Guest Column | July 18, 2023

FDA Finalizes Guidance On Nonclinical Evaluation Of Pharmaceutical Immunotoxicity

By Tim Sandle, Ph.D.

FDA headquarter iStock-1213293784

The U.S. FDA has now finalized a guidance document on the immunotoxic potential of pharmaceuticals1 after issuing the draft guidance in February 2020. The aim is to provide advice in terms of pharmaceutical product development for the purposes of nonclinical evaluation. There are several pharmacological impurities that can appear, through unintended routes, in formulated final products. These can arise through ingredients, as the result of reactions between product and materials, and as the result of contamination.

The agency defines immunotoxicity as “unintended immunosuppression or stimulation (including hypersensitivity), which can include adverse effects of exaggerated pharmacology of pharmaceuticals that are intended to function as immunomodulators.” Any applicable type of pharmaceutical, including most biologics, are within scope (however, cell and gene therapies, adjuvanted vaccines, and blood products are excluded from this guidance document).

Immunotoxic Potential

Immunotoxicology is an evolving scientific discipline, although allergic responses to various chemicals have long been recognized. Chemicals classed as immunotoxic will either suppress or enhance the immune system. Immunosuppression leads to the host being susceptible to infectious agents that the healthy body can otherwise resist, or it leads to a weakened immune response to pathogens. An enhanced immune system can lead to:

  • immunosuppression
  • hypersensitivity
  • adverse immunostimulation

When considering pharmaceuticals, immunotoxic pharmaceuticals will either be:

  • Medicines intended to modulate the immune function for therapeutic purposes, where adverse immunosuppression can be considered exaggerated pharmacodynamics. An example is drugs intended to avoid organ rejection following an organ transplant.
  • Drugs that are not intended to affect immune function but that cause immunotoxicity. This can lead to necrosis (passive accidental cell death) or apoptosis (alterations to the normal pattern of programmed cell death) of immune cells or an unintended interaction with cellular receptors shared by both the drug target tissues and non-target immune system cells.

Understanding the immunotoxic potential of different agents is not straightforward. The immune system of humans is complex, and various cells that comprise the immune system interact with one another along complex pathways to produce the desired effect.2 Often, studies are conducted in animal models, and for many agents the effects of autoimmunity cannot be reliably screened using laboratory assays. This is because autoimmunity in response to immunotoxic agents remains dependent upon the observations of individuals. Despite this, assays, including cytokine release assays, complement-dependent cytotoxicity assays, antibody-dependent cell-mediated cytotoxicity assays, complement activation assays, and proliferation/activation assays, can support the necessary assessment.

FDA’s Approach

The FDA builds on current ICH guidance3 and proposes a two-stage approach. The first tier is to determine if an immunotoxicity hazard is present. This is based on an assessment of:

  1. the pharmacological properties of the pharmaceutical,
  2. the intended patient population,
  3. a review of the structural similarities to compounds known to affect the immune system (note: with immunomodulators, the role of the intended target in immune function needs to be considered),
  4. the pharmaceutical disposition, and
  5. findings in clinical studies.

If the first-tier assessment identifies a potential immunotoxicity hazard, then a second-tier evaluation is required. This is a specific assessment of the immune function and the data from this will need to be used to characterize the specific risk to human subjects and patients.

Risk-Based Approach

Central to the FDA guidance is assessing immunotoxic effects using a risk framework. This is based on the weight of evidence (WoE) approach. This approach is a method of decision-making that involves consideration of multiple sources of information and lines of evidence. The risk assessment should include:

  • the mechanism of action of the drug (the assessment of how a drug produces an effect in the body, such as how it affects a specific target in a cell),
  • an assessment of nonclinical studies,
  • the predicted extent and duration of human exposure, including dosing periods,
  • the clinical population, where special consideration needs to be given to fetal, neonatal, and juvenile stages of development and to implantation and pregnancy,
  • the anticipated disease status of the population, and
  • application of concomitant medication.

