**S8 IMMUNOTOXICITY STUDIES FOR HUMAN PHARMACEUTICALS**

For questions regarding this draft document contact (CDER) Kenneth L.
Hastings at

301-827-5922.

This draft guidance, when finalized, will represent the Food and Drug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.

**IMMUNOTOXICITY STUDIES FOR HUMAN PHARMACEUTICALS**

**ICH Harmonised Tripartite Guideline**

Having reached *Step 2* of the ICH Process at the ICH Steering Committee
meeting

on 18 November 2004, the Committee releases this document for
consultation.

**TABLE OF CONTENTS**

1.  **INTRODUCTION
    ........................................................................1**

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1.  Objectives of the Guideline
    .......................................................1

2.  Background
    ....................................................\...................1

3.  Scope of the Guideline
    .......................................\...\...................1

4.  Overview
    ...\.........\.................................................................2

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2.  **GUIDELINE
    ..........................................................\...................2**

> 2.1 Assessment of Potential Immunotoxicity
> ....................\...................2

2.1.1 Standard Toxicity Studies
............................................2

2.1.2 Other Causes-for-Concern in the

Weight-of-Evidence Review ...\....................3

2.  Selection and Design of Additional

> Immunotoxicity Studies .............................4

1.  Selection of Assays
    ......................................................4

2.  Study Design
    ..........................................................\....4

3.  Evaluation of Need for Follow-up Testing
    ......................\....5

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3.  **FOLLOW-UP IMMUNOTOXICITY STUDIES**
    ..........\..........................5

4.  **Timing of Immunotoxicity Testing in Relation to**

**Clinical Studies** ........5

> Flow Diagram for Recommended
>
> Immunotoxicity Evaluation
> ...\.........................................6

Appendix 1 -- Methods to Evaluate Immunotoxicity
..................................7

**INTRODUCTION**

Evaluation of potential adverse effects of human pharmaceuticals on the
immune system should be incorporated into standard drug development.
Toxicity to the immune system encompasses a variety of adverse effects.
These include suppression or enhancement of the immune response.
Suppression of the immune response can lead to decreased host resistance
to infectious agents or tumor cells, whereas enhancing the immune
response can stimulate the expansion of autoreactive immune cells and
lead to autoimmune diseases. Drug or drug-protein adducts might also be
recognized as foreign and stimulate an anti-drug response. Subsequent
exposures to the drug can lead to hypersensitivity (allergic) reactions.
Much of the science and method development and validation efforts in the
past have been focused on evaluating drug development candidates for
their potential to be either immunosuppressive or contact (dermal)
sensitizers.

1.  **Objectives of the guideline**

The objectives of this guideline are to provide (1) recommendations on
nonclinical testing approaches to identify compounds which have the
potential to be immunosuppressive, and (2) guidance on a
weight-of-evidence decision making approach for immunotoxicity testing.

2.  **Background**

The most robust methods available for practical use in the assessment of
adverse drug effects on immune function are those designed to detect and
evaluate immunosuppression. Historically, unintended immunosuppression
has been causally related to antiproliferative drugs primarily used to
treat cancer. In such instances, adverse findings in nonclinical studies
are predictive of human immunotoxicity in a rather straightforward
manner. That is, specific assays to determine immunotoxicity are
probably not valuable in drug risk assessment since the target tissues
are usually rapidly dividing cell types, such as bone marrow-derived
immune system progenitor cells. Hence, the adverse effects on immune
function can be predicted based on pharmacologic activity and can
usually be reliably modelled in animals.

It has become apparent in recent years that immunosuppression can be
associated with other types of drugs. It is possible to divide these
drugs into two distinct groups: Those that are intended to modulate
immune function for therapeutic purposes (e.g. drugs intended to prevent
organ transplant rejection) where adverse immunosuppression can be
considered exaggerated pharmacodynamics, and those that are not intended
to affect immune function but can cause unintended immunosuppression due
for instance, to induced necrosis, apoptosis of immune cells or
interaction with cellular receptors shared by both target tissues and
non-target immune system cells. Although this difference is relatively
obvious, distinction between exaggerated pharmacodynamics and non-target
toxicity can be less obvious for certain classes of drugs (e.g.
anti-inflammatory drugs).

