Nonclinical Evaluation of 
the Immunotoxic Potential 
of Pharmaceuticals 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
June 2023 
Pharmacology/Toxicology 
 
   
Nonclinical Evaluation of 
the Immunotoxic Potential 
of Pharmaceuticals 
Guidance for Industry 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information 
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor 
Silver Spring, MD 20993-0002 
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov 
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
June 2023 
Pharmacology/Toxicology 
 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
I.  INTRODUCTION ............................................................................................................. 1 
II.  BACKGROUND ................................................................................................................ 1 
III.  ASSESSING THE POTENTIAL TO SUPPRESS IMMUNE SYSTEM ACTIVITY 3 
A.  General Immunosuppression Assessment ................................................................................... 3 
B.  Immunosuppression and Carcinogenicity ................................................................................... 3 
C.  Immunosuppression and Opportunistic Infections .................................................................... 4 
VI.  ASSESSING THE POTENTIAL TO INCREASE IMMUNE SYSTEM ACTIVITY 5 
A.  Immunostimulation ....................................................................................................................... 5 
B.  Dermal Sensitization ...................................................................................................................... 7 
C.  Systemic Hypersensitivity-Based Reactions ................................................................................ 8 
V.  ASSESSING ADVERSE EFFECTS ON IMPLANTATION AND PREGNANCY .... 8 
VI.  ASSESSING DEVELOPMENTAL IMMUNOTOXICITY .......................................... 9 
VII.  RISK ASSESSMENT ........................................................................................................ 9 
VIII.  ADMINISTRATIVE CONSIDERATIONS .................................................................. 10 
REFERENCES ............................................................................................................................ 11 
 
  Contains Nonbinding Recommendations 
Nonclinical Evaluation of the Immunotoxic 
Potential of Pharmaceuticals 
Guidance for Industry1 
 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or 
Agency) on this topic.  It does not establish any rights for any person and is not binding on FDA 
or the public.  You can use an alternative approach if it satisfies the requirements of the 
applicable statutes and regulations.  To discuss an alternative approach, contact the FDA office 
responsible for this guidance as listed on the title page.   
 
 
 
I.  INTRODUCTION 
 
The purpose of this guidance is to assist sponsors in the nonclinical evaluation of the 
immunotoxic potential of pharmaceuticals. Immunotoxicity is, for the purposes of this guidance, 
defined as unintended immunosuppression or stimulation (including hypersensitivity), which can 
include adverse effects of exaggerated pharmacology of pharmaceuticals that are intended to act 
as immunomodulators.2 This guidance applies to drug products, including small molecule drugs 
and oligonucleotides, as well as certain biological products such as biotechnology-derived 
therapeutic proteins (referred to herein as biopharmaceuticals). For the purposes of this 
guidance, the term pharmaceutical will be used as a general term that encompasses all of these 
product types. Cell and gene therapies, adjuvanted vaccines, and blood products are not within 
the scope of this guidance. 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities. 
Instead, guidances describe the Agencys 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. 
 
 
II.  BACKGROUND 
 
The immune system is a complex and tightly regulated system that involves multiple biological 
components (e.g., circulating peptides, proteins and cells, tissue-resident cells, as well as 
 
1 This guidance has been prepared by the Office of New Drugs in the Center for Drug Evaluation and Research at 
the Food and Drug Administration. 
 
2 For the purposes of this guidance, an immunomodulator is a pharmaceutical that is intended to alter the 
performance of a discrete component of the immune system to either stimulate or suppress specific immune system 
activities. 
  1  Contains Nonbinding Recommendations 
specific tissues and organs distributed throughout the body) and mediates complex 
physiological responses, with the primary purpose of protecting the body from infections, 
tumors, and foreign substances. Perturbations of the function of the immune system have the 
potential to result in adverse consequencesimmunotoxicitywhich can in some cases be 
severe. Therefore, an assessment of the ability of pharmaceuticals to adversely affect the 
activity of the immune system is an essential component of evaluating the safety of 
pharmaceuticals. 
 
