Guidance for Industry
S6 Preclinical Safety Evaluation of
Biotechnology-Derived
Pharmaceuticals
July 1997
ICH Guidance for Industry
S6 Preclinical Safety Evaluation of
Biotechnology-Derived
Pharmaceuticals
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July 1997
ICH Table of Contents
I. INTRODUCTION (1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
A. Background (1.1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
B. Objectives (1.2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
C. Scope (1.3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
II. SPECIFICATION OF THE TEST MATERIAL (2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
III. PRECLINICAL SAFETY TESTING (3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
A. General Principles (3.1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
B. Biological Activity/Pharmacodynamics (3.2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
C. Animal Species/Model Selection (3.3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
D. Number/Gender of Animals (3.4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
E. Administration/Dose Selection (3.5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
F. Immunogenicity (3.6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
IV. SPECIFIC CONSIDERATIONS (4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
A. Safety Pharmacology (4.1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
B. Exposure Assessment (4.2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
C. Single Dose Toxicity Studies (4.3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
D. Repeated Dose Toxicity Studies (4.4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
E. Immunotoxicity Studies (4.5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
F. Reproductive Performance and Developmental Toxicity Studies (4.6) . . . . . . . . . 9
G. Genotoxicity Studies (4.7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
H. Carcinogenicity Studies (4.8) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
I. Local Tolerance Studies (4.9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
NOTES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
i GUIDANCE FOR INDUSTRY1
S6 Preclinical Safety Evaluation of
Biotechnology-Derived Pharmaceuticals
I. INTRODUCTION (1)
A. Background (1.1) 
Biotechnology-derived pharmaceuticals (biopharmaceuticals) were initially developed in the early
1980's.  The first marketing authorizations were granted later in the decade. Several guidelines
and points-to-consider documents have been issued by various regulatory agencies regarding
safety assessment of these products.  Review of such documents, which are available from
regulatory authorities, may provide useful background in developing new biopharmaceuticals.
Considerable experience has now been gathered with submission of applications for
biopharmaceuticals.  Critical review of this experience has been the basis for development of this
guidance, which is intended to provide general principles for designing scientifically acceptable
preclinical safety evaluation programs.  
B. Objectives (1.2)
Regulatory standards for biotechnology-derived pharmaceuticals have generally been comparable
among the European Union, Japan, and the United States.  All three regions have adopted a
flexible, case-by-case, science-based approach to preclinical safety evaluation needed to support
clinical development and marketing authorization. In this rapidly evolving scientific area, there is a
need for common understanding and continuing dialogue among the regions. 
The primary goals of preclinical safety evaluation are:  (1) To identify an initial safe dose and
subsequent dose escalation schemes in humans; (2) to identify potential target organs for toxicity
and for the study of whether such toxicity is reversible; and (3) to identify safety parameters for
clinical monitoring.  Adherence to the principles presented in this document should improve the
1 This guidance was developed within the Expert Working Group (Safety) 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, July 1997.  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.  This guidance
was published in the Federal Register on November 18, 1997 (62 FR 61515), and is applicable to drug and biological
products.  This guidance represents the Agencys current thinking on preclinical safety evaulation of biotechnology-
derived pharmaceuticals.  It does not create or confer any rights for or on any person and does not operate to bind FDA
or the public.  An alternative approach may be used if such approach satisfies the requirements of the applicable statute,
regulations, or both. quality and consistency of the preclinical safety data supporting the development of
biopharmaceuticals.
C. Scope (1.3)
This guidance is intended primarily to recommend a basic framework for the preclinical safety
evaluation of biotechnology-derived pharmaceuticals.  It applies to products derived from
characterized cells through the use of a variety of expression systems including bacteria, yeast,
insect, plant, and mammalian cells.  The intended indications may include in vivo diagnostic,
therapeutic, or prophylactic uses.  The active substances include proteins and peptides, their
derivatives, and products of which they are components; they could be derived from cell cultures
or produced using recombinant deoxyribonucleic acid (DNA) technology, including production by
transgenic plants and animals.  Examples include but are not limited to:  Cytokines,  plasminogen
activators, recombinant plasma factors, growth factors, fusion proteins, enzymes, receptors,
hormones, and monoclonal antibodies.