3.  **Scope of the Guideline**

This guideline is focused on providing recommendations on nonclinical
testing for immunosuppression induced by low molecular weight drugs
(non-biologicals). This guideline applies to new pharmaceuticals
intended for use in humans, as well as to marketed drug products
proposed for different indications or other variations on the current
product label in which the change could result in unaddressed and
relevant toxicologic issues. In addition, the guideline might also apply
to drugs in which clinical signs of immunosuppression are observed
during clinical trials and following approval to market. The term
immunotoxicity in this guideline will primarily refer to
immunosuppression, i.e. a state of increased susceptibility to
infections or the development of tumors.

It is beyond the scope of this guideline to provide specific guidance on
how each immunotoxicity study should be performed. General guidance is
provided in Appendix 1.

4.  **Overview**

The general principles that apply to this guideline are:

1)  All new investigational drugs should be evaluated for the potential
    to produce immunosuppression.

2)  Methods include standard toxicity studies (STS) and additional
    immunotoxicity studies conducted as appropriate. Whether additional
    immunotoxicity studies are appropriate should be determined by a
    weight of evidence review of cause(s) for concern.

The description of the guideline below will follow the flow diagram
shown in Figure 1. More detailed descriptions of the testing methods are
described in Appendix 1.

2.  **GUIDELINE**

**2.1 Assessment of potential immunotoxicity**

The initial screen for potential immunotoxicity involves the standard
toxicity studies. Data from rodent and non-rodent studies from early
short term to more chronic repeat-dose studies should be taken into
consideration. Additional details on the parameters that should be
evaluated and the reporting of histopathology findings are provided in
Appendix 1.

In addition to the findings from the STS, other causes for concern that
might prompt additional immunotoxicity studies include: (1) the
pharmacological properties of the drug, (2) the intended patient
population, (3) known drug class effects, and (4) the disposition of the
drug.

The flow diagram (Figure 1) illustrates the recommended decision process
in immunotoxicity evaluation.

**2.1.1 Standard toxicity studies**

Data from STS should be evaluated for signs of immunotoxic potential.
Signs that should be taken into consideration are the following.

(1) Hematological changes - Evidence of myelosuppression, usually seen
    in peripheral blood changes (e.g. pancytopenia, leukopenia,
    lymphopenia, or other blood dyscrasias);

(2) Alterations in immune system organ weights and histology (e.g.
    changes in thymus, spleen, lymph nodes, and/or bone marrow);

(3) Decreased basal serum immunoglobulins -- serum globulins are a
    rather insensitive marker of immunotoxicity due to the long half
    life of immunoglobulins. However, changes in globulins that occur
    without a plausible explanation can indicate potential
    immunotoxicity.

(4) Increased incidence of infections.

(5) Evidence of carcinogenicity, especially in the absence of
    genotoxicity.

If the findings from the STS indicate that there are signs of
immunotoxicity, the decision to conduct additional immunotoxicity
testing should be considered in a weight-of-evidence review of the data.
Similar to the assessment of risk with toxicities in other organ
systems, the assessment of immunotoxicity should include the following:

-   statistical and biological significance of the changes,

-   severity of the effects,

-   dose dependency,

-   safety factor above the expected clinical dose,

-   study duration,

-   number of species and endpoints affected,

-   changes that may occur secondarily to other factors (eg. stress, see
    appendix 1),

-   possible cellular targets and/or mechanism of action,

-   doses which produces these changes in relation to doses which
    produce other toxicities and

-   reversibility of effect(s).