There are internationally harmonized guidances that provide recommendations related to the 
assessment of immunotoxicity risk. The primary guidances concerning immunotoxicity 
assessment of small molecule drugs and biopharmaceuticals are the ICH guidances for industry 
S8 Immunotoxicity Studies for Human Pharmaceuticals (April 2006)3 and S6(R1) Preclinical 
Safety Evaluation of Biotechnology-Derived Pharmaceuticals (May 2012). The guiding principle 
of both guidances is that the assessment of immunotoxic potential relies primarily upon 
integration of the findings of the general toxicity studies in combination with other data, such as 
(1) pharmacological properties of the pharmaceutical, (2) intended patient population, (3) 
structural similarities to compounds known to affect the immune system, (4) pharmaceutical 
disposition, (5) and findings in clinical studies. For immunomodulators, the role of the intended 
target in immune function should also be considered. When this first-tier assessment identifies a 
potential immunotoxicity hazard, more specific assessments of immune function may be 
warranted to further characterize the risk to human subjects and patients. 
 
In addition to these aforementioned ICH guidances, FDA maintained guidance for 
investigational new drugs that was published as final guidance in 2002.4 The guidance provided 
immunotoxicity guidance that was outside of the scope of ICH S8 (e.g., hypersenstitivity, 
adverse immunostimulation). This present guidance expands upon the previously withdrawn 
2002 guidance by broadening the scope to also include pharmaceuticals intended to affect the 
immune system, biopharmaceuticals, and oligonucleotides. Expanded guidance is also provided 
for assessing the carcinogenicity risk of immunomodulators, methods to assess the risk for 
adverse immunostimulation (e.g., cytokine release assays), nonanimal methods for assessing 
dermal sensitization, approaches for assessing the effects of immunotoxicants on pregnancy, and 
developmental immunotoxicity. 
 
This guidance finalizes the draft guidance for industry Nonclinical Safety Evaluation of the 
Immunotoxic Potential of Drugs and Biologics (February 2020).5 
 
 
3 We update guidances periodically. For the most recent version of a guidance, check the FDA guidance web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
 
4 See the guidance for industry Immunotoxicology Evaluation of Investigational New Drugs (October 2002)
withdrawn. 
 
5 When final, this guidance will represent the FDAs current thinking on this topic. For the most recent version of a 
guidance, check the FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-
documents. 
 
  2  Contains Nonbinding Recommendations 
For product-specific recommendations related to this guidance, FDA recommends that sponsors 
contact the appropriate review division.6 
 
 
III.  ASSESSING THE POTENTIAL TO SUPPRESS IMMUNE SYSTEM ACTIVITY 
 
A.  General Immunosuppression Assessment 
 
As noted above, internationally harmonized guidance regarding nonclinical immunotoxicity 
assessment of small molecule drugs and biopharmaceuticals is provided by ICH S8 and ICH 
S6(R1), respectively. The following concepts for the assessment of immunosuppression risk 
should be considered in conjunction with these guidances. 
 
For pharmaceuticals (including biopharmaceuticals) that are intended to act via a suppressive 
immunomodulatory mechanism of action (MoA), sponsors should fully assess the known 
immunobiology of the intended mode of action as it relates to the potential for adverse 
consequences of exaggerated pharmacology. For less well-characterized immune targets, this 
assessment may warrant one or more dedicated nonclinical studies7 to better inform the risk of 
exaggerated target engagement. In addition, dedicated assays may be warranted to characterize 
the effects of the pharmaceutical on other related immune functions. The specifics of on- and off-
target assay selection should be dictated by the intended MoA of the pharmaceutical along with 
scientific evaluation of how the intended immune suppression may affect related activities of the 
immune system. 
 
For biopharmaceuticals that are not intended to affect the immune system, sponsors may 
consider performing an integrated evaluation of the potential for unintended immunosuppression. 
This evaluation could be similar in principle to the weight-of-evidence (WoE) evaluation as 
described in ICH S8. Whether any additional studies are warranted would be guided by the 
findings of the integrated evaluation.  
 