The principles outlined in this guidance may also be applicable to recombinant DNA protein
vaccines, chemically synthesized peptides, plasma derived products, endogenous proteins
extracted from human tissue, and oligonucleotide drugs.  
This document does not cover antibiotics, allergenic extracts, heparin, vitamins, cellular blood
components,  conventional bacterial or viral vaccines, DNA vaccines, or cellular and gene
therapies.
II. SPECIFICATION OF THE TEST MATERIAL (2)
Safety concerns may arise from the presence of impurities or contaminants.  It is preferable to rely
on purification processes to remove impurities and contaminants rather than to establish a
preclinical testing program for their qualification.  In all cases, the product should be sufficiently
characterized to allow an appropriate design of preclinical safety studies. 
There are potential risks associated with host cell contaminants derived from bacteria, yeast,
insect, plants, and mammalian cells.  The presence of cellular host contaminants can result in
allergic reactions and other immunopathological effects.  The adverse effects associated with
nucleic acid contaminants are theoretical but include potential integration into the host genome. 
For products derived from insect, plant, and mammalian cells, or transgenic plants and animals,
there may be an additional risk of viral infections.  
In general, the product that is used in the definitive pharmacology and toxicology studies should
be comparable to the product proposed for the initial clinical studies.  However, it is appreciated
that during the course of development programs, changes normally occur in the manufacturing
2 process in order to improve product quality and yields.  The potential impact of such changes for
extrapolation of the animal findings to humans should be considered.
The comparability of the test material during a development program should be demonstrated
when a new or modified manufacturing process is developed or other significant changes in the
product or formulation are made in an ongoing development program.  Comparability can be
evaluated on the basis of biochemical and biological characterization (i.e., identity, purity,
stability, and potency).  In some cases, additional studies may be needed (i.e., pharmacokinetics,
pharmacodynamics and/or safety).  The scientific rationale for the approach taken should be
provided.  
III. PRECLINICAL SAFETY TESTING (3)
A. General Principles (3.1)
The objectives of the preclinical safety studies are to define pharmacological and toxicological
effects not only prior to initiation of human studies but throughout clinical development.  Both in
vitro and in vivo studies can contribute to this characterization.  Biopharmaceuticals that are
structurally and pharmacologically comparable to a product for which there is wide experience in
clinical practice may need less extensive toxicity testing.
Preclinical safety testing should consider:  (1) Selection of the relevant animal species; (2) age; (3)
physiological state; (4) the manner of delivery, including dose, route of administration, and
treatment regimen; and (5) stability of the test material under the conditions of use.  
Toxicity studies are expected to be performed in compliance with Good Laboratory Practice
(GLP); however, it is recognized that some studies employing specialized test systems, which are
often needed for biopharmaceuticals, may not be able to comply fully with GLP.  Areas of
noncompliance should be identified and their significance evaluated relative to the overall safety
assessment.  In some cases, lack of full GLP compliance does not necessarily mean that the data
from these studies cannot be used to support clinical trials and marketing authorizations.  
Conventional approaches to toxicity testing of pharmaceuticals may not be appropriate for
biopharmaceuticals due to the unique and diverse structural and biological properties of the latter
that may include species specificity, immunogenicity, and unpredicted pleiotropic activities.  
B. Biological Activity/Pharmacodynamics (3.2)
Biological activity may be evaluated using in vitro assays to determine which effects of the
product may be related to clinical activity.  The use of cell lines and/or primary cell cultures can be
useful to examine the direct effects on cellular phenotype and proliferation.  Due to the species
specificity of many biotechnology-derived pharmaceuticals, it is important to select relevant
3 animal species for toxicity testing.  In vitro cell lines derived from mammalian cells can be used to
predict specific aspects of in vivo activity and to assess quantitatively the relative sensitivity of
various species (including human) to the biopharmaceutical.  Such studies may be designed to
determine, for example, receptor occupancy, receptor affinity, and/or pharmacological effects, and
to assist in the selection of an appropriate animal species for further in vivo pharmacology and
toxicology studies.  The combined results from in vitro and in vivo studies assist in the
extrapolation of the findings to humans.  In vivo studies to assess pharmacological activity,
including defining mechanism(s) of action, are often used to support the rationale of the proposed
use of the product in clinical studies.  