**2.1.2 Other Causes for Concern in the Weight-of-Evidence Review**

> The following factors should also be considered:
>
> \(1\) If the pharmacological properties of a test compound indicate it
> has the potential to produce significant immunosuppression, additional
> immunotoxicity testing should be considered. For example,
> anti-inflammatory drugs are known to affect the function of certain
> types of cells of the immune system; however, their ability to
> suppress adaptive and/or innate immune responses is not clear.
> Information on the ability of the compound to affect the immune system
> can be gathered as part of the pharmacological studies conducted
> during the discovery or early development phases. These non-GLP
> pharmacology studies could be used in deciding if additional
> immunotoxicity studies are needed. The decision to conduct additional
> immunotoxicity studies should be based on a weight of evidence
> approach.
>
> \(2\) The targeted patient population should also be considered. For
> instance, additional immunotoxicity testing might be needed if the
> majority of the targeted patient population is immunocompromised.
>
> \(3\) Compounds structurally similar to compounds with known
> immunosuppressive properties should also be considered for additional
> immunotoxicity testing.
>
> \(4\) If the compound and/or its metabolites are known to be retained
> at high concentrations in cells of the immune system, additional
> immunotoxicity testing should be considered.

If signs of immunotoxicity are observed in STS and/or one of the above
four factors apply, it is recommended that the sponsor conduct studies
of drug effect on immune function or provide justification for not
performing these evaluations.

**2.2 Selection and Design of Additional Immunotoxicity Studies**

**2.2.1 Selection of assays**

If the weight-of-evidence approach indicates that additional
immunotoxicity studies are needed, there are a number of animal models
which can be used. If there are changes in standard toxicity testing
data suggesting immunosuppression, the type of additional immunotoxicity
testing that is appropriate will depend on the nature of the
immunological changes observed and concerns raised by the class of
compound.

It is recommended that an immune function study be conducted. Where a
specific target is not identified, an immune function study such as the
T-cell dependent antibody response (TDAR) is recommended. If specific
cell types are affected in STS, assays that measure function of that
specific cell type might be conducted (see appendix 1).

Immunophenotyping of leukocyte populations, a non-functional assay, can
be conducted to identify the specific cell populations affected and
useful clinical biomarkers.

2.  **Study Design**

It is a generally accepted study design to assess drug-induced
immunosuppression in studies with 28 consecutive daily oral doses in
mice or rats. The species, dose, duration, and route of administration
used in immune function assays should be consistent, where possible,
with the nonclinical toxicology study in which an adverse immune effect
was observed. The high dose should be above the no observed adverse
effect level (NOAEL) but below a level inducing changes secondary to
stress. Multiple dose levels are recommended in order to determine
dose-response relationships and the dose at which no immunotoxicity is
observed. Adaptations of immune function assays developed in rodents
have been described using non-rodent species. Under most circumstances,
immunological test methods can be appropriately modified for these other
species.

3.  **Evaluation of Need for Follow-up Testing**

Results of the entire data set should be evaluated as to whether
sufficient data are available to reasonably determine the risk of
immunotoxicity. If the overall risk-benefit analysis suggests that the
risk of immunotoxicity is acceptable, then no follow-up testing might be
called for.

**3. FOLLOW-UP IMMUNOTOXICITY STUDIES**

If changes are observed with immunotoxicity testing, further studies
should be considered to help determine the cell type affected and the
mechanism of action. This type of information can provide more insight
into potential risk and possibly lead to biomarker selection for
clinical studies. These assays can include natural killer cell, host
resistance or macrophage function assays. The findings from the STS
and/or additional nonclinical immunotoxicity testing will help determine
the need for, feasibility of, and type of clinical monitoring that is
appropriate. In situations where the development candidate might have a
pharmacological effect on the immune system, that specific component or
associated function could be monitored. Additional guidance is beyond
the scope of this guideline.

