity studies 
generally should be evaluated before initiating human clinical trials.  Further information on 
pharmacokinetics (PK) (e.g., absorption, distribution, metabolism and excretion) in test 
species and in vitro biochemical information relevant to potential drug interactions should be 
available before exposing large numbers of human subjects or treating for long duration 
(generally before phase 3).  These data can be used to compare human and animal metabolites 
and for determining if any additional testing is warranted. 
 
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. Such studies should be conducted to support phase 3 clinical trials. For drugs for 
which the daily administered dose is <10 mg, greater fractions of the drug related material 
might be more appropriate triggers for testing. Some metabolites are not of toxicological 
concern (e.g., most glutathione conjugates) and do not warrant testing. The nonclinical 
characterization of metabolites with an identified cause for concern (e.g., a unique human 
metabolite) should be considered on a case-by-case basis. 
 
IV.  ACUTE TOXICITY STUDIES (4) 
 
Historically, acute toxicity information has been obtained from single-dose toxicity studies in 
two mammalian species using both the clinical and a parenteral route of administration.  
However, such information can be obtained from appropriately conducted dose-escalation 
studies or short-duration dose-ranging studies that define an MTD in the general toxicity test 
species (Refs. 8 and 9).  
 
  6 Contains Nonbinding Recommendations 
 
When this acute toxicity information is available from any study, separate single-dose studies 
are not recommended.  Studies providing acute toxicity information can be limited to the 
clinical route only and such data can be obtained from non-GLP studies if clinical 
administration is supported by appropriate GLP repeated-dose toxicity studies. Lethality 
should not be an intended endpoint in studies assessing acute toxicity. 
 
In some specific situations (e.g., microdose trials; see section VII (7) acute toxicity or single-
dose studies can be the primary support for studies in humans.  In these situations, the high 
dose selection can be different from that described in section I.E (1.5), but should be 
appropriate for supporting the intended clinical dose and route. These studies should be 
performed in compliance with GLP.  
 
Information on the acute toxicity of pharmaceutical agents could be useful to predict the 
consequences of human overdose situations and should be available to support phase 3.  An 
earlier assessment of acute toxicity could be important for therapeutic indications for which 
patient populations are at higher risk for overdosing (e.g., depression, pain, and dementia) in 
out-patient clinical trials. 
 
V.  REPEATED-DOSE TOXICITY STUDIES (5) 
 
The recommended duration of the repeated-dose toxicity studies is usually related to the 
duration, therapeutic indication, and scope of the proposed clinical trial.  In principle, the 
duration of the animal toxicity studies conducted in two mammalian species (one nonrodent) 
should be equal to or exceed the duration of the human clinical trials up to the maximum 
recommended duration of the repeated-dose toxicity studies (Table 1).  Limit doses/exposures 
that are considered appropriate in repeated-dose toxicity studies are described in section I.E 
(1.5). 
In circumstances where significant therapeutic gain has been shown, trials can be extended 
beyond the duration of supportive repeated-dose toxicity studies on a case-by-case basis. 
 
A.  Clinical Development Trials (5.1) 
 
Repeated-dose toxicity studies in two species (one nonrodent) for a minimum duration of 2 
weeks (Table 1) would generally support any clinical development trial up to 2 weeks in 
duration. Clinical trials of longer duration should be supported by repeated-dose toxicity 
studies of at least equivalent duration.  Six-month rodent and 9-month nonrodent studies 
generally support dosing for longer than 6 months in clinical trials (for exceptions, see Table 
1 footnotes). 
  7 Contains Nonbinding Recommendations 
 
Table 1  Recommended Duration of Repeated-Dose Toxicity Studies to Support the 
Conduct of Clinical Trials  
 
Maximum Duration of  Recommended Minimum Duration of Repeated-
Clinical Trial  Dose Toxicity Studies to Support Clinical Trials 
  Rodents  Nonrodents 
Up to 2 weeks   2 weeksa   2 weeksa 
Between 2 weeks and 6  Same as clinical trialb  Same as clinical trialb 
months  
> 6 months  6 monthsb, c  9 months b, c, d 
 
a.  In the United States, as an alternative to 2-week studies, extended single-dose toxicity studies 
(see footnote c in Table 3) can support single-dose human trials. Clinical studies of less than 
14 days can be supported with toxicity studies of the same duration as the proposed clinical 
study. 
b.   In some circumstances, clinical trials of longer duration than 3 months can be initiated, 
provided that the data are available from a 3-month rodent and a 3-month nonrodent study, 
and that complete data from the chronic rodent and nonrodent study are made available, 
consistent with local clinical trial regulatory procedures, before extending dosing beyond 3 
months in the clinical trial.  For serious or life-threatening indications or on a case-by-case 
basis, this extension can be supported by complete chronic rodent data and in-life and 
necropsy data for the nonrodent study. Complete histopathology data from the nonrodent 
should be available within an additional 3 months.   
c.    There can be cases where a pediatric population is the primary population, and existing animal 
studies (toxicology or pharmacology) have identified potential developmental concerns for 
target organs. In these cases, long-term toxicity testing starting in juvenile animals can be 
appropriate in some circumstances (see section XII (12)).   
d.    In the EU, studies of 6 months duration in nonrodents are considered acceptable.  However, 
where studies with a longer duration have been conducted, it is not appropriate to conduct an 
additional study of 6 months.   
The following are examples where nonrodent studies of up to 6 months duration can also be 
appropriate for Japan and the United States: 
  When immunogenicity or intolerance confounds conduct of longer term studies 
  Repeated short-term drug exposure even if clinical trial duration exceeds 6 months, such 
as intermittent treatment of migraine, erectile dysfunction, or herpes simplex 
  Drugs administered on a chronic basis to reduce the risk of recurrence of cancer  
  Drugs for indications for which life expectancy is short 
 
  8 Contains Nonbinding Recommendations 
 
B.  Marketing Authorization (5.2) 
 
Because of the size of the population at risk and the relatively less controlled conditions in 
clinical practice in contrast to clinical trials, longer durations of nonclinical testing can be 
valuable. The durations of repeated-dose toxicity studies to support marketing for different 
treatment durations are outlined in Table 2.  However, for a small number of conditions in 
which the indicated use is between 2 weeks and 3 months, but for which there is extensive 
clinical experience suggesting both widespread and long-term use beyond that recommended 
(e.g., anxiety, seasonal allergic rhinitis, pain), the duration of testing might more appropriately 
be equivalent to that recommended for treatment of greater than 3 months. 
 