For monoclonal antibodies, the immunological properties of the antibody should be described in
detail, including its antigenic specificity, complement binding, and any unintentional reactivity
and/or cytotoxicity towards human tissues distinct from the intended target.  Such cross-reactivity
studies should be carried out by appropriate immunohistochemical  procedures using a range of
human tissues.  
C. Animal Species/Model Selection (3.3)
The biological activity together with species and/or tissue specificity of many biotechnology-
derived pharmaceuticals often preclude standard toxicity testing designs in commonly used
species (e.g., rats and dogs).  Safety evaluation programs should include the use of relevant
species.  A relevant species is one in which the test material is pharmacologically active due to the
expression of the receptor or an epitope (in the case of monoclonal antibodies).  A variety of
techniques (e.g., immunochemical or functional tests) can be used to identify a relevant species. 
Knowledge of receptor/epitope distribution can provide greater understanding of potential in vivo
toxicity.
Relevant animal species for testing of monoclonal antibodies are those that express the desired
epitope and demonstrate a similar tissue cross-reactivity profile as for human tissues.  This would
optimize the ability to evaluate toxicity arising from the binding to the epitope and any
unintentional tissue cross-reactivity.  An animal species that does not express the desired epitope
may still be of some relevance for assessing toxicity if comparable unintentional tissue cross-
reactivity to humans is demonstrated.  
Safety evaluation programs should normally include two relevant species. However, in certain
justified cases one relevant species may suffice (e.g., when only one relevant species can be
identified or where the biological activity of the biopharmaceutical is well understood).  In
addition, even where two species may be necessary to characterize toxicity in short term studies,
it may be possible to justify the use of only one species for subsequent long-term toxicity studies
(e.g., if the toxicity profile in the two species is comparable in the short term). 
Toxicity studies in nonrelevant species may be misleading and are discouraged. When no relevant
species exists, the use of relevant transgenic animals expressing the human receptor or the use of
4 homologous proteins should be considered.  The information gained from use of a transgenic
animal model expressing the human receptor is optimized when the interaction of the product and
the humanized receptor has similar physiological consequences to those expected in humans. 
While useful information may also be gained from the use of homologous proteins, it should be
noted that the production process, range of impurities/contaminants, pharmacokinetics, and exact
pharmacological mechanism(s) may differ between the homologous form and the product
intended for clinical use.  Where it is not possible to use transgenic animal models or homologous
proteins, it may still be prudent to assess some aspects of potential toxicity in a limited toxicity
evaluation in a single species, e.g., a repeated dose toxicity study of < 14 days duration that
includes an evaluation of important functional endpoints (e.g., cardiovascular and respiratory). 
In recent years, there has been much progress in the development of animal models that are
thought to be similar to the human disease.  These animal models include induced and
spontaneous models of disease, gene knockout(s), and transgenic animals.  These models may
provide further insight, not only in determining the pharmacological action of the product,
pharmacokinetics, and dosimetry, but may also be useful in the determination of safety (e.g.,
evaluation of undesirable promotion of disease progression).  In certain cases, studies performed
in animal models of disease may be used as an acceptable alternative to toxicity studies in normal
animals (Note 1).  The scientific justification for the use of these animal models of disease to
support safety should be provided.  
D. Number/Gender of Animals (3.4)
The number of animals used per dose has a direct bearing on the ability to detect toxicity.  A small
sample size may lead to failure to observe toxic events due to observed frequency alone regardless
of severity.  The limitations that are imposed by sample size, as often is the case for nonhuman
primate studies, may be in part compensated by increasing the frequency and duration of
monitoring.  Both genders should generally be used or justification given for specific omissions. 
E. Administration/Dose Selection (3.5)
The route and frequency of administration should be as close as possible to that proposed for
clinical use.  Consideration should be given to pharmacokinetics and bioavailability of the product
in the species being used and to the volume which can be safely and humanely administered to the
test animals.  For example, the frequency of administration in laboratory animals may be increased
compared to the proposed schedule for the human clinical studies in order to compensate for
faster clearance rates or low solubility of the active ingredient.  In these cases, the level of
exposure of the test animal relative to the clinical exposure should be defined.  Consideration
should also be given to the effects of volume, concentration, formulation, and site of
administration.  The use of routes of administration other than those used clinically may be
acceptable if the route must be modified due to limited bioavailability, limitations due to the route
of administration, or to size/physiology of the animal species.  