**4. Timing of Immunotoxicity Testing in Relation to Clinical Studies**

If the weight-of-evidence review indicates the need for additional
immunotoxicity studies, these should be completed before exposure of a
large population of patients to the drug. This will allow for the
incorporation of immunotoxicity testing in the clinical studies if
appropriate. The timing of the additional immunotoxicity testing might
be determined by the nature of the effect by the test compound and the
type of clinical testing that would be needed if a positive finding is
observed with the additional immunotoxicity testing. If the target
patient population is immunocompromised, immunotoxicity testing can be
initiated at an earlier time point in the development of the drug.

> **Flow Diagram for Recommended Immunotoxicity Evaluation**
>
> ![](media/image1.wmf)
>
> **Appendix 1**

**Methods to Evaluate Immunotoxicity**

**1. Standard Toxicity Studies**

The following table lists the parameters that should be evaluated in
standard toxicity studies for signs of immunotoxicity. These parameters
(excluding hematology and serum chemistry) and methods in obtaining
samples and evaluating tissue sections are described in more detail in
documents from professional toxicological pathology societies.

  ----------------- -----------------------------------------------------
  **Parameter**     **Specific Component**

  Hematology        Total leukocyte counts and absolute differential
                    leukocyte counts

  Clinical          Globulin levels and A/G ratios
  Chemistry         

  Gross pathology   Lymphoid organs / tissues

  Organ weights     thymus, spleen, (optional: lymph nodes)

  Histology         thymus, spleen, draining lymph node and at least one
                    additional lymph node, bone marrow, Peyer's patch
  ----------------- -----------------------------------------------------

**1.1 Hematology and Clinical Chemistry**

Total leukocyte counts (white blood cells) and absolute differential
leukocyte counts are recommended to assess for immunotoxicity. When
evaluating changes in globulin levels, other factors should be taken
into account (e.g. nephrotoxicity). Changes in serum globulins can be an
indication that there are changes in serum immunoglobulins. Although
serum immunoglobulins are an insensitive indicator of immunosuppression,
it can be useful in certain situations in order to better understand
target cell populations or mechanism of action.

**1.2 Gross Pathology and Organ Weights**

All lymphoid tissues should be evaluated for gross changes at necropsy.
However, this can be more difficult for the Peyer's patches of rats due
to the small size. Spleen and thymus weights should be recorded. To
minimize variability of spleen weights in dogs and monkeys, bleeding the
animals thoroughly at necropsy is recommended. Atrophy of the thymus
with ageing may preclude obtaining accurate thymus weight.

**1.3 Histopathological Examination**

Histopathological changes of the spleen and thymus should be evaluated
as an indicator of systemic immunosuppression. The lymphoid tissue that
drains or contacts the site of drug administration (and therefore is
exposed to the highest concentration of the drug) should be examined.
These sites include the Peyer's patches and mesenteric lymph nodes for
orally administered drugs, bronchus-associated lymphoid tissues (BALT)
for drugs administered by the inhalation route, nasal-associated
lymphoid tissues (NALT) for drugs administered by the inhalation or
nasal route, and the most proximal regional draining lymph nodes for
drugs administered by the dermal, intramuscular, intradermal,
intrathecal, or subcutaneous routes. The specific node selected should
be at the discretion of the sponsor based on the sponsors experience
with the nodes. For intravenously administered drugs, the spleen can be
considered the draining lymphoid tissue.

It is recommended that a "semi-quantitative" description of changes in
compartments of lymphoid tissues should be used in recording changes and
reporting treatment-related changes in lymphoid tissues.

**1.4 Interpretation of Stress Related Changes**

With standard toxicity studies, doses near or at the maximum tolerated
dose can result in changes to the immune system related to stress. These
effects on the immune system are most likely mediated by increased
corticosterone or cortisol release. Commonly observed stress-related
immune changes include increases in circulating neutrophils, decreases
in circulating lymphocytes, decreases in thymus weight, decreases in
thymic cortical cellularity and associated histopathologic changes
("starry sky" appearance), and changes in spleen and lymph node
cellularity. Increases in adrenal gland weight can also be observed. In
situations with clear clinical observations (eg. decrease body weight
gain, decreased activity), some or all of the changes to lymphoid tissue
and hematology parameters might be attributable to stress rather than to
a direct immunotoxic effect. The evidence of stress should be
compelling.

