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
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Guidance for Industry1

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. 
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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 drugspecific 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 antiinflammatory 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 
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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 
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(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. 
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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. 
Contains Nonbinding Recommendations

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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. 
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V. REFERENCES (5) 

1. ICH S6 Preclinical Safety Evaluation of Biotechnology-derived Pharmaceuticals (FDA, 

1997). 

2. ICH E2E Pharmacovigilance Planning (FDA, 2005). 
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FIGURE 1: FLOW DIAGRAM FOR RECOMMENDED IMMUNOTOXICITY 

EVALUATION 

 (2.1) Identify factors to consider

(2.2) Weight of evidence (WoE) review

Additional nonclinical

immunotoxicity testing

not needed

NO

(3.0) Conduct additional immunotoxicity studies

YES

WoE review

 warrants additional

immunotoxicity

Testing ?

(3.4) Significant

changes

observed?

YES

(3.4 Pt 1) Further

nonclinical

immunotoxicity testing

not needed

NO

All human pharmaceuticals (non-biologicals)

(3.4) Sufficient

data for risk

assessment / risk

management?

NO

YES

(3.4 Pt 2) Consider further immunotoxicity testing

(3.4 Pt 3) Further

nonclinical

immunotoxicity testing

not needed
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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, Peyer’s patch,3 BALT,4

NALT4

1

Unexplained alterations in globulin levels could call for measurement of immunoglobulins. 

2

Unexplained alterations in peripheral blood cell lines or histopathologic findings might suggest that cytologic 

evaluation of the bone marrow would be appropriate. 

3

Oral administration only. 

4

For 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 Peyer’s 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 
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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 (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 
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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 interanimal 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. 
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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. 
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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.