Table 2  Recommended Duration of Repeated-Dose Toxicity Studies to Support 
Marketing 
 
Duration of Indicated  Rodent  Nonrodent 
Treatment 
Up to 2 weeks  1 month   1 month 
>2 weeks to 1 month  3 months  3 months 
>1 month to 3 months  6 months  6 months 
>3 months  6 monthsc  9 months c, d  
 
N.B. See footnotes c and d in Table 1. 
 
 
VI.  ESTIMATION OF THE FIRST DOSE IN HUMANS (6) 
 
The estimation of the first dose in humans is an important element to safeguard subjects 
participating in first-in-human studies.  All of the relevant nonclinical data, including the 
pharmacological dose response, the pharmacological/toxicological profile, and 
pharmacokinetics, should be considered when determining the recommended starting dose in 
humans. 
 
In general, the no observed adverse effect level (NOAEL) determined in nonclinical safety 
studies performed in the most appropriate animal species gives the most important 
information.  The proposed clinical starting dose will also depend on various factors, 
including pharmacodynamics (PD), particular aspects of the molecule, and the design of the 
clinical trials.  See available regional guidance for specific approaches that can be used. 
 
Exploratory clinical trials (see section VII (7)) in humans can be initiated with less, or 
different, nonclinical support than is generally warranted for clinical development trials (see 
section V.A (5.1)); therefore, the estimation of the clinical starting (and maximal) dose can 
differ.  The recommended criteria for starting doses for various exploratory clinical trial 
designs are described in Table 3. 
 
 
  9 Contains Nonbinding Recommendations 
 
VII.  EXPLORATORY CLINICAL TRIALS (7) 
 
It is recognized that in some cases, earlier access to human data can provide improved insight 
into human physiology/pharmacology, knowledge of drug candidate characteristics and 
therapeutic target relevance to disease. Streamlined early exploratory approaches can 
accomplish this end.  Exploratory clinical studies for the purpose of this guidance are those 
intended to be conducted early in phase 1, involve limited human exposure, have no 
therapeutic intent, and are not intended to examine clinical tolerability.  They can be used to 
investigate a variety of parameters such as PK, PD, and other biomarkers, which could 
include positron emission tomography (PET) receptor binding and displacement or other 
diagnostic measures. The subjects included in these studies can be patients from selected 
populations or healthy individuals. 
 
The amount and type of nonclinical supporting data that is appropriate in these situations will 
be dependent on the extent of proposed human exposure, both with respect to the maximum 
clinical dose used and the duration of dosing.  Five different examples of exploratory clinical 
approaches are summarized below and in more detail in Table 3, together with the nonclinical 
testing programs that would be recommended in these particular approaches.  However, 
alternative approaches not described in this guidance can also be used, including strategies to 
support biotechnology-derived products. It is recommended that these alternative approaches 
be discussed and agreed upon with the appropriate regulatory authority.  The use of any of 
these approaches can reduce overall animal use in drug development. 
 
Recommended starting doses and maximal doses for the five approaches are included in Table 
3.  In all cases, characterization of PD and pharmacology using in vivo and/or in vitro models 
as noted in Table 3 and section II (2) is important and should be used in support of human 
dose selection. 
 
A.  Microdose Trials (7.1) 
 
Two different microdose approaches are described below with details provided in Table 3. 
 
The first approach would involve not more than a total dose of 100 micrograms (g) that can 
be administered as a single dose or divided doses in any subject.  This could be useful to 
investigate target receptor binding or tissue distribution in a PET study.  A second use could 
be to assess PK with or without the use of an isotopically labelled agent.  
  
A second microdose approach is one that involves < 5 administrations of a maximum of 100 
g per administration (a total of 500 g per subject).  This can be useful for applications 
similar to the first microdose approach described above, but with less active PET ligands.  
 
In some situations, it could be appropriate to carry out a clinical microdose study using the 
intravenous (i.v.) route on a product intended for oral administration and for which an oral 
nonclinical toxicology package already exists.  In this case the i.v. microdose can be qualified 
by the existing oral toxicity studies as described in Table 1 or Table 3, Approach 3, where 
adequate exposure margins have been achieved.  It is not recommended to investigate i.v. 
local tolerance of the drug substance in this situation because the administered dose is very 
  10 Contains Nonbinding Recommendations 
 
low (100 g maximum).  If a novel i.v. vehicle is being employed, then local tolerance of the 
vehicle should be assessed. 
 
B.  Single-Dose Trials at Subtherapeutic Doses or Into the Anticipated 
Therapeutic Range (7.2) 
 
The third approach involves a single-dose clinical study typically starting at subtherapeutic 
doses and possibly escalating into the pharmacological or anticipated therapeutic range (see 
Table 3).  The maximum allowable dose should be based on the nonclinical data, but could be 
further limited based on emerging clinical information obtained during the course of the 
study.  This approach could allow, for example, determination of PK parameters with non-
radiolabeled drug at or near the predicted pharmacodynamically active dose. Another example 
could be assessment of target engagement or pharmacology after a single dose. This approach 
is not intended to support the determination of the maximum tolerated clinical dose (see 
exception, Table 1, footnote a). 
 