5 Dosage levels should be selected to provide information on a dose-response relationship,
including a toxic dose and a no observed adverse effect level (NOAEL).  For some classes of
products with little to no toxicity, it may not be possible to define a specific maximum dose.  In
these cases, a scientific justification of the rationale for the dose selection and projected multiples
of human exposure should be provided.  To justify high dose selection, consideration should be
given to the expected pharmacological/physiological effects, availability of suitable test material,
and the intended clinical use. Where a product has a lower affinity to or potency in the cells of the
selected species than in human cells, testing of higher doses may be important.  The multiples of
the human dose that are needed to determine adequate safety margins may vary with each class of
biotechnology-derived pharmaceutical and its clinical indication(s). 
F. Immunogenicity (3.6)
Many biotechnology-derived pharmaceuticals intended for humans are immunogenic in animals. 
Therefore, measurement of antibodies associated with administration of these types of products
should be performed when conducting repeated dose toxicity studies in order to aid in the
interpretation of these studies.  Antibody responses should be characterized (e.g., titer, number of
responding animals, neutralizing or non-neutralizing) and their appearance should be correlated
with any pharmacological and/or toxicological changes.  Specifically, the effects of antibody
formation on pharmacokinetic/pharmacodynamic parameters, incidence and/or severity of adverse
effects, complement activation, or the emergence of new toxic effects should be considered when
interpreting the data.  Attention should also be paid to the evaluation of possible pathological
changes related to immune complex formation and deposition.
The detection of antibodies should not be the sole criterion for the early termination of a
preclinical safety study or modification in the duration of the study design unless the immune
response neutralizes the pharmacological and/or toxicological effects of the biopharmaceutical in
a large proportion of the animals.  In most cases, the immune response to biopharmaceuticals is
variable, like that observed in humans.  If the interpretation of the data from the safety study is not
compromised by these issues, then no special significance should be ascribed to the antibody
response.   
The induction of antibody formation in animals is not predictive of a potential for antibody
formation in humans. Humans may develop serum antibodies against humanized proteins, and
frequently the therapeutic response persists in their presence.  The occurrence of severe
anaphylactic responses to recombinant proteins is rare in humans.  In this regard, the results of
guinea pig anaphylaxis tests, which are generally positive for protein products, are not predictive
for reactions in humans; therefore, such studies are considered of little value for the routine
evaluation of these types of products.
6 IV. SPECIFIC CONSIDERATIONS (4)
A. Safety Pharmacology (4.1)
It is important to investigate the potential for undesirable pharmacological activity in appropriate
animal models and, where necessary, to incorporate particular monitoring for these activities in
the toxicity studies and/or clinical studies.  Safety pharmacology studies measure functional
indices of potential toxicity.  These functional indices may be investigated in separate studies or
incorporated in the design of toxicity studies. The aim of the safety pharmacology studies should
be to reveal any functional effects on the major physiological systems (e.g., cardiovascular,
respiratory, renal, and central nervous systems).  Investigations may also include the use of
isolated organs or other test systems not involving intact animals.  All of  these studies may allow
for a mechanistically-based explanation of specific organ toxicities, which should be considered
carefully with respect to human use and indication(s).  
B. Exposure Assessment (4.2)
1. Pharmacokinetics and Toxicokinetics (4.2.1)
It is difficult to establish uniform guidances for pharmacokinetic studies for biotechnology-
derived pharmaceuticals.  Single and multiple dose pharmacokinetics,  toxicokinetics, and
tissue distribution studies in relevant species are useful; however, routine studies that
attempt to assess mass balance are not useful.  Differences in pharmacokinetics among
animal species may have a significant impact on the predictiveness of animal studies or on
the assessment of dose-response relationships in toxicity studies. Alterations in the
pharmacokinetic profile due to immune-mediated clearance mechanisms may affect the
kinetic profiles and the interpretation of the toxicity data.  For some products, there may
also be inherent, significant delays in the expression of pharmacodynamic effects relative
to the pharmacokinetic profile (e.g., cytokines) or there may be prolonged expression of
pharmacodynamic effects relative to plasma levels.  