For small molecule drugs that act via an antiproliferative MoA, such as those used to treat 
cancer, follow-up assays such as those discussed in ICH S8 are generally not warranted. 
 
B.  Immunosuppression and Carcinogenicity 
 
Immunosuppression has the potential to increase the risk of certain tumor types in humans, 
depending on which components of the immune system are suppressed and to what extent. 
Tumors arising from immunosuppression are primarily associated with loss of control of 
chronic/latent pathogen infections, although direct interference with tumor surveillance could 
 
6 See the draft guidance for industry Formal Meetings Between the FDA and Sponsors or Applicants of PDUFA 
Products (December 2017). When final, this guidance will represent the FDAs current thinking on this topic. 
 
7 FDA supports the principles of the 3Rs (replace/reduce/refine) for animal use in testing when feasible. FDA 
encourages sponsors to consult with review divisions when considering a nonanimal testing method believed to be 
suitable, adequate, and feasible. FDA will consider whether the alternative method is adequate to meet the 
nonclinical regulatory need. 
 
  3  Contains Nonbinding Recommendations 
also result in an increased risk for tumors. As such, sponsors should consider the effects of a 
drug on the immune system when assessing its carcinogenic potential. 
 
Sponsors should follow the recommendations in the ICH guidances for industry S1A The Need 
for Long-term Rodent Carcinogenicity Studies of Pharmaceuticals (March 1996), ICH S6(R1), 
S9 Nonclinical Evaluation for Anticancer Pharmaceuticals (March 2010), and S9 Nonclinical 
Evaluation for Anticancer PharmaceuticalsQuestions and Answers (June 2018) regarding the 
need for a carcinogenicity assessment as well as which experimental approaches may be 
warranted. To date, animal models, including rodent carcinogenicity studies, have been shown 
to be of limited help in identifying an increased cancer risk that may arise in patients as a 
consequence of immunosuppression. This is particularly true when the increased tumor risk is 
caused by recrudescence of latent viral oncogenes, infectious agents, or chronic inflammatory 
states, for which significant species differences exist that make clinical translatability 
challenging. For small molecule drugs for which an increased risk of carcinogenicity is 
anticipated as a result of profound drug-induced immunosuppression (e.g., antirejection 
pharmaceuticals), a written, WoE-based risk assessment may be adequate to assess 
carcinogenicity risk. A WoE-based risk assessment is also particularly relevant for 
pharmaceuticals that lack the intended pharmacological activity in rodents and/or when standard 
carcinogenicity studies in rodents are not technically feasible. A WoE-based risk assessment 
may also be adequate for most immunomodulatory biopharmaceuticals. 
 
This WoE-based risk assessment should address relevant attributes of the drug and drug 
target(s), including an evaluation of the impact on immune cell subpopulations and the potential 
for a drug to increase tumor promotion, growth, and metastasis, with an emphasis on clinical 
translatability. The effects of the pharmaceutical on key immune components thought to be 
involved in tumor surveillance (e.g., natural killer cells, T cells, antigen-presenting cells), such 
as downregulation or functional impairment of key immune cell populations, should be 
considered. For small molecule drug products in particular, the WoE-based risk assessment 
should also address the carcinogenic relevance of any compound-specific toxicology findings 
not related to the products intended effect on the immune system (e.g., off-target activity).  
 
For small molecule drugs that act through a more targeted modulation of the immune system, 
such that profound immunosuppression is unlikely, the conduct of one or more rodent 
carcinogenicity studies may be warranted.8 
 
FDA recommends that sponsors discuss their proposed approach for assessing carcinogenic 
potential of their product with the appropriate review division. 
 