 C.  Multiple Dose Trials (7.3) 
 
Two different nonclinical approaches (numbers 4 and 5) to support multiple dose clinical 
trials are provided in Table 3. These approaches support up to 14 days of dosing for 
determination of PK and PD in human in the therapeutic dose range, but are not intended to 
support the determination of maximum tolerated clinical dose. 
 
Approach 4 involves 2-week repeated-dose toxicity studies in rodents and nonrodents where 
dose selection in animals is based on exposure multiples of anticipated AUC at the maximum 
clinical dose.  
 
Approach 5 involves a 2-week toxicity study in a rodent species and a confirmatory nonrodent 
study that is designed to investigate whether the NOAEL in the rodent is also not a toxic dose 
in the nonrodent. If toxic effects are observed in the nonrodent at the rodent NOAEL 
exposure, clinical administration should be deferred until further nonclinical studies in this 
species have been conducted (usually a standard toxicity study (see section V(5))). 
 
 
 
  11 Contains Nonbinding Recommendations 
 
Table 3    Recommended Nonclinical Studies to Support Exploratory Clinical Trials 
Clinical:  Nonclinical: 
Dose to be Administered  Start and Maximum Doses  Pharmacology  General Toxicity Studiesa  Genotoxicityb / Other 
Approach 1:  Maximal and starting doses can be the  In vitro target/ receptor  Extended single dose toxicity  Genotoxicity studies are 
Total dose  100 g (no  same but not exceed a total accumulated  profiling should be  study (see footnotes c and d) in  not recommended, but 
inter-dose interval  dose of 100 g  conducted   one species, usually rodent, by  any studies or structure-
limitations)    intended route of administration  activity relationship 
Appropriate characterization  with toxicokinetic data, or via  (SAR) assessments 
AND  
of primary pharmacology  the i.v. route.  A maximum dose  conducted should be 
Total dose  1/100th NOAEL  (mode of action and/or  of 1000-fold the clinical dose on  included in the clinical 
and 1/100th  effects) in a  a mg/kg basis for i.v. and mg/m2  trial application.   
pharmacologically active  pharmacodynamically  for oral administration can be   
dose (scaled on mg/kg for i.v.  relevant model should be  used.  For highly radioactive 
and mg/m2 for oral)  available to support human  agents (e.g., PET 
dose selection.  imaging agents), 
appropriate PK and 
dosimetry estimates 
should be submitted.  
Approach 2:   Maximal daily and starting doses can be  In vitro target/receptor  7-day repeated-dose toxicity  Genotoxicity studies are 
Total cumulative dose  500  the same, but not exceed 100 g.  profiling should be  study in one species, usually  not recommended, but 
g, maximum of 5  conducted   rodent, by intended route of  any studies or SAR 
administrations with a    administration with toxicokinetic  assessments conducted 
washout between doses (6 or  Appropriate characterization  data, or via the i.v. route.  should be included in the 
more actual or predicted half- of primary pharmacology  Hematology, clinical chemistry,  clinical trial application. 
lives)  (mode of action and/or  necropsy, and histopathology   
effects) in a  data should be included.  A  For highly radioactive 
AND  
pharmacodynamically  maximum dose of 1000-fold the  agents (e.g., PET 
each dose  100 g   relevant model should be  clinical dose on a mg/kg basis  imaging agents), 
AND   available to support human  for i.v. and mg/m2 for oral  appropriate PK and 
each dose  1/100th of the  dose selection.  administration can be used.  dosimetry estimates 
NOAEL and  1/100th of the    should be submitted.  
pharmacologically active 
dose 
  12 Contains Nonbinding Recommendations 
 
Table 3    Recommended Nonclinical Studies to Support Exploratory Clinical Trials (contd) 
Clinical:  Non clinical: 
Dose to be Administered  Start and Maximum Doses  Pharmacology  General Toxicity Studiesa  Genotoxicityb 
Approach 3  Starting dose should be based on the types  In vitro target/receptor  Extended single dose toxicity  Ames assay (or an 
Single Dose Studies at  of toxicity findings observed in the most  profiling should be conducted  studies in both the rodent and  alternative assay if Ames 
Subtherapeutic Doses or  sensitive species and a consideration of the    nonrodent (see footnote c) by  is inappropriate, for 
into the Anticipated  pharmacologically active dose.  For other  Appropriate characterization  intended clinical route of  example, for an 
Therapeutic Range  considerations on initial dosing in humans,  of primary pharmacology  administration with  antibacterial product). 
regional guidances should be consulted.  (mode of action and/or  toxicokinetics, hematology, 
  effects) in a  clinical chemistry, necropsy, 
Maximum dose can be that yielding up to 12  pharmacodynamically  and histopathology data.  For 
NOAEL exposure in the more sensitive  relevant model should be  this situation the top dose 
species, in cases where any relevant toxicity  available to support human  should be MTD, MFD or limit 
observed in animals is anticipated to be  dose selection.  dose (see section I.E (1.5)). 
monitorable and reversible in humans.   
Core battery of safety 
pharmacology (see section II 
(2)). 
  13 Contains Nonbinding Recommendations 
 
