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

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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
Additional copies are available from:
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Division of Drug Information
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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

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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

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XVII. 	COMBINATION DRUG TOXICITY TESTING (17) ............................................. 21
XVIII. 	CONTINUING EFFORTS TO IMPROVE HARMONIZATION (18) ................ 23
XIX. 	ENDNOTES (19) ............................................................................................................. 24
XX. 	REFERENCES (20) ........................................................................................................ 25

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LIST OF ABBREVIATIONS
AUC 	Area Under the Curve
Cmax 	Maximum Plasma Concentration
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

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Guidance for Industry1
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 (FDA’s) current thinking on this topic.
It does not create or confer any rights for or on any person and does not operate to bind FDA or the
public. You can use an alternative approach if the approach satisfies the requirements of the
applicable statutes and regulations. If you want to discuss an alternative approach, contact the FDA
staff responsible for implementing this guidance. If you cannot identify the appropriate FDA staff,
call the appropriate number listed on the title page of this guidance.
I. 	INTRODUCTION (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].

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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.

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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

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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.

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Figure 1. Recommended High Dose Selection for General Toxicity Studies
Are/were any
of the above situations
met? 
Yes
1000 mg/kg
limit dose
reached?
Mean exposure
margin ≥10x clinical?
Human Dose
<1 gram/day?
2000 mg/kg
dose level tested?
1. MTD
2. Exposure saturation
3. MFD
4. Mean exposure margin 50x clinical*
MFD or mean
exposure margin
≥10x clinical?
Exposure
animals ≥ humans?
Consider testing up to MFD
* For the US: see additional recommendations in section 1.5
No
Yes 	Yes
No
Yes
Yes 	Yes
Yes
In completed or subsequent studies:
Additional studies might
be warranted
No
No
No 	No
No
Tested dose level
is considered
adequate
Tested dose level
is considered
adequate
Additional studies might
be warranted

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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).

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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).

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Table 1 	Recommended Duration of Repeated-Dose Toxicity Studies to Support the
Conduct of Clinical Trials
Maximum Duration of
Clinical Trial
Recommended Minimum Duration of Repeated-
Dose Toxicity Studies to Support Clinical Trials
Rodents 	Nonrodents
Up to 2 weeks 	2 weeks a 	2 weeks a
Between 2 weeks and 6
months
Same as clinical trialb 	Same as clinical trialb
> 6 months 	6 months b, 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

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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
Treatment
Rodent 	Nonrodent
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 months c 	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.

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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 (μg) 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
μg per administration (a total of 500 μg 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

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low (100 μg 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))).

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Table 3 	Recommended Nonclinical Studies to Support Exploratory Clinical Trials
Clinical: 	Nonclinical:
Dose to be Administered 	Start and Maximum Doses 	Pharmacology 	General Toxicity Studies a 	Genotoxicityb / Other
Approach 1:
Total dose ≤ 100 μg (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/m 2 for oral)
Maximal and starting doses can be the
same but not exceed a total accumulated
dose of 100 μg
In vitro target/ receptor
profiling should be
conducted
Appropriate characterization
of primary pharmacology
(mode of action and/or
effects) in a
pharmacodynamically
relevant model should be
available to support human
dose selection.
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.
Genotoxicity studies are
not recommended, but
any studies or structure-
activity relationship
(SAR) assessments
conducted should be
included in the clinical
trial application.
For highly radioactive
agents (e.g., PET
imaging agents),
appropriate PK and
dosimetry estimates
should be submitted.
Approach 2:
Total cumulative dose ≤ 500
μg, maximum of 5
administrations with a
washout between doses (6 or
more actual or predicted half-
lives)
AND
each dose ≤ 100 μg
AND
each dose ≤ 1/100 th of the
NOAEL and ≤ 1/100th of the
pharmacologically active
dose
Maximal daily and starting doses can be
the same, but not exceed 100 μg.
In vitro target/receptor
profiling should be
conducted
Appropriate characterization
of primary pharmacology
(mode of action and/or
effects) in a
pharmacodynamically
relevant model should be
available to support human
dose selection.
7-day repeated-dose toxicity
study in one species, usually
rodent, by intended route of
administration with toxicokinetic
data, or via the i.v. route.
Hematology, clinical chemistry,
necropsy, and histopathology
data should be included. 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.
Genotoxicity studies are
not recommended, but
any studies or SAR
assessments conducted
should be included in the
clinical trial application.
For highly radioactive
agents (e.g., PET
imaging agents),
appropriate PK and
dosimetry estimates
should be submitted.