C.  Immunosuppression and Opportunistic Infections 
 
Profound immunosuppression is known to increase the risk of opportunistic infections, which 
are typically associated with a wide range of bacteria, viruses, fungi, and parasites. The standard 
general toxicology studies are not designed to be able to reliably predict the risk of these 
 
8 See the ICH draft guidance for industry S1B(R1) Addendum to S1B Testing for Carcinogenicity of 
Pharmaceuticals (November 2022). When final, this guidance will represent the FDAs current thinking on this 
topic. 
  4  Contains Nonbinding Recommendations 
infections occurring in humans. If the WoE review suggests a cause for concern, then an 
appropriately focused host resistance model, which may provide additional insight, could be 
considered. The design of such a study, and whether it is warranted, should be discussed with 
the appropriate review division. Regardless, the risk of opportunistic infections in humans as a 
result of drug-induced immunosuppression should be evaluated clinically.   
 
 
VI.  ASSESSING THE POTENTIAL TO INCREASE IMMUNE SYSTEM ACTIVITY 
 
A.  Immunostimulation 
 
Assessment of the risks associated with a heightened immune response normally requires a 
safety evaluation paradigm that differs from that of immunosuppression, and may use specific 
assays or alternative methodologies for translation to first-in-human trials.  
 
Except where indicated, immunostimulatory pharmaceuticals are defined in this section as 
products that are intended to either directly stimulate signaling in an immune cell subtype or 
indirectly enhance the immune system response by blocking or activating an endogenous 
regulator of the immune system response. 
 
Toxicities of these products are often the result of exaggerated pharmacological activity but 
may also be caused by expression of the target receptor in untargeted immune cell 
subpopulations or tissues. It is therefore important to understand the MoA of the 
pharmaceutical when determining which assays are most appropriate to evaluate the potential 
hazards. For example, assays used when evaluating the immunotoxicity of T cell-engaging 
(direct activation) molecules would be different from assays used when evaluating checkpoint 
inhibitors or receptor agonists. The MoA, cellular distribution of the molecule, and potential to 
induce direct immunostimulation in key cell types (e.g., T cells, dendritic cells) should guide 
the approach to assay selection.  
 
The following are examples of immunostimulatory effects: 
 
  Excessive release of cytokines that can cause severe adverse reactions9 as shown by 
the near-fatal clinical responses to the monoclonal antibody TGN1412 (Duff 2006). It 
should be noted that the intended MoA of TGN1412 was immunosuppression through 
activation of regulatory T cells, but its administration resulted in strong 
immunostimulation of effector memory T cells. There are now commonly used in vitro 
models available to evaluate the potential for this hazard (Bugelski et al. 2009; Finco et 
al. 2014; Grimaldi et al. 2016).  
 
  General immune activation of T cells, T regulatory cells, or antigen-presenting cells 
caused by checkpoint inhibitor engagement resulting in immune-mediated tissue damage 
in nontarget organs. 
 
 
9 See guidance for industry Immunogenicity Assessment for Therapeutic Protein Products (August 2014). 
 
  5  Contains Nonbinding Recommendations 
  Rapid, generalized depletion of target cells via immune clearance mechanisms resulting 
in generalized immune activation (with or without cytokine release) as can be observed 
with rapid Fc receptorbased clearance. 
 
  Activation of pathogen-associated molecular pattern receptors (e.g., toll-like receptors) 
and/or complement by oligonucleotides. 
 
Below are examples of assays that may be considered for the evaluation of these types of 
hazards. This list is not intended to be all-inclusive, as new assays and technologies are 
constantly emerging and evolving. Sponsors should use the criteria defined above to determine 
which assays will best identify the hazards associated with their molecule and what dosing and 
monitoring will be most appropriate for the first-in-human clinical trial. All assays should 
include appropriate positive and negative controls and have clear, measurable outcomes. 
Information should be provided to support that the conducted assay(s) is fit-for-purpose (e.g., 
validation study results, literature citation). 
 
  Cytokine release assay  Standard assays to date include use of soluble and immobilized 
formats using peripheral blood mononuclear cells, whole blood, and other matrices. Other 
formats that may be considered include endothelial cell/peripheral blood mononuclear 
cell co-culture assay, tumor and endothelial cell co-cultures, and use of patient-derived 
cells.  
 