Table 3    Recommended Nonclinical Studies to Support Exploratory Clinical Trials (contd) 
Clinical:  Non clinical: 
Dose to be Administered  Start and Maximum Dose  Pharmacology  General toxicity studiesa  Genotoxicityb 
Approach 4:  With toxicity in both species, follow  In vitro target/receptor  2-week repeated-dose  Ames assay (or an 
Dosing up to 14 days into  appropriate regional guidance for clinical  profiling should be conducted  toxicity studies in rodent and  appropriate 
the therapeutic range but  starting dose.  If toxicity is not seen in    nonrodent with standard  alternative assay if 
not intended to evaluate  either species (i.e., the NOAELs are the  Appropriate characterization  parameters assessed and  Ames is 
clinical MTD   highest dose tested and doses used were not  of primary pharmacology  where dose selection in  inappropriate, for 
otherwise limited, e.g., not an MFD), or is  (mode of action and/or  animals is based on  example, for an 
seen only in one species, the clinical  effects) in a  exposure multiples of  antibacterial product) 
starting dose should be one that gives a  pharmacodynamically  anticipated clinical AUC at  and an assay (in vitro 
predicted clinical AUC value (based on  relevant model should be  maximum dose.  or in vivo) capable of 
either interspecies PK modeling or mg/m2  available to support human    detecting 
conversion) that is approximately 1/50th of  dose selection.  chromosomal damage 
the AUC at the NOAEL from the species  Core battery of safety  in a mammalian 
yielding the lower exposure. For other  pharmacology (see section  system    
considerations on initial dosing in humans,  II (2)) using doses similar   
e.g., predicted PD activity, regional  to those used for the   
guidance should be consulted.  toxicity studies. 
   
Without toxicity in both 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.  
 
When only one species demonstrates 
toxicity, the maximum clinical dose should 
not be higher than the NOAEL in the 
species showing toxicity, or 1/2 the AUC at 
the highest dose tested in the species not 
showing toxicity, whichever is lower. 
With toxicity in both species, the maximum 
clinical dose should be based on standard 
risk assessment approaches and, in this 
specific case, the clinical MTD can be 
explored. 
  14 Contains Nonbinding Recommendations 
 
Table 3    Recommended Nonclinical Studies to Support Exploratory Clinical Trials (contd) 
Clinical:  Non clinical: 
Dose to be Administered  Start and Maximum Doses  Pharmacology  General Toxicity Studiesa  Genotoxicityb 
Approach 5:  Starting dose predicted exposures should  In vitro target/receptor  Standard 2-week repeated- Ames assay (or an 
Dosing up to 14 days and  not exceed 1/50th the NOAEL in the more  profiling should be conducted  dose toxicity study in  appropriate alternative 
not to exceed duration of  sensitive species on a mg/m2 basis.  For    rodents (with justification of  assay if Ames is 
dosing in nonrodent; into  other considerations on initial dosing in  Appropriate characterization  the rodent as an appropriate  inappropriate, for 
therapeutic range but not  humans, regional guidance should be  of primary pharmacology  species). The top dose  example, for an 
intended to evaluate  consulted.  (mode of action and/or  should be the MTD, MFD or  antibacterial product) 
clinical MTD.    effects) in a  limit dose (see section I.E  and an assay (in vitro or 
The maximum exposure in humans should  pharmacodynamically  (1.5)).  in vivo) capable of 
not be higher than the AUC at the NOAEL  relevant model should be  Confirmatory study in  detecting chromosomal 
in the nonrodent species or higher than 12  available to support human  nonrodent (n=3) at the  damage in a mammalian 
the AUC at the NOAEL in the rodent  dose selection.  anticipated NOAEL  system. If an in vivo 
species, whichever is lowere.    exposure in rodent, with  assessment is used then 
Core battery of safety  duration of a minimum of 3  this could be part of the 
pharmacology (see section  days and at least the  rodent toxicity study. 
II (2)) using doses similar  intended clinical study   
to those used for the  duration.   
toxicity studies.  
Alternatively, an escalating 
dose study in the nonrodent 
with duration of a minimum 
of 3 days and at least the 
intended clinical study 
duration at the anticipated 
NOAEL exposure in the 
rodent.   
a.  General toxicity studies should be conducted according to GLP regulations. 
b.  See Ref. 10 for genotoxicity study design and dose selection. 
c.  Generally, extended single dose toxicity studies should be designed to evaluate hematology, clinical chemistry, necropsy, and histopathology data (control and 
high dose only if no treatment-related pathology is seen at the high dose) after a single administration, with further evaluations conducted 2 weeks later to assess 
delayed toxicity and/or recovery.  The usual design for rodents consists of 10 animals/sex/group to be assessed on the day following dosing, and 5 animals/sex at 
the dose level(s) selected to be assessed on day 14 post-dose. The usual design for nonrodents consists of 3/sex/group for all groups on day 2 and 2/sex for the 
dose level(s) assessed on day 14.  
d.  A single dose level to assess reversibility/delayed toxicity on day 14 can support the microdose approach. The dose level used need not be the high dose but 
should be a dose that is at least 100 times the clinical dose. 
e.  In the absence of adverse effects in the clinical trial, escalation above this AUC can be appropriate if the findings in the toxicity studies are anticipated to be 
monitorable, reversible, and of low severity in humans.  
  15 Contains Nonbinding Recommendations 
 
VIII.  LOCAL TOLERANCE STUDIES (8) 
 
It is preferable to evaluate local tolerance by the intended therapeutic route as part of the general 
toxicity studies; stand alone studies are generally not recommended. 
 
To support limited human administration by nontherapeutic routes (e.g., a single i.v. dose to 
assist in the determination of absolute bioavailability of an oral drug), a single dose local 
tolerance study in a single species is considered appropriate.  In cases where the anticipated 
systemic exposure (AUC and maximum plasma concentration (C )) from the nontherapeutic 
max
administration is covered by the existing toxicology package, the endpoints in the local tolerance 
study can be confined to clinical signs and macroscopic and microscopic examination of the 
application site. The formulation delivered for local tolerance need not be identical but should be 
similar to the clinical formulation. 
 
For an i.v. microdose study that is supported by an oral toxicology package (see section VII (7)), 
evaluation of local tolerance of the drug substance is not warranted. If a novel i.v. vehicle is 
being employed, then local tolerance of the vehicle should be assessed. 
 
