Guidance for Industry 
 
 
S8 Immunotoxicity Studies 
for Human 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) 
 
April 2006 
ICH 
 
 
   
Guidance for Industry 
 
 
S8 Immunotoxicity Studies 
for Human Pharmaceuticals
  
 
 
 
 
 
Additional copies are available from: 
 
Office of Training and Communication 
Division of Drug Information, HFD-240 
Center for Drug Evaluation and Research 
Food and Drug Administration 
5600 Fishers Lane 
Rockville, MD  20857 
(Tel) 301-827-4573 
 http://www.fda.gov/cder/guidance/index.htm 
 
Office of Communication, Training and  
Manufacturers Assistance, HFM-40  
Center for Biologics Evaluation and Research 
 Food and Drug Administration 
1401 Rockville Pike, Rockville, MD 20852-1448  
 http://www.fda.gov/cber/guidelines.htm.   
 
 
 
 
 
 
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) 
 
April 2006 
ICH 
   
TABLE OF CONTENTS 
 
 
 
I.  INTRODUCTION (1, 1.1)................................................................................................1 
A.  Background (1.2)............................................................................................................................2 
B.  Scope of the Guidance (1.3)...........................................................................................................2 
C.  Overview (1.4)................................................................................................................................3 
II.  GUIDANCE (2).................................................................................................................3 
A.  Factors to Consider in the Evaluation of Potential Immunotoxicity (2.1)................................3 
1. Standard Toxicity Studies (2.1.1).....................................................................................................3 
2. Pharmacological Properties (2.1.2)................................................................................................4 
3. Intended Patient Population (2.1.3).................................................................................................4 
4. Structural Similarity (2.1.4).............................................................................................................5 
5. Disposition of the Drug (2.1.5)........................................................................................................5 
6. Signs Observed in Clinical Trials or Clinical Use (2.1.6)...............................................................5 
B.  Weight-of-Evidence Review (2.2).................................................................................................5 
III.  SELECTION AND DESIGN OF ADDITIONAL  IMMUNOTOXICITY 
STUDIES (3)..................................................................................................................................5 
A.  Objectives (3.1)...............................................................................................................................5 
B.  Selection of Assays (3.2).................................................................................................................5 
C.  Study Design (3.3)..........................................................................................................................6 
D.  Evaluation of Additional Immunotoxicity Studies and Recommendations Concerning 
Further Studies (3.4)..............................................................................................................................6 
IV.   TIMING OF IMMUNOTOXICITY TESTING IN RELATION TO CLINICAL 
STUDIES (4)..................................................................................................................................6 
V.  REFERENCES (5)............................................................................................................7 
FIGURE 1:   FLOW DIAGRAM FOR RECOMMENDED IMMUNOTOXICITY 
EVALUATION.............................................................................................................................8 
APPENDIX:   METHODS TO EVALUATE IMMUNOTOXICITY......................................9 
  Contains Nonbinding Recommendations 
 
1
Guidance for Industry  
 
S8 Immunotoxicity Studies for Human 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, 1.1)2 
 
The objectives of this guidance are to provide (1) recommendations on nonclinical testing 
approaches to identify compounds that have the potential to be immunotoxic and (2) guidance on 
a weight-of-evidence decisionmaking approach for immunotoxicity testing. Immunotoxicity is, 
for the purpose of this guidance, defined as unintended immunosuppression or enhancement. 
Drug-induced hypersensitivity and autoimmunity are excluded. 
 
FDA's guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the Agency's current thinking on a topic and should 
be viewed only as recommendations, unless specific regulatory or statutory requirements are 
cited.  The use of the word should in Agency guidances means that something is suggested or 
recommended, but not required. 
 
 
 
 
                                                 
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, August 2005.  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, August 2005.  
 
  1 Contains Nonbinding Recommendations 
 
A.  Background (1.2) 
 
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. Enhancing the immune response can exaggerate autoimmune diseases or 
hypersensitivity. Drug or drug-protein adducts might also be recognized as foreign and stimulate 
an antidrug 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 for either 
immunosuppression or contact sensitization. No standard approaches for human pharmaceuticals 
are currently available for testing for respiratory or systemic allergenicity (antigenicity) or drug-
specific autoimmunity; testing for these endpoints is not currently required in any region.  There 
are no regional differences in testing approaches of skin sensitization.  
 