**2. Additional Immunotoxicity Studies**

**2.1 Assay Characterization and Validation**

In general, the immunotoxicity test selected should be widely used and
have been demonstrated to be adequately sensitive and specific for known
immunosuppressive agents. However, in certain situations, extensive
validation might have not been completed and/or the assay might not be
widely used. In these situations, a scientific / mechanistic basis for
use of the assay is needed and if feasible, appropriate positive
controls should be incorporated.

There can be variations of response for each type of immunotoxicity test
used by different labs. In most situations, these changes do not affect
the ability of the assay to assess immunotoxicity. However, to assure
proper assay performance and lab proficiency, several standard technical
validation parameters should be observed. These parameters can include
determining intra- and inter-assay precision, technician-to-technician
precision, limit of quantitation, linear region of quantitation and test
sample stability. In addition, assay sensitivity to known
immunosuppressive agents should be established. It is recommended that
each laboratory conduct a positive control study periodically in order
to demonstrate proficiency of performance, except for studies with
non-human primates.

**2.2 T-cell Dependent Antibody Response (TDAR)**

The TDAR should be performed using a recognized T-cell dependent antigen
(e.g. sheep red blood cells, SRBC or keyhole limpet hemocyanin, KLH)
that result in a robust antibody response. The endpoint selected should
be shown to be the most appropriate for the chosen assay. For the SRBC
assay, IgM is considered the most appropriate endpoint.

Antigens for immunization should not be used with adjuvants without
justification. Alum might be acceptable for use only in non-human
primate studies. The relative TDAR response can be strain-dependent,
especially in mice. With outbred rats, there can be significant
variability among rats within the same group. Inbred rat strains should
not be used unless sufficient exposure data are provided.

Antibody can be measured by using an ELISA or other immunoassay methods.
One advantage of this method over the antibody forming cell response is
that samples can be collected serially during the study. In monkeys,
serial blood collection can be important due to the high inter-animal
variability in the kinetics of the response. For these studies, data may
be expressed as the sum of the antibody response over several collection
dates (eg. area under the curve).

ELISA results should be expressed either as concentration or titer, but
expression as optical densities is not recommended.

When SRBC antigens are used for an ELISA, the preparation of the capture
antigen that is coated on the plates is considered critical. SRBC
capture antigen may be used as whole fixed erythrocytes or as membrane
preparations.

For the rat TDAR and immunophenotyping assays, the addition of positive
controls for each study with test compound might not be needed if the
method used has been demonstrated to be adequately sensitive to
immunosuppressive compounds.

**2.3 Immunophenotyping**

Immunophenotyping is the identification and/or enumeration of leukocyte
subsets using antibodies. Immunophenotyping is usually conducted by flow
cytometric analysis or by immunohistochemistry. With flow cytometry the
percentage and absolute counts of a specific cell type or activation
markers can be determined from large numbers of cells analyzed.

One of the advantages of immunohistochemistry over flow cytometry is
that tissues from standard toxicity studies can be analyzed
retrospectively if signs of immunotoxicity are observed. In addition,
changes in cell types within a specific compartment within the lymphoid
tissue can be observed. Some of the lymphocyte markers for certain
species are sensitive to formalin fixation and can only be localized in
tissue that are either fixed with certain fixatives or flash frozen. In
addition, quantitation of leukocytes among subsets and intensity of
staining is much more difficult with immunohistochemistry.