For parenteral products, evaluation for local tolerance at unintended injection sites, when 
appropriate, should be conducted before exposure of large numbers of patients (e.g., phase 3 
clinical trials).  The approach to such studies differs in the various regions.  Such studies are 
generally not recommended in the United States (an example of an exception would be 
intrathecal for the epidural route).  Japan and the EU recommend single dose paravenous 
administration for the i.v. route. Other parenteral routes should be evaluated on a case-by-case 
basis.   
 
 
IX.  GENOTOXICITY STUDIES (9) 
 
An assay for gene mutation is generally considered sufficient to support all single dose clinical 
development trials.  To support multiple dose clinical development trials, an additional 
assessment capable of detecting chromosomal damage in a mammalian system(s) should be 
completed (Ref. 10). A complete battery of tests for genotoxicity should be completed before 
initiation of phase 2 trials (Ref. 10). 
 
If a positive finding occurs, an assessment, and then possibly additional testing (Ref. 10), should 
be conducted to determine if further administration to humans is still appropriate. 
 
The genotoxicity studies recommended to support Exploratory Clinical Study approaches are 
discussed in section VII (7). 
 
 
X.  CARCINOGENICITY STUDIES (10) 
Conditions relevant for carcinogenicity testing are discussed in the ICH S1A document (Ref. 11). 
If carcinogenicity studies are recommended for the clinical indication, they should be conducted 
to support the marketing application.  Only in circumstances where there is a significant cause 
for concern for carcinogenic risk should the study results be submitted to support clinical trials.  
A long clinical study duration alone is not considered to be a significant cause for concern. 
  16 Contains Nonbinding Recommendations 
 
For pharmaceuticals developed to treat certain serious diseases for adults or pediatric patients, 
carcinogenicity testing, if recommended, can be concluded postapproval. 
 
 
IX.  REPRODUCTION TOXICITY STUDIES (11) 
 
Reproduction toxicity studies (Ref. 3) should be conducted as is appropriate for the population 
that is to be exposed. 
 
A.  Men (11.1) 
 
Men can be included in phase 1 and 2 trials before the conduct of the male fertility study since an 
evaluation of the male reproductive organs is performed in the repeated-dose toxicity studies 
(Note 2). 
 
A male fertility study (Ref. 3) should be completed before the initiation of large scale or long 
duration clinical trials (e.g., phase 3 trials). 
 
B.  Women Not of Childbearing Potential (11.2) 
 
Women not of childbearing potential (i.e., permanently sterilized, postmenopausal) can be 
included in clinical trials without reproduction toxicity studies if the relevant repeated-dose 
toxicity studies (which include an evaluation of the female reproductive organs) have been 
conducted.  Postmenopausal is defined as 12 months with no menses without an alternative 
medical cause. 
   
C.  Women of Childbearing Potential (11.3) 
 
For women of childbearing potential (WOCBP), there is a high level of concern for the 
unintentional exposure of an embryo or fetus before information is available concerning the 
potential benefits versus potential risks. The recommendations on timing of reproduction toxicity 
studies to support the inclusion of WOCBP in clinical trials are similar in all ICH regions. 
 
It is important to characterize and minimize the risk of unintentional exposure of the embryo or 
fetus when including WOCBP in clinical trials.   One approach to achieve this objective is to 
conduct reproduction toxicity studies to characterize the inherent risk of a drug and take 
appropriate precautions during exposure of WOCBP in clinical trials.  A second approach is to 
limit the risk by taking precautions to prevent pregnancy during clinical trials. Precautions to 
prevent pregnancy include pregnancy testing (e.g., based on the -subunit of human chorionic 
gonadotropin (HCG)), use of highly effective methods of birth control (Note 3), and study entry 
only after a confirmed menstrual period. Testing for pregnancy during the trial and subject 
education should be sufficient to ensure compliance with the measures designed to prevent 
pregnancy during the period of drug exposure (which could exceed the length of study). To 
support these approaches, informed consent should be based on any known pertinent information 
related to reproduction toxicity, such as a general assessment of potential toxicity of 
pharmaceuticals with related structures or pharmacological effects. If no relevant reproductive 
information is available, the potential for unidentified risks to the embryo or fetus should be 
communicated. 
  17 Contains Nonbinding Recommendations 
 
 
In all ICH regions, WOCBP can be included in early clinical trials without nonclinical 
developmental toxicity studies (e.g., embryo-fetal studies) in certain circumstances.  One 
circumstance could be intensive control of pregnancy risk over short duration (e.g., 2 weeks) 
clinical trials.  Another circumstance could be where there is a predominance of the disease in 
women and the objectives of the clinical trial cannot be effectively met without inclusion of 
WOCBP and there are sufficient precautions to prevent pregnancy (see above).  
 
Additional considerations for the conduct of studies in WOCBP without the nonclinical 
developmental toxicity studies include knowledge of the mechanism of action of the agent, the 
type of pharmaceutical agent, and the extent of fetal exposure or the difficulty of conducting 
developmental toxicity studies in an appropriate animal model. For example, for monoclonal 
antibodies for which embryo-fetal exposure during organogenesis is understood to be low in 
humans based on current scientific knowledge, the developmental toxicity studies can be 
conducted during phase 3. The completed reports should be submitted with the marketing 
application.  
  
Generally, where appropriate preliminary reproduction toxicity data are available (see Note 4) 
from two species, and where precautions to prevent pregnancy in clinical trials (see above) are 
used, inclusion of WOCBP (up to 150) receiving investigational treatment for a relatively short 
duration (up to 3 months) can occur before conduct of definitive reproduction toxicity testing. 
This is based on the very low rate of pregnancy in controlled clinical trials of this size and 
duration (see Note 5), and the ability of adequately designed preliminary studies to detect most 
developmental toxicity findings that could raise concern for enrolment of WOCBP in clinical 
trials.  The number of WOCBP and the duration of the study can be influenced by characteristics 
of the population that alter pregnancy rates (e.g., age, disease). 
 