Immunosuppression or enhancement can be associated with two distinct groups:  
 
(1) Drugs intended to modulate immune function for therapeutic purposes (e.g., to prevent 
organ transplant rejection) where adverse immunosuppression can be considered 
exaggerated pharmacodynamics  
(2) Drugs not intended to affect immune function but cause immunotoxicity due, for instance, 
to necrosis or apoptosis of immune cells or interaction with cellular receptors shared by 
both target tissues and nontarget immune system cells  
 
Antiproliferative agents used to treat cancer are an example of drugs that produce unintended 
immunosuppression. 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 evaluated in nonclinical studies. For other types of 
compounds not intended to suppress the immune response, distinction between exaggerated 
pharmacodynamics and nontarget effects can be less obvious. As an example, some anti-
inflammatory compounds have an effect on certain innate immune functions but do not 
necessarily affect the adaptive immune response.  
 
B.  Scope of the Guidance (1.3) 
 
This guidance is focused on providing recommendations on nonclinical testing for 
immunotoxicity induced by human pharmaceuticals. It is restricted to unintended 
immunosuppression and immunoenhancement, excluding allergenicity or drug-specific 
autoimmunity.  
 
This guidance applies to new pharmaceuticals intended for use in humans, as well as to marketed 
pharmaceuticals proposed for different indications or other variations on the current product 
  2 Contains Nonbinding Recommendations 
 
label in which the change could result in unaddressed and relevant immunotoxicity issues. In 
addition, the guidance might also apply to drugs for which clinical signs of immunotoxicity are 
observed during clinical trials and following approval to market. The guidance does not apply to 
biotechnology-derived pharmaceutical products covered by ICH S6 (1) and other biologicals.  
 
Existing guidance documents on sensitization or hypersensitivity remain in force and are not 
affected by this document. It is beyond the scope of this guidance to provide specific guidance 
on how each immunotoxicity study should be performed. General methodology guidance is 
provided in the Appendix. 
 
C.  Overview (1.4) 
 
The general principles that apply to this guidance are: 
 
(1) All new human pharmaceuticals should be evaluated for the potential to produce 
immunotoxicity. 
(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 factor(s) in section II.A (2.1). 
 
The description of the guidance below will follow the recommended decision process in 
immunotoxicity evaluation as shown in the flow diagram (Figure 1).  More detailed descriptions 
of the testing methods are in the Appendix.   
   
II.  GUIDANCE (2) 
 
A.  Factors to Consider in the Evaluation of Potential Immunotoxicity (2.1) 
 
Factors to consider that might prompt additional immunotoxicity studies can be identified in the 
following areas: (1) findings from STS, (2) the pharmacological properties of the drug, (3) the 
intended patient population, (4) structural similarities to known immunomodulators, (5) the 
disposition of the drug, and (6) clinical information. 
 
The initial screen for potential immunotoxicity involves standard toxicity studies. Data from 
rodent and nonrodent 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 the appendix.   
 
1.  Standard Toxicity Studies (2.1.1) 
 
Data from STS should be evaluated for signs of immunotoxic potential. Signs that should be 
taken into consideration are the following: 
 
(1) Hematological changes such as leukocytopenia/leukocytosis, granulocytopenia/ 
granulocytosis, or lymphopenia/ lymphocytosis 
  3 Contains Nonbinding Recommendations 
 
(2) Alterations in immune system organ weights and/or histology (e.g., changes in thymus, 
spleen, lymph nodes, and/or bone marrow) 
(3) Changes in serum globulins that occur without a plausible explanation, such as effects on 
the liver or kidney, can be an indication that there are changes in serum immunoglobulins 
(4) Increased incidence of infections 
(5) Increased occurrence of tumors can be viewed as a sign of immunosuppression in the 
absence of other plausible causes such as genotoxicity, hormonal effects, or liver enzyme 
induction.  
 
Changes in these parameters could reflect immunosuppression or enhanced activation of the 
immune system. Immunosuppression is usually reflected by reduced values of immune 
parameters, whereas immunoenhancement is usually reflected by increased values. However, 
these relationships are not absolute and can be inverted in some cases. 
 