  Complement-dependent cytotoxicity/antibody-dependent cell-mediated cytotoxicity 
assays  Complement-dependent cytotoxicity and antibody-dependent cell-mediated 
cytotoxicity assays evaluate cytolytic processes using either complement or antibody-
dependent interactions for cell lysis to occur.   
 
  Complement activation assays  Measurement of the levels in serum of activated 
complement fragments (C3a, C5a) and/or the Bb split product. 
 
  Proliferation/activation assays  Many assays exist to assess proliferation, including 
carboxyfluorescein succinimidyl ester (CFSE) proliferation assay, 5-bromo-2-
deoxyuridine incorporation assay, flow cytometric assessment of activation markers, and 
the enzyme-linked immune absorbent spot (ELISpot) assay, among others. Such assays 
will evaluate the ability of T cells to activate and proliferate and in some cases assess 
innate immune activation of antigen-presenting cells. 
 
  Other assays  Assays typically used to assess vaccine effectiveness or cellular 
proliferation such as MIMIC (minimal methylation classifier) and other new technologies 
can be considered depending on the molecule being evaluated. Novel methods, including 
microphysiological systems or immune-humanized mice, may also be options depending 
on the validation of these techniques. 
 
If a product has already demonstrated a clear potential to directly cause cytokine release (e.g., a 
CD3 bispecific T cell redirector) or induce other forms of immune activation, then additional 
  6  Contains Nonbinding Recommendations 
assays may not be necessary, as the hazard has already been identified. Similarly, if one assay is 
positive, then an assay in another format may not be warranted. 
 
Although a positive response in a cytokine release assay may not preclude further development 
of a drug, depending on the indication, magnitude/duration of the effect, and/or the number and 
functions of cytokines affected, it may impact the selection of the appropriate starting dose and 
inform clinical monitoring, the need for potential interventions, and dose escalation and 
stopping criteria.  
 
For biopharmaceuticals intended to stimulate an immune response either directly or indirectly, a 
starting dose based on a minimal anticipated biologic effect level or a pharmacologically active 
dose may be more appropriate than a starting dose based on toxicology endpoints, such as the 
no-observed-adverse-effect level. The same approach may apply to drug products intended to 
stimulate specific immune system outcomes, depending on the relevance of available animal 
models.  
 
The above approaches may also be suitable for assessing the immunotoxicity risk of products 
whose intended pharmacology does not involve immune cell activation but nonetheless are 
expected to activate components of the immune system (e.g., oligonucleotides). 
 
B.  Dermal Sensitization 
 
Topical pharmaceuticals should be assessed for their dermal sensitization potential. Dermal 
sensitization is an immune response to a previously encountered chemical or substance that 
results in an inflammatory dermal reaction upon reexposure. As an assessment of skin 
sensitization for individual chemicals, FDA will consider a battery of studies (e.g., in silico, in 
chemico, in vitro) that have been shown to adequately predict human skin sensitization with an 
accuracy similar to existing in vivo methods. Several in chemico and in vitro assays for skin 
sensitization and defined approaches for combining data are described in the Organisation for 
Economic Co-operation and Development (OECD) test guidelines.10 Additionally, FDA also 
currently accepts the guinea pig maximization test using the clinical formulation to assess the 
sensitization potential of a topical drug product. FDA will also accept the murine local lymph 
node assay but no longer recommends this assay because of its technical limitations (Basketter 
and Kimber 2011). 
 
 
10 See OECD (2021), Test No. 442C: In Chemico Skin Sensitisation: Assays addressing the Adverse Outcome 
Pathway key event on covalent binding to proteins, OECD Guidelines for the Testing of Chemicals, Section 4, OECD 
Publishing, Paris, https://doi.org/10.1787/9789264229709-en; OECD (2018), Test No. 442D: In Vitro Skin 
Sensitisation: ARE-Nrf2 Luciferase Test Method, OECD Guidelines for the Testing of Chemicals, Section 4, OECD 
Publishing, Paris, https://doi.org/10.1787/9789264229822-en; OECD (2018), Test No. 442E: In Vitro Skin 
Sensitisation: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the 
Adverse Outcome Pathway for Skin Sensitisation, OECD Guidelines for the Testing of Chemicals, Section 4, OECD 
Publishing, Paris, https://doi.org/10.1787/9789264264359-en; OECD (2021), Guideline No. 497: Defined 
Approaches on Skin Sensitisation, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, 
Paris, https://doi.org/10.1787/b92879a4-en. 
 