In the United States, assessment of embryo-fetal development can be deferred until before phase 
3 for WOCBP using precautions to prevent pregnancy in clinical trials (see above).  In the EU 
and Japan, other than the situations described in the above paragraphs, definitive nonclinical 
developmental toxicity studies should be completed before exposure of WOCBP.   
In all ICH regions, WOCBP can be included in repeated-dose phase 1 and 2 trials before conduct of 
the female fertility study since an evaluation of the female reproductive organs is performed in the 
repeated-dose toxicity studies (Note 2). Nonclinical studies that specifically address female fertility 
(Ref. 3) should be completed to support inclusion of WOCBP in large-scale or long-duration clinical 
trials (e.g., phase 3 trials). 
 
In all ICH regions, the pre-postnatal development study should be submitted for marketing 
approval.   
 
All female reproduction toxicity studies (Ref. 3) and the standard battery of genotoxicity tests 
(Ref. 10) should be completed before inclusion, in any clinical trial, of WOCBP not using highly 
effective birth control (see Note 3) or whose pregnancy status is unknown. 
 
D.  Pregnant Women (11.4) 
 
  18 Contains Nonbinding Recommendations 
 
Before the inclusion of pregnant women in clinical trials, all female reproduction toxicity studies 
(Refs. 3) and the standard battery of genotoxicity tests (Ref. 10) should be conducted.  In 
addition, safety data from previous human exposure should be evaluated. 
 
XII.  CLINICAL TRIALS IN PEDIATRIC POPULATIONS (12) 
 
When pediatric patients are included in clinical trials, safety data from previous adult human 
experience would usually represent the most relevant information and should generally be 
available before initiation of pediatric clinical trials.  The appropriateness and extent of adult 
human data should be determined on a case-by-case basis.  Extensive adult experience might not 
be available before pediatric exposures (e.g., for pediatric-specific indications).   
 
Results from repeated-dose toxicity studies of appropriate duration in adult animals (see Table 
1), the core safety pharmacology package, and the standard battery of genotoxicity tests should 
be available before initiation of trials in pediatric populations.  Reproduction toxicity studies 
relevant to the age and gender of the pediatric patient populations under study can also be 
important to provide information on direct toxic or developmental risks (e.g., fertility and pre-
postnatal developmental studies).  Embryo-fetal developmental studies are not critical to support 
clinical studies for males or prepubescent females. 
 
The conduct of any juvenile animal toxicity studies should be considered only when previous 
animal data and human safety data, including effects from other drugs of the pharmacological 
class, are judged to be insufficient to support pediatric studies. If a study is warranted, one 
relevant species, preferably rodent, is generally considered adequate. A study in a nonrodent 
species can be appropriate when scientifically justified.  
  
Generally, juvenile animal toxicity studies are not considered important for short-term PK 
studies (e.g., 1 to 3 doses) in pediatric populations. 
 
Depending on the therapeutic indication, age of the pediatric population, and safety data from 
adult animal and human exposure, the appropriateness of obtaining juvenile animal study results 
before initiation of short-duration multiple-dose efficacy and safety trials should be considered. 
The age of the trial participants in relation to the duration of the clinical study (i.e., the fraction 
of a developmental period of concern during which clinical study participants are exposed) is 
among the most important considerations. This evaluation can determine whether juvenile 
animal studies are warranted and, if warranted, their timing in relation to clinical trials.  
 
For long-term clinical trials in pediatric populations when an assessment of juvenile animal 
toxicity is recommended, the nonclinical studies should be completed before the initiation of the 
trials. 
 
There can be cases where a pediatric population is the primary population and existing animal 
studies have identified potential developmental concerns for target organs (toxicology or 
pharmacology). In some of these cases, long-term juvenile animal toxicity testing can be 
appropriate.  A chronic study initiated in the appropriate age and species with the relevant end 
points to address this developmental concern (e.g., 12 months duration in dog or 6 months in 
rodent) can be appropriate.  A 12-month study can cover the full development period in the dog. 
  19 Contains Nonbinding Recommendations 
 
For either species, this design could be adapted to replace the corresponding standard chronic 
study and a separate juvenile animal study in some circumstances. 
 
The appropriateness of carcinogenicity testing should be addressed before long-term exposure in 
pediatric clinical trials.  However, unless there is a significant cause for concern (e.g., evidence 
of genotoxicity in multiple tests, or concern for pro-carcinogenic risk based on mechanistic 
considerations or findings from general toxicity studies), carcinogenicity studies are not 
recommended to support the conduct of pediatric clinical trials.   
 
 
XIII.  IMMUNOTOXICITY (13) 
 
As stated in the ICH S8 guidance (Ref. 14), all new human pharmaceuticals should be evaluated 
for the potential to produce immunotoxicity using standard toxicity studies and additional 
immunotoxicity studies conducted as appropriate based on a weight-of-evidence review, 
including immune-related signals from standard toxicity studies.  If additional immunotoxicity 
studies are indicated, these should be completed before exposure of a large population of patients 
(e.g., phase 3). 
 
 
XIV.  PHOTOSAFETY TESTING (14) 
 
The appropriateness or timing of photosafety testing in relation to human exposure should be 
influenced by: (1) the photochemical properties (e.g., photoabsorption and photostability) of the 
molecule, (2) information on the phototoxic potential of chemically related compounds, (3) 
tissue distribution, and (4) clinical or nonclinical findings indicative of phototoxicity.   
 