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/exposure relationship 
  Safety factor above the expected clinical dose 
  Treatment duration 
  Number of species and endpoints affected 
  Changes that may occur secondarily to other factors (e.g., stress, see the appendix, 
section A.4 (1.4)),  
  Possible cellular targets and/or mechanism of action  
  Doses that produce these changes in relation to doses that produce other toxicities 
  Reversibility of effect(s)   
 
2.  Pharmacological Properties (2.1.2) 
 
If the pharmacological properties of a test compound indicate it has the potential to affect 
immune function (e.g., anti-inflammatory drugs), additional immunotoxicity testing should be 
considered.  Information obtained from the nonclinical pharmacology studies on the ability of the 
compound to affect the immune system could be used in a weight-of-evidence approach to 
decide if additional immunotoxicity studies are needed.  
 
3.  Intended Patient Population (2.1.3) 
 
Additional immunotoxicity studies might be warranted if the majority of the patient population 
for whom the drug is intended is immunocompromised by a disease state or concurrent therapy. 
  4 Contains Nonbinding Recommendations 
 
 
4.  Structural Similarity (2.1.4) 
 
Compounds structurally similar to compounds with known immunosuppressive properties should 
also be considered for additional immunotoxicity testing. 
 
5.  Disposition of the Drug (2.1.5) 
 
If the compound and/or its metabolites are retained at high concentrations in cells of the immune 
system, additional immunotoxicity testing should be considered. 
 
6.  Signs Observed in Clinical Trials or Clinical Use (2.1.6) 
 
Clinical findings suggestive of immunotoxicity in patients exposed to the drug could call for 
additional nonclinical immunotoxicity testing. 
 
B.  Weight-of-Evidence Review (2.2) 
 
A weight-of-evidence review should be performed on information from all the factors outlined 
above to determine whether a cause for concern exists. A finding of sufficient magnitude in a 
single area should trigger additional immunotoxicity studies. Findings from two or more factors, 
each one of which would not be sufficient on its own, could trigger additional studies.  If 
additional immunotoxicity studies are not performed, the sponsor should provide justification.  
 
III.  SELECTION AND DESIGN OF ADDITIONAL  IMMUNOTOXICITY 
STUDIES (3) 
 
A.  Objectives (3.1) 
 
If a cause for concern is identified, additional immunotoxicity studies should be performed to 
verify the immunotoxic potential of the compound. These studies can also help determine the 
cell type affected, reversibility, and the mechanism of action.  This type of information can also 
provide more insight into potential risk and possibly lead to biomarker selection for clinical 
studies. 
 
B.  Selection of Assays (3.2) 
 
If the weight-of-evidence review indicates that additional immunotoxicity studies are called for, 
there are a number of assays that can be used. If there are changes in standard toxicity testing 
data suggesting  immunotoxicity, the type of additional immunotoxicity testing that is considered 
appropriate will depend on the nature of the immunological changes observed and the concerns 
raised by the class of compound. It is recommended that an immune function study be 
conducted, such as a T-cell dependent antibody response (TDAR). If specific cell types that are 
affected in STS are not known to participate in a TDAR, assays that measure function of that 
specific affected cell type might be conducted (see the appendix). Where a specific target is not 
identified, an immune function study such as the TDAR is recommended.  
  5 Contains Nonbinding Recommendations 
 
 
In addition, immunophenotyping of leukocyte populations, a nonfunctional assay, can be 
conducted to identify the specific cell populations affected and might provide useful clinical 
biomarkers.   
 
C.  Study Design (3.3) 
 
To assess drug-induced immunotoxicity, a generally accepted study design in rodents is a 28-day 
study with consecutive daily dosing. Adaptations of immunotoxicity assays have been described 
using nonrodent species. The species, strain, dose, duration, and route of administration used in 
additional immunotoxicity studies should be consistent, where possible, with the standard 
toxicity study in which an adverse immune effect was observed. Usually both sexes should be 
used in these studies, excluding nonhuman primates. Rationale should be given when one sex is 
used in other species. The high dose should be above the no observed adverse effect level 
(NOAEL) but below a level inducing changes secondary to stress (see appendix, section A.4 
(1.4)). Multiple dose levels are recommended in order to determine dose-response relationships 
and the dose at which no immunotoxicity is observed.  
 
D.  Evaluation of Additional Immunotoxicity Studies and Recommendations 
Concerning Further Studies (3.4)  
 
Results from additional immunotoxicity studies should be evaluated as to whether sufficient data 
are available to reasonably determine the risk of immunotoxicity. 
1.  Additional studies might show that no risk of immunotoxicity can be detected and no 
further testing is called for. 
2.  Additional studies might demonstrate a risk of immunotoxicity but fail to provide 
sufficient data to make a reasonable risk-benefit decision. In this case, further testing 
might help provide sufficient information for the risk-benefit decision. 
3.  If the overall risk-benefit analysis suggests that the risk of immunotoxicity is considered 
acceptable and/or can be addressed in a risk management plan (see ICH E2E (2)), then no 
further testing in animals might be called for. 
 