Pharmacokinetic studies should, whenever possible, utilize preparations that are
representative of those intended for toxicity testing and clinical use and employ a route of
administration that is relevant to the anticipated clinical studies.  Patterns of absorption
may be influenced by formulation, concentration, site, and/or volume.  Whenever possible,
systemic exposure should be monitored during the toxicity studies.  
When using radiolabeled proteins, it is important to show that the radiolabeled test
material maintains activity and biological properties equivalent to that of the unlabeled
material.  Tissue concentrations of radioactivity and/or autoradiography data using
radiolabeled proteins may be difficult to interpret due to rapid in vivo metabolism or
unstable radiolabeled linkage.  Care should be taken in the interpretation of studies using
7 radioactive tracers incorporated into specific amino acids because of recycling of amino
acids into nondrug related proteins/peptides.
Some information on absorption, disposition, and clearance in relevant animal models
should be available prior to clinical studies in order to predict margins of safety based
upon exposure and dose.  
2. Assays (4.2.2)
The use of one or more assay methods should be addressed on a case-by-case basis and
the scientific rationale should be provided.  One validated method is usually considered
sufficient.  For example, quantitation of TCA-precipitable radioactivity following
administration of a radiolabeled protein may provide adequate information, but a specific
assay for the analyte is preferred.  Ideally, the assay methods should be the same for
animals and humans.  The possible influence of plasma binding proteins and/or antibodies
in plasma/serum on the assay performance should be determined.
3 Metabolism (4.2.3)
The expected consequence of metabolism of biotechnology-derived pharmaceuticals is the
degradation to small peptides and individual amino acids.  Therefore, the metabolic
pathways are generally understood.  Classical biotransformation studies as performed for
pharmaceuticals are not needed.  
Understanding the behavior of the biopharmaceutical in the biologic matrix (e.g., plasma,
serum, cerebral spinal fluid) and the possible influence of binding proteins is important for
understanding the pharmacodynamic effect.  
C. Single Dose Toxicity Studies (4.3)
Single dose studies may generate useful data to describe the relationship of dose to systemic
and/or local toxicity.  These data can be used to select doses for repeated dose toxicity studies. 
Information on dose-response relationships may be gathered through the conduct of a single dose
toxicity study or as a component of pharmacology or animal model efficacy studies.  The
incorporation of safety pharmacology parameters in the design of these studies should be
considered.
D. Repeated Dose Toxicity Studies (4.4)
For consideration of the selection of animal species for repeated dose studies, see section III.C
(3.3).  The route and dosing regimen (e.g., daily versus intermittent dosing) should reflect the
intended clinical use or exposure.  When feasible,  these studies should include toxicokinetics.
8 A recovery period should generally be included in study designs to determine the reversal or
potential worsening of pharmacological/toxicological effects, and/or potential delayed toxic
effects.  For biopharmaceuticals that induce prolonged pharmacological/toxicological effects,
recovery group animals should be monitored until reversibility is demonstrated.  The duration of
repeated dose studies should be based on the intended duration of clinical exposure and disease
indication.  This duration of animal dosing has generally been 1-3 months for most biotechnology-
derived pharmaceuticals.  For biopharmaceuticals intended for short-term use (e.g., < to 7 days)
and for acute life-threatening diseases, repeated dose studies up to 2 weeks duration have been
considered adequate to support clinical studies as well as  marketing authorization. For those
biopharmaceuticals intended for chronic indications, studies of 6 months duration have generally
been appropriate, although in some cases shorter or longer durations have supported marketing
authorizations.  For biopharmaceuticals intended for chronic use, the duration of long-term
toxicity studies should be scientifically justified.
E. Immunotoxicity Studies (4.5)
One aspect of immunotoxicological evaluation includes assessment of potential immunogenicity
(see section III.F (3.6)).  Many biotechnology-derived pharmaceuticals are intended to stimulate
or suppress the immune system and, therefore, may affect not only humoral but also cell-mediated
immunity.  Inflammatory reactions at the injection site may be indicative of a stimulatory
response.  It is important, however, to recognize that simple injection trauma and/or specific toxic
effects caused by the formulation vehicle may also result in toxic changes at the injection site.  In
addition, the expression of surface antigens on target cells may be altered, which has implications
for autoimmune potential.  Immunotoxicological testing strategies may require screening studies
followed by mechanistic studies to clarify such issues.  Routine tiered testing approaches or
standard testing batteries, however, are not recommended for biotechnology-derived
pharmaceuticals.