An initial assessment of phototoxic potential based on a drugs photochemical properties and 
pharmacological/chemical class should be performed.  If assessment of all the available data and 
the proposed clinical plan indicates a potential for a significant human phototoxicity risk, 
appropriate protective measures should be taken during outpatient clinical studies. In addition, a 
subsequent evaluation of the nonclinical drug distribution to skin and eye should be completed to 
inform further on the human risk and the need for further testing.  Then, if appropriate, an 
experimental evaluation (nonclinical, in vitro or in vivo, or clinical) of phototoxic potential 
should be undertaken before exposure of large numbers of subjects (phase 3). 
 
Alternatively, instead of the above stepwise approach, a direct assessment of phototoxic potential 
in a nonclinical or clinical study can be undertaken. If this study is negative, an early assessment 
of eye/skin distribution studies and clinical protective measures are not called for. 
 
If the phototoxicity assessment indicates a potential photocarcinogenic risk, the risk can usually 
be adequately managed in patients by protective measures including a warning statement in the 
informed consent for clinical trials and in product information for marketing (Note 6). 
 
 
XV.  NONCLINICAL ABUSE LIABILITY (15) 
 
  20 Contains Nonbinding Recommendations 
 
For drugs that produce central nervous system activity, regardless of therapeutic indication, it 
should be considered whether or not an evaluation of abuse liability is warranted. Nonclinical 
studies should support the design of clinical evaluations of abuse potential, 
classification/scheduling by regulatory agencies, and product information.  There are regional 
guidance documents on the conduct of nonclinical abuse liability assessment that can be helpful 
in designing specific abuse liability packages.   
 
Nonclinical data collected early in the drug development process can be useful in identification 
of early indicators of abuse potential.  These early indicators would typically be available before 
first human dose and include the PK/PD profile to identify the duration of action, similarity of 
chemical structure to known drugs of abuse, receptor binding profile, and behavioural/clinical 
signs from in vivo nonclinical studies.  When no abuse potential is apparent from these early 
studies, extensive testing in nonclinical abuse liability models might not be warranted.  
Generally, if the active substance shows signals associated with known abuse liability patterns or 
the active substance has a novel mechanism of action on the central nervous system, further 
nonclinical studies are recommended to support large clinical trials (e.g., phase 3).   
 
When the metabolite profile and the target for drug activity in rodent are consistent with that of 
human, the nonclinical abuse liability evaluations should be conducted in rodents.  Nonhuman 
primates should be reserved only for those limited cases where there is clear evidence that they 
would be predictive of human abuse liability and the rodent model is inadequate.  Three types of 
studies are often completed to evaluate the potential for abuse liability: drug discrimination, self-
administration of the compound, and an assessment of withdrawal.  When conducted, studies of 
drug discrimination and self-administration are generally stand-alone. Assessments of 
withdrawal can sometimes be incorporated within the design of the reversibility arm of a 
repeated-dose toxicity study.  A maximum dose that produces a plasma concentration several-
fold higher than that obtained at the therapeutic clinical dose is considered appropriate for these 
nonclinical abuse assessments. 
 
 
XVI.  OTHER TOXICITY STUDIES (16) 
 
Additional nonclinical studies (e.g., to identify potential biomarkers, to provide mechanistic 
understanding) can be useful if previous nonclinical or clinical findings with the product or 
related products have indicated special safety concerns. 
 
The approaches for qualifying impurities and degradants are outlined in ICH Q3A and Q3B 
(Refs. 12 and 13). If specific studies are warranted to qualify an impurity or degradant, generally 
these studies are not warranted before phase 3 unless there are changes that result in a significant 
new impurity profile (e.g., a new synthetic pathway, a new degradant formed by interactions 
between the components of the formulation). In these latter cases, appropriate qualification 
studies can be warranted to support phase 2 or later stages of development. 
 
   
XVII.     COMBINATION DRUG TOXICITY TESTING (17) 
 
This section covers combination drugs that are intended to be co-packaged or administered in a 
single dosage form (fixed formulation).  The principles outlined can also apply when 
  21 Contains Nonbinding Recommendations 
 
developing products that will have product information recommendations for co-use with a 
specific drug, even if not in a fixed combination, and for which there is minimal clinical 
information regarding the combination.   
 
Combinations covered might involve: (1) two or more late stage entities (defined as compounds 
with significant clinical experience (i.e., from phase 3 studies and/ or post marketing)); (2) one or 
more late stage entity(ies) and one or more early stage entities (defined as compounds with 
limited clinical experience (i.e., phase 2 studies or less)); or (3) more than one early stage entity. 
For most combinations which involve two late stage entities and for which there is adequate 
clinical experience with co-administration, combination toxicity studies would generally not be 
recommended to support clinical studies or marketing unless there is significant toxicological 
concern (e.g., similar target organ toxicity).  This concern would be modified depending on the 
margins of safety and the ability to monitor the adverse effects in humans. If a study is being 
conducted to address a cause for significant toxicological concern, it should generally be 
completed before carrying out clinical studies with the combination. 
 
Where there are two late stage products for which there is not adequate clinical experience with 
co-administration, but there are no causes for significant toxicological concern based on the 
available data, nonclinical combination studies generally are not recommended to support small-
scale, relatively short-duration clinical studies (e.g., phase 2 studies of up to 3 months duration). 
Nonclinical combination studies, however, are recommended before large-scale or long-term 
combination trials, as well as for marketing.  
 
For combinations of an early stage entity(ies) with clinical experience with a late stage 
entity(ies), for which there is no significant toxicological concern, combination toxicity studies 
are not recommended to support clinical proof-of-concept studies of up to one months duration. 
The clinical study of the combination should not be longer than the clinical experience of the 
individual entities. Later stage or longer duration clinical studies should be supported by a 
nonclinical combination toxicity study. 
 