IV.   TIMING OF IMMUNOTOXICITY TESTING IN RELATION TO CLINICAL 
STUDIES (4) 
 
If the weight-of-evidence review indicates that additional immunotoxicity studies are 
appropriate, these should be completed before exposure of a large population of patients, usually 
phase 3.  This will allow for the incorporation of monitoring immune system parameters 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 called for 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. 
 
  6 Contains Nonbinding Recommendations 
 
V.  REFERENCES (5) 
 
1.  ICH S6 Preclinical Safety Evaluation of Biotechnology-derived Pharmaceuticals (FDA, 
1997). 
 
2.  ICH E2E Pharmacovigilance Planning (FDA, 2005). 
  7 Contains Nonbinding Recommendations 
 
FIGURE 1:   FLOW DIAGRAM FOR RECOMMENDED IMMUNOTOXICITY 
EVALUATION 
 
 
 
All human pharmaceuticals (non-biologicals)
 (2.1) Identify factors to consider
(2.2) Weight of evidence (WoE) review
WoE review
Additional nonclinical
 warrants additional
NO immunotoxicity testing
immunotoxicity
not needed
Testing ?
YES
(3.0) Conduct additional immunotoxicity studies
(3.4 Pt 1) Further
(3.4) Significant
nonclinical
changes NO
immunotoxicity testing
observed?
not needed
YES
(3.4) Sufficient (3.4 Pt 3) Further
data for risk nonclinical
YES
assessment / risk immunotoxicity testing
management? not needed
NO
(3.4 Pt 2) Consider further immunotoxicity testing
  8 Contains Nonbinding Recommendations 
 
APPENDIX:   METHODS TO EVALUATE IMMUNOTOXICITY 
 
 
A.  Standard Toxicity Studies (1) 
 
The following table lists the parameters that should be evaluated in standard toxicity studies for 
signs of immunotoxicity. These parameters (excluding hematology and clinical chemistry) and 
methods for obtaining samples and evaluating tissue sections are described in more detail in 
documents from professional toxicological pathology societies. 
 
Parameter  Specific Component 
Hematology  Total and absolute differential leukocyte counts  
Clinical Chemistry  Globulin levels1 and A/G ratios 
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 marrow2, Peyers patch,3 BALT,4 
NALT4 
1Unexplained alterations in globulin levels could call for measurement of immunoglobulins.   
2Unexplained alterations in peripheral blood cell lines or histopathologic findings might suggest that cytologic 
evaluation of the bone marrow would be appropriate.  
3Oral administration only. 
4For inhalation or nasal route only. BALT: bronchus-associated lymphoid tissues. NALT: nasal-associated lymphoid 
tissues 
 
1.  Hematology and Clinical Chemistry (1.1) 
 
Total leukocyte counts and absolute differential leukocyte counts are recommended to assess 
immunotoxicity. When evaluating changes in globulin levels, other factors should be taken into 
account (e.g., liver toxicity, 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, changes in immunoglobulins levels can be useful in 
certain situations in order to better understand target cell populations or mechanism of action. 
 
2.  Gross Pathology and Organ Weights (1.2) 
 
All lymphoid tissues should be evaluated for gross changes at necropsy.  However, this can be 
more difficult for the Peyers patches of rodents 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 aging 
can preclude obtaining accurate thymus weight.   
 
3.  Histopathological Examination (1.3) 
 
Histopathological changes of the spleen and thymus should be evaluated as an indicator of 
systemic immunotoxicity.  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 
  9 Contains Nonbinding Recommendations 
 
examined. These sites include the Peyers 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 (if possible), and the most proximal regional draining lymph nodes for 
drugs administered by the dermal, intramuscular, intradermal, intrathecal, or subcutaneous 
routes. The specific node selected and the additional lymph node should be at the discretion of 
the sponsor based on the sponsor's experience. 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 be used in recording changes and reporting treatment-related changes in 
lymphoid tissues. 
 