When immunophenotyping studies are used to characterize or identify
alterations in specific leukocyte populations, the choice of the
lymphoid organs to be evaluated should be based on changes observed.
Immunophenotyping can be easily added to standard repeat dose toxicity
studies and changes can be followed during the dosing phase and periods
without drug exposure (reversal period). When flow cytometry is employed
to enumerate specific cell populations, it is not a functional assay.
However, flow cytometry can be used to measure antigen-specific immune
responses of lymphocytes. Data obtained from peripheral blood can be
useful as a bridge for clinical studies in which peripheral blood
leukocytes are also evaluated. It is recommended that absolute numbers
of lymphocyte subsets rather than percentages be used in evaluating
treatment-related changes.

**2.4 Natural Killer Cell Activity Assays**

Natural killer (NK) cell activity assays can be conducted if
immunophenotyping studies demonstrate a decrease in number, or if STS
studies demonstrate increased viral infection rates, or in response to
other causes for concern. In general, all NK cell assays are ex vivo
assay in which tissues (e.g. spleen) or blood are obtained from animals
that have been treated with the test compound. Cell preparations are
co-incubated with target cells that have been labeled with chromium. New
methods that involve of non-radioactive labels can be used if adequately
validated. Different effector to target cell ratios should be evaluated
for each assay to obtain a sufficient level of cytotoxicity.

**2.5 Host Resistance Studies**

Host resistance studies involve challenging groups of mice or rats
treated with the different doses of test compound with varying
concentrations of a pathogen (bacteria, viral, parasitic) or tumor
cells. Infectivity of the pathogens or tumor burden observed in vehicle
versus test compound treated animals is used to determine if the test
compound is able to alter host resistance. Models have been developed to
evaluate a wide range of pathogens such as *Listeria monocytogenes*,
*Streptoccous pneumoninae*, influenza virus, cytomegalovirus,
*Plasmodium yoelii* and *Trichinella spiralis*. Tumor host resistance
models in mice have used the B16F10 melanoma and PYB6 sarcoma tumor cell
lines. Since the host defense mechanisms in these models are relatively
well understood, it is recommended to choose a host resistance assay
suitable for a specific target affected in standard toxicity studies or
other immune toxicity tests.

Host resistance assays have an important role in identifying or
confirming the cell type affected by a test compound. Since the host
defense mechanisms against some microbes or tumor cells are well
defined, test compound-related changes in these host resistance models
would demonstrate that those cells types are targets. In addition, host
resistance assay involve innate immune mechanisms in which specific
immune function assays have not been developed. In conducting host
resistance studies, the investigator should carefully consider the
direct or indirect (non-immune mediated) effects of the test compound on
the growth and pathogenicity of the organism or tumor cell. For
instance, compounds that inhibit the proliferation of certain tumor
cells can seem to increase host resistance.

**2.6 Macrophage / Neutrophil Function**

In vitro macrophage and neutrophil function assays (phagocytosis,
oxidative burst) have been published for several species. These assays
assess macrophage/neutrophil function of cells exposed to the test
compound in vitro or obtained from animals treated with the test
compound (ex vivo assay). In vitro exposure to test compound can also be
investigated. An in vivo assay can also be used to assess the effects on
the reticuloendothelial cell to phagocytize chromium-labeled sheep red
blood cells. Animals should be treated with the test compound and
injected with chromium-labeled sheep red blood cells. Animals should be
necropsied and tissues (eg. liver, spleen, lung, kidney) removed and
radioactivity is counted.

**2.7 Assays to Measure Cell-Mediated Immunity**

Assays to measure cell-mediated immunity have not been as well
established as those used for the antibody response. Delayed-type
hypersensitivity (DTH) reactions with KLH immunization and challenge
have been reported for mice. Cytotoxic T cell response can be generated
in mice, however, since these involve the administration of a tumor
cells line or graft, these assays are not considered toxicological
studies. Reports on monkey DTH reactions have also been reported.
However, these reactions in monkeys are very difficult to consistently
reproduce. In addition, one should make sure that the DTH response is
not mistaken for an antibody and complement mediated arthus reaction.
Models in which contact sensitizers are used have been explored in mice
but have not been well validated or extensively used.