For combinations of two early stage entities, nonclinical combination toxicity studies are 
recommended to support clinical trials. 
 
Provided complete nonclinical development programs are being conducted on the individual 
entities and a nonclinical combination toxicity study is warranted to support combination clinical 
trials, the duration of the combination study should be equivalent to that of the clinical trial, up to 
a maximum duration of 90 days. A 90-day combination toxicity study would also support 
marketing. A combination toxicity study of shorter duration can also support marketing, 
depending on the duration of the intended clinical use. 
 
The design of the nonclinical studies recommended to characterize the combination will depend 
on the pharmacological, toxicological and PK profiles of the individual entities, the treatment 
indication(s), the intended patient population, and the available clinical data. 
 
Combination nonclinical studies should generally be limited to a single relevant species. If 
unexpected toxicity is identified, additional testing can be appropriate. 
 
  22 Contains Nonbinding Recommendations 
 
When complete nonclinical development programs are not conducted on the individual entities, 
then a complete nonclinical toxicology program with the combination only can be appropriate, 
provided that the individual agents are only intended for use in combination. 
 
Combination genotoxicity, safety pharmacology, or carcinogenicity studies generally are not 
recommended to support clinical trials or marketing if the individual agents have been tested 
according to current standards.  In those cases where the patient population includes WOCBP 
and studies with the 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.  If 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.  In circumstances when the individual agents have been tested 
in embryo-fetal studies but embryo-fetal studies of the drug combination are warranted, the 
study(ies) of the combination should be available to support the marketing application.  
 
 
XVIII.      CONTINUING EFFORTS TO IMPROVE HARMONIZATION (18) 
 
It is recognized that significant advances in harmonization of the timing of nonclinical safety 
studies for the conduct of human clinical trials for pharmaceuticals have already been achieved 
and are detailed in this guidance.  However, differences remain in a few areas.  Regulators and 
industry will continue to consider these differences and work towards further improving the drug 
development process.   
 
  23 Contains Nonbinding Recommendations 
 
XIX.  ENDNOTES (19) 
 
Note 1: In this document, exposure generally means group mean AUC. In some circumstances 
(e.g., if the compound or compound class is known to produce acute functional cardiovascular 
changes or central nervous system-related clinical signs), it might be appropriate to base the 
exposure margin on group mean Cmax values rather than AUC. 
 
Note 2: An assessment of male and female fertility by thorough standard histopathological 
examination on the testis and ovary in a repeated-dose toxicity study (generally rodent) of at 
least 2-week duration is considered to be as sensitive as fertility studies in detecting toxic effects 
on male and female reproductive organs (Refs. 3, 15, 16).  
 
Note 3: Highly effective methods of birth control are defined as those, alone or in combination, 
that result in a low failure rate (i.e., less than 1 percent per year) when used consistently and 
correctly.  For subjects using a hormonal contraceptive method, information regarding the 
product under evaluation and its potential effect on the contraceptive should be addressed. 
 
Note 4: A preliminary embryo-fetal study useful for this purpose is one with adequate dose 
levels; that includes assessment of fetal survival, body weight, and external and visceral 
examinations; that uses a minimum of six dams per group; and that has dams treated over the 
period of organogenesis. This preliminary nonclinical study should be conducted under high-
quality scientific standards with data collection records readily available or under GLP 
conditions.  
 
Note 5: The pregnancy rate of women initially attempting to become pregnant is ~17 percent per 
menstrual cycle.  Pregnancy rates estimated from phase 3 studies conducted in WOCBP were 
observed to be <0.1 percent per menstrual cycle.  During these studies, subjects were encouraged 
to avoid pregnancy and measures were instituted to prevent pregnancy.  Survey information from 
earlier phase 2 studies suggests that the pregnancy rates were lower than in phase 3 studies but 
the extent of further reduction could not be estimated due to the limited number of women 
enrolled. Based on the above phase 3 experience, phase 2 trials enrolling 150 WOCBP for 3 
months are estimated to result in significantly less than 0.5 pregnancies per pharmaceutical under 
development. 
 
Note 6: Testing for photocarcinogenicity in rodents using currently available models (e.g., 
hairless rodent) is not considered useful in support of pharmaceutical development and generally 
is not recommended.  If the phototoxicity assessment suggests a potential photocarcinogenic risk 
and an appropriate assay becomes available, the study should usually be completed before 
marketing and the results should be considered in the human risk assessment. 
 
 
  24 Contains Nonbinding Recommendations 
 
XX.  REFERENCES (20) 
 
1.  ICH S6 Safety Studies for Biotechnological Products 
2.  ICH E8 General Considerations for Clinical Trials 
3.  ICH S5(R2) Detection of Toxicity to Reproduction for Medicinal Products and Toxicity to 
Male Fertility 
4.  ICH S1C(R2) Dose Selection for Carcinogenicity Studies of Pharmaceuticals 
5.  ICH S7A Safety Pharmacology Studies for Human Pharmaceuticals 
6.  ICH S7B Nonclinical Evaluation of QT Interval Prolongation 
7.  ICH S3A Toxicokinetics:  The Assessment of Systemic Exposure in Toxicity Studies 
8.  National Centre for the Replacement, Refinement and Reduction of Animals in Research. 
Challenging Requirements for Acute Toxicity Studies: Workshop Report; May 2007. 
9.  Robinson S, Delongeas JL, Donald E, Dreher D, Festag M, Kervyn S et al.  A European 
pharmaceutical company initiative challenging the regulatory requirement for acute 
toxicity studies in pharmaceutical drug development. Regul Toxicol Pharmacol 
2008;50:345-352. 
10.  ICH S2B Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals 
11.  ICH S1A The Need for Carcinogenicity Studies for Pharmaceuticals 
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