4.  Interpretation of Stress-Related Changes (1.4) 
 
With standard toxicity studies, doses near or at the maximum tolerated dose can result in changes 
to the immune system related to stress (e.g., by exaggerated pharmacodynamic action).  These 
effects on the immune system might be mediated by increased corticosterone or cortisol release 
or other mediators.  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, and changes in 
spleen and lymph node cellularity.  Increases in adrenal gland weight and/or histologic evidence 
of adrenal cortical hyperplasia can also be observed.  Thymic weight decreases in the presence of 
clinical signs, such as decreased body weight and physical activity, are too often attributed to 
stress. These findings on their own should not be considered sufficient evidence of stress-related 
immunotoxicity. The evidence of stress should be compelling in order to justify not conducting 
additional immunotoxicity studies. 
 
B.  Additional Immunotoxicity Studies (2) 
 
1.  Assay Characterization and Validation (2.1) 
 
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 called 
for 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 ensure 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 test a positive control 
concomitantly with an investigational compound or periodically in order to demonstrate 
  10 Contains Nonbinding Recommendations 
 
proficiency of performance, except for studies with nonhuman primates. For 
immunophenotyping, if properly validated technically, the addition of positive controls for each 
study might not be needed. 
 
Immunotoxicity studies are expected to be performed in compliance with good laboratory 
practice (GLP). It is recognized that some specialized assays, such as those described below, 
might not comply fully with GLP. 
 
2.  T-cell Dependent Antibody Response (TDAR) (2.2) 
 
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 results in a robust antibody 
response.  The endpoint selected should be justified as the most appropriate for the chosen assay 
and the selected species.  
 
Antigens for immunization should not be used with adjuvants without justification. Alum might 
be considered acceptable for use only in nonhuman 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 could be used with provision of sufficient 
exposure data to bridge to the strain used in the STS. 
 
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 can be expressed as the 
sum of the antibody response over several collection dates (e.g., area under the curve).  
 
When SRBC antigens are used for an ELISA, the preparation of the capture antigen that is 
coated on the plates is considered critical. Whole fixed erythrocytes or membrane preparations 
can be used as the SRBC capture antigen. ELISA results should be expressed either as 
concentration or as titer, but expression as optical densities is not recommended.  
 
3.  Immunophenotyping (2.3) 
 
Immunophenotyping is the identification and/or enumeration of leukocyte subsets using 
antibodies.  Immunophenotyping is usually conducted by flow cytometric analysis or by 
immunohistochemistry.  
 
Flow cytometry, when employed to enumerate specific cell populations, 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 as well as percentages be used in evaluating treatment-related 
changes. 
 
  11 Contains Nonbinding Recommendations 
 
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.  Quantitation of leukocytes 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 and/or peripheral blood 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).  
 
4.  Natural Killer Cell Activity Assays (2.4) 
 
Natural killer (NK) cell activity assays can be conducted if immunophenotyping studies 
demonstrate a change in number, or if STS demonstrate increased viral infection rates, or in 
response to other factors.  In general, all NK cell assays are ex vivo assays 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 51Cr.  New methods 
that involve nonradioactive 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 
and generate a curve.  
 
5.  Host Resistance Studies (2.5) 
 
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, fungal, 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, Streptococcus pneumoniae, Candida albicans, 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. 
 
Host resistance assays can provide information on the susceptibility to particular classes of 
infectious agents or tumor cells and can have an impact on the risk management plan. In 
addition, they can have an important role in identifying or confirming the cell type affected by a 
test compound. Moreover, host resistance assays involve innate immune mechanisms for which 
specific immune function assays have not been developed.  In conducting host resistance studies, 
the investigator should carefully consider the direct or indirect (nonimmune 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.  An in vitro assay to test direct effects on the organism is recommended. 
 
  12 Contains Nonbinding Recommendations 
 
6.  Macrophage/Neutrophil Function (2.6) 
 
In vitro macrophage and neutrophil function assays (phagocytosis, oxidative burst, chemotaxis, 
and cytolytic activity) 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 radioactively or fluorescently labeled targets. 
 
7.  Assays to Measure Cell-Mediated Immunity (2.7) 
 
Assays to measure cell-mediated immunity have not been as well established as those used for 
the antibody response. These are in vivo assays where antigens are used for sensitization. The 
endpoint is the ability of drugs to modulate the response to challenge. Delayed-type 
hypersensitivity (DTH) reactions with protein immunization and challenge have been reported 
for mice and rats. Models in which contact sensitizers are used have been explored in mice but 
have not been well validated or extensively used. Cytotoxic T-cell response can be generated in 
mice using a virus, tumor cell line, or allograft as the antigenic challenge. 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.   
 
  13