  7  Contains Nonbinding Recommendations 
For topical pharmaceuticals, data from the dermal sensitization assessment should be submitted 
at the time of investigational new drug application submission.  
 
C.  Systemic Hypersensitivity-Based Reactions 
 
Systemic hypersensitivity reactions are significant potential pharmaceutical-induced adverse 
reactions. To date, however, there are no standard nonclinical assays to adequately evaluate the 
potential risks. Fit-for-purpose assays and/or a WoE assessment may be considered as long as 
they are appropriate and scientifically justified. For example, for drug-induced anaphylactoid 
reactions, in vitro assays such as complement activation or mast cell/basophil activation assays 
may have value in assessing risk. FDA recommends that sponsors discuss their proposed 
approaches for assessing systemic hypersensitivity risk, and whether such studies are warranted, 
with the appropriate review division. 
 
 
V.  ASSESSING ADVERSE EFFECTS ON IMPLANTATION AND PREGNANCY  
 
Pharmaceuticals that affect the maternal immune system have the potential to adversely affect 
implantation of the embryo, fetal development, and the ability to maintain the pregnancy. 
 
For pharmaceuticals that are not intended to affect the immune system, the risk for adverse 
effects on the maternal immune system that can affect implantation and gestation would typically 
be identified in the fertility and early embryonic development and/or embryo-fetal development 
studies conducted on most pharmaceuticals.11 These studies would generally be considered 
adequate for assessing this risk. 
 
Similarly, for pharmaceuticals that are intended to affect the immune system, fertility and early 
embryonic development and embryo-fetal development studies may have utility in characterizing 
the risk for adverse effects on the maternal immune system that can affect implantation and 
gestation. However, if the MoA of a pharmaceutical is known to be incompatible with fertility or 
maintenance of pregnancy, it may be appropriate to assess the risk based on a WoE 
approach.12,13 
 
Fertility and early embryonic development studies are not generally warranted for 
pharmaceuticals intended to treat patients with advanced cancer.14 
 
 
 
 
11 See the ICH guidance for industry S5(R3) Detection of Reproductive and Developmental Toxicity for Human 
Pharmaceuticals (May 2021). 
 
12 Ibid. 
 
13 See the guidance for industry Oncology Pharmaceuticals: Reproductive Toxicity Testing and Labeling 
Recommendations (May 2019). 
 
14 See ICH S9. 
  8  Contains Nonbinding Recommendations 
VI.  ASSESSING DEVELOPMENTAL IMMUNOTOXICITY 
 
Pharmaceuticals that have no appreciable adverse effect on the mature immune system 
nonetheless have the potential to adversely affect the developing immune system. Given the 
extended period over which the immune system develops, the immune system can be sensitive 
to adverse effects of pharmaceuticals throughout the fetal, neonatal, and juvenile stages of 
development. 
 
As with the assessment of immunotoxicity of the mature immune system, an evaluation of the 
potential of a pharmaceutical to adversely affect the developing immune system relies on a 
WoE integration of multiple factors, as described in ICH S8. 
 