F. Reproductive Performance and Developmental Toxicity Studies (4.6)
The need for reproductive/developmental toxicity studies is dependent upon the product, clinical
indication and intended patient population (Note 2).  The specific study design and dosing
schedule may be modified based on issues related to species specificity, immunogenicity,
biological activity, and/or a long elimination half-life.  For example, concerns regarding potential
developmental immunotoxicity, which may apply particularly to certain monoclonal antibodies
with prolonged immunological effects, could be addressed in a study design modified to assess
immune function of the neonate.  
G. Genotoxicity Studies (4.7)
The range and type of genotoxicity studies routinely conducted for pharmaceuticals are not
applicable to biotechnology-derived pharmaceuticals and therefore are not needed.  Moreover, the
administration of large quantities of peptides/proteins may yield uninterpretable results.  It is not
9 expected that these substances would interact directly with DNA or other chromosomal material
(Note 3). 
Studies in available and relevant systems, including newly developed systems, should be
performed in those cases where there is cause for concern about the product (e.g., because of the
presence of an organic linker molecule in a conjugated protein product).  The use of standard
genotoxicity studies for assessing the genotoxic potential of process contaminants is not
considered appropriate.  If performed for this purpose, however, the rationale should be provided.
H. Carcinogenicity Studies (4.8)
Standard carcinogenicity bioassays are generally inappropriate for biotechnology-derived
pharmaceuticals.  However, product-specific assessment of carcinogenic potential may still be
needed depending upon duration of clinical dosing, patient population, and/or biological activity
of the product (e.g., growth factors, immunosuppressive agents, etc.).  When there is a concern
about carcinogenic potential, a variety of approaches may be considered to evaluate risk. 
Products that may have the potential to support or induce proliferation of transformed cells and
clonal expansion possibly leading to neoplasia should be evaluated with respect to receptor
expression in various malignant and normal human cells that are potentially relevant to the patient
population under study.  The ability of the product to stimulate growth of normal or malignant
cells expressing the receptor  should be determined.  When in vitro data give cause for concern
about carcinogenic potential, further studies in relevant animal models may be needed. 
Incorporation of sensitive indices of cellular proliferation in long-term repeated dose toxicity
studies may provide useful information.
In those cases where the product is biologically active and nonimmunogenic in rodents and other
studies have not provided sufficient information to allow an assessment of carcinogenic potential,
then the utility of a single rodent species should be considered.  Careful consideration should be
given to the selection of doses.  The use of a combination of pharmacokinetic  and
pharmacodynamic endpoints with consideration of comparative receptor characteristics and
intended human exposures represents the most scientifically based approach for defining the
appropriate doses.  The rationale for the selection of doses should be provided.  
I. Local Tolerance Studies (4.9)
Local tolerance should be evaluated.  The formulation intended for marketing should be tested;
however, in certain justified cases, the testing of representative formulations may be acceptable. 
In some cases, the potential adverse effects of the product can be evaluated in single or repeated
dose toxicity studies, thus obviating the need for separate local tolerance studies.
10 NOTES
Note 1. Animal models of disease may be useful in defining toxicity endpoints, selection of clinical
indications, and determination of appropriate formulations, route of administration, and treatment
regimen.  It should be noted that with these models of disease there is often a paucity of historical
data for use as a reference when evaluating study results.  Therefore, the collection of concurrent
control and baseline data is critical to optimize study design.
Note 2.  There may be extensive public information available regarding potential reproductive
and/or developmental effects of a particular class of compounds (e.g., interferons) where the only
relevant species is the nonhuman primate.  In such cases, mechanistic studies indicating that
similar effects are likely to be caused by a new but related molecule may obviate the need for
formal reproductive/developmental toxicity studies.  In each case, the scientific basis for assessing
the potential for possible effects on reproduction/development should be provided.
Note 3.  With some biopharmaceuticals, there is a potential concern about accumulation of
spontaneously mutated cells (e.g., via facilitating a selective advantage of proliferation) leading to
carcinogenicity.  The standard battery of genotoxicity tests is not designed to detect these
conditions.  Alternative in vitro or in vivo models to address such concerns may have to be
developed and evaluated.
11