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Table 3 	Recommended Nonclinical Studies to Support Exploratory Clinical Trials (cont’d)
Clinical: 	Non clinical:
Dose to be Administered 	Start and Maximum Doses 	Pharmacology 	General Toxicity Studiesa 	Genotoxicityb
Approach 3
Single Dose Studies at
Subtherapeutic Doses or
into the Anticipated
Therapeutic Range
Starting dose should be based on the types
of toxicity findings observed in the most
sensitive species and a consideration of the
pharmacologically active dose. For other
considerations on initial dosing in humans,
regional guidances should be consulted.
Maximum dose can be that yielding up to ½
NOAEL exposure in the more sensitive
species, in cases where any relevant toxicity
observed in animals is anticipated to be
monitorable and reversible in humans.
In vitro target/receptor
profiling should be conducted
Appropriate characterization
of primary pharmacology
(mode of action and/or
effects) in a
pharmacodynamically
relevant model should be
available to support human
dose selection.
Core battery of safety
pharmacology (see section II
(2)).
Extended single dose toxicity
studies in both the rodent and
nonrodent (see footnote c) by
intended clinical route of
administration with
toxicokinetics, hematology,
clinical chemistry, necropsy,
and histopathology data. For
this situation the top dose
should be MTD, MFD or limit
dose (see section I.E (1.5)).
Ames assay (or an
alternative assay if Ames
is inappropriate, for
example, for an
antibacterial product).

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Table 3 	Recommended Nonclinical Studies to Support Exploratory Clinical Trials (cont’d)
Clinical: 	Non clinical:
Dose to be Administered 	Start and Maximum Dose 	Pharmacology 	General toxicity studiesa 	Genotoxicityb
Approach 4:
Dosing up to 14 days into
the therapeutic range but
not intended to evaluate
clinical MTD
With toxicity in both species, follow
appropriate regional guidance for clinical
starting dose. If toxicity is not seen in
either species (i.e., the NOAELs are the
highest dose tested and doses used were not
otherwise limited, e.g., not an MFD), or is
seen only in one species, the clinical
starting dose should be one that gives a
predicted clinical AUC value (based on
either interspecies PK modeling or mg/m2
conversion) that is approximately 1/50th of
the AUC at the NOAEL from the species
yielding the lower exposure. For other
considerations on initial dosing in humans,
e.g., predicted PD activity, regional
guidance should be consulted.
Without toxicity in both species, it is
recommended that the maximum clinical
dose not exceed 1/10 th 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.
In vitro target/receptor
profiling should be conducted
Appropriate characterization
of primary pharmacology
(mode of action and/or
effects) in a
pharmacodynamically
relevant model should be
available to support human
dose selection.
Core battery of safety
pharmacology (see section
II (2)) using doses similar
to those used for the
toxicity studies.
2-week repeated-dose
toxicity studies in rodent and
nonrodent with standard
parameters assessed and
where dose selection in
animals is based on
exposure multiples of
anticipated clinical AUC at
maximum dose.
Ames assay (or an
appropriate
alternative assay if
Ames is
inappropriate, for
example, for an
antibacterial product)
and an assay (in vitro
or in vivo) capable of
detecting
chromosomal damage
in a mammalian
system

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Table 3 	Recommended Nonclinical Studies to Support Exploratory Clinical Trials (cont’d)
Clinical: 	Non clinical:
Dose to be Administered 	Start and Maximum Doses 	Pharmacology 	General Toxicity Studiesa 	Genotoxicityb
Approach 5:
Dosing up to 14 days and
not to exceed duration of
dosing in nonrodent; into
therapeutic range but not
intended to evaluate
clinical MTD.
Starting dose predicted exposures should
not exceed 1/50 th the NOAEL in the more
sensitive species on a mg/m 2 basis. For
other considerations on initial dosing in
humans, regional guidance should be
consulted.
The maximum exposure in humans should
not be higher than the AUC at the NOAEL
in the nonrodent species or higher than ½
the AUC at the NOAEL in the rodent
species, whichever is lower e
.
In vitro target/receptor
profiling should be conducted
Appropriate characterization
of primary pharmacology
(mode of action and/or
effects) in a
pharmacodynamically
relevant model should be
available to support human
dose selection.
Core battery of safety
pharmacology (see section
II (2)) using doses similar
to those used for the
toxicity studies.
Standard 2-week repeated-
dose toxicity study in
rodents (with justification of
the rodent as an appropriate
species). The top dose
should be the MTD, MFD or
limit dose (see section I.E
(1.5)).
Confirmatory study in
nonrodent (n=3) at the
anticipated NOAEL
exposure in rodent, with
duration of a minimum of 3
days and at least the
intended clinical study
duration.
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.
Ames assay (or an
appropriate alternative
assay if Ames is
inappropriate, for
example, for an
antibacterial product)
and an assay (in vitro or
in vivo) capable of
detecting chromosomal
damage in a mammalian
system. If an in vivo
assessment is used then
this could be part of the
rodent toxicity study.
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.

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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 (Cmax )) from the nontherapeutic
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.

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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 β-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.

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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)

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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 sex 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.

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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 drug’s 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)

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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

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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.

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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.

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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.

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XX. 	REFERENCES (20)
1. 	ICH S6 Safety Studies for Biotechnological Products
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