If there is a concern that immune system development could be adversely affected by a 
pharmaceutical, and the existing data do not adequately characterize the risk to the exposed 
subject (pediatric patient or in utero or lactationally exposed infant), then sponsors should 
provide additional data to characterize this risk. As recommended in the ICH guidance for 
industry S11 Nonclinical Safety Testing in Support of Development of Pediatric 
Pharmaceuticals (May 2021), this can include the evaluation of immunotoxicology endpoints 
in the offspring of treated dams in otherwise warranted developmental toxicology studies (i.e., 
pre- and postnatal development (PPND) study or enhanced PPND (ePPND) study) if adequate 
exposure to the pharmaceutical is demonstrated in the offspring. A juvenile animal study in 
which juveniles are directly exposed to the pharmaceutical may be warranted if the 
PPND/ePPND study is unable to adequately characterize the risk. Juvenile and PPND/ePPND 
studies are generally not warranted for pharmaceuticals intended to treat patients with advanced 
cancer. 
 
Should a study be warranted to assess the risk of developmental immunotoxicity, the test 
species should be appropriate for the endpoints being assessed. The endpoints to be included 
should be scientifically justified and appropriate for assessing the concern, and could include 
enumeration of specific immune cell populations (immunophenotyping), the function of the 
immune system and its components, and/or the anatomical integrity of the immune system. As 
there are differences in the timing of immune system developmental landmarks across species 
(Skaggs et al. 2019), sponsors should ensure that the dosing interval covers the intended 
developmental period. 
 
To avoid the potentially unwarranted use of animals, sponsors should consult with the 
appropriate review division before conducting ePPND or juvenile animal studies, particularly if 
the studies are being conducted solely for assessing developmental immunotoxicity risk. 
 
 
VII.  RISK ASSESSMENT 
 
There is limited understanding of the extent of reduced (or increased) immune function required 
to have a significant biological effect in humans (e.g., increased risk of infection, tumor 
development, or autoimmunity). A WoE approach where all immunotoxicity data are 
considered as a whole (e.g., consideration of the MoA of the drug, the translatability of 
  9  Contains Nonbinding Recommendations 
nonclinical findings, the predicted extent and duration of human exposure, the clinical 
population, disease status, concomitant medication, etc.) is recommended when interpreting the 
findings of immunotoxicity assays and when considering the risk of clinically significant 
immunotoxicity occurring in humans. 
 
 
VIII.  ADMINISTRATIVE CONSIDERATIONS 
 
Sponsors should, to the extent practicable, follow existing guidance on placing 
immunotoxicology studies in the electronic common technical document (eCTD) format.15 
Stand-alone immunotoxicology studies should be included in the eCTD in section 4.2.3.7.2, 
with the exception of stand-alone assessments of antigenicity (allergenicity), which should be 
included in section 4.2.3.7.1. WoE assessments of immunotoxicology should be submitted to 
eCTD section 4.2.3.7.2. Data evaluating the immune system, which are part of a general repeat-
dose toxicity study, including immunogenicity (antidrug antibody formation) data, should be 
included with the repeat-dose toxicity study in section 4.2.3.2. Please refer to the FDA eCTD 
technical specification The Comprehensive Table of Contents Headings and Hierarchy16 for 
further details.
 
15 See the ICH guidance for industry M2 eCTD: Electronic Common Technical Document Specification (April 
2003). 
 
16 Accessible at https://www.fda.gov/downloads/drugs/ucm163175.pdf. 
 
  10  Contains Nonbinding Recommendations 
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8  499521. 
9   
10  Duff, GW, Expert Scientific Group on Phase One Clinical Trials, 2006, available at 
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13   
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15  Schneider, A Ibraghimov, S Scesney, D Serna, R Prell, R Stebbings, and PK Narayanan, 2014, 
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17   
18  Grimaldi, C, D Finco, MM Fort, D Gliddon, K Harper, WS Helms, JA Mitchell, R OLone, ST 
19  Parish, M-S Piche, DM Reed, G Reichmann, PC Ryan, R Stebbings, and M Walker, Cytokine 
20  Release: A Workshop Proceedings on the State-of-the-Science, Current Challenges and Future 
21  Directions, 2016, Cytokine, 85, 101108. 
22   
23  Skaggs, H, GJ Chellman, M Collinge, B Enright, CL Fuller, J Krayer, L Sivaraman, and GF 
24  Weinbauer, 2019, Comparison of Immune System Development in Nonclinical Species and 
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27   
  11