The risk-based approach will need to evaluate the benefits of the medicine against the predicted effects. Where reactions are observed, discussions with regulators need to be held. For example, a positive response in a cytokine release assay does not necessarily preclude the next stage of drug development, where a careful, risk-based assessment can be made as to the magnitude and duration of the effect and the number and functions of cytokines affected. Alternative courses of action include lowering the starting dose, considering the need for other interventions, controlling dose escalation, and determining when to stop the active effect of the medicine.

Examples Of Immunotoxic Agents


Under certain conditions, fluorine presents a theoretical risk in terms of immune system disruption. When pharmaceuticals contain fluorine or a product may come into contact with perfluorinated compounds, an evaluation is required.

Fluorine-containing pharmaceuticals (fluoro-pharmaceuticals) have been attracting attention for more than half a century. Presently, about 20% of commercial pharmaceuticals are fluoro-pharmaceuticals.4

Perfluorinated compounds are a large group of compounds that are defined by a fluorinated hydrophobic linear carbon chain attached to one or more hydrophilic head groups. Materials produced using these chemicals are designed to repel both water and oil, and this makes them common chemicals for surface treatments.5 A common domestic example is Teflon.


One specific class of agents that is capable of exerting immunotoxicity is mycotoxins. Mycotoxins are metabolic products of filamentous fungi, with distinctive characteristics attributed to each species. More than 400 different mycotoxins have been identified to date. Mycotoxins can arise in pharmaceuticals either as an unintended byproduct from manufacturing where fungi are used in biotechnological manufacturing or as an extraneous contaminant.6 The immune responses are highly variable, with some of the most active immunosuppression caused by aflatoxin B1 (produced by molds like Aspergillus flavus and Aspergillus parasiticus).


A further example of an impurity that can arise is beta glucan. β-glucans are soluble polysaccharides of glucose. As an impurity, one source derives from the use of cellulosic-based filters in pharmaceutical processing, because the heterogeneous molecules of beta glucan constitute a major carbohydrate fraction of plant cell walls. Another source is fungal contamination.

High concentrations of ß-D-glucans can bind to and activate macrophages, neutrophils, monocytes, and NK cells. This can lead to the production of cytokines, which are important components in inflammation. Glucans are therefore considered pro-inflammatory molecules and they are capable of immuno-modulatory activity.7

Other Considerations

The FDA guidance refers to the need to assess the carcinogenicity risk of immunomodulators as an additional consideration in the development process. Not included in the FDA guidance, but a matter of wider societal importance, is the presence of immunomodulators in wastewater as a result of chemical discharges from pharmaceutical plants or from the discarding of unused medicines by consumers.


  1. FDA. Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals. Guidance for Industry, June 2023, U.S. Department of Health and Human Services,
  2. Blalock, J. E., and E. M. Smith. (1985) A complete regulatory loop between the immune and neuroendocrine systems. Fed. Proc. 44: 108
  3. ICH. Note for guidance on immunotoxicity studies for human pharmaceuticals, International Conference on Harmonization S8, CHMP/167235/2004, May 2006:
  4. Inoue M, Sumii Y, Shibata N. Contribution of Organofluorine Compounds to Pharmaceuticals. ACS Omega. 2020 Apr 22;5(19):10633-10640
  5. Steenland, Kyle; Fletcher, Tony; Savitz, David A. (2010) Epidemiologic Evidence on the Health Effects of Perfluorooctanoic Acid (PFOA) Environmental Health Perspectives. 118 (8): 1100–8
  6. Sandle, T. (2021) Mycological Assessment of Pharmaceutical Grade Raw Materials, American Pharmaceutical Review, March 2021,
  7. Sandle, T. (2013) Pharmaceutical Product Impurities: Considering Beta Glucans, American Pharmaceutical Review, August 2013,

About The Author:

Tim Sandle, Ph.D., is a pharmaceutical professional with wide experience in microbiology and quality assurance. He is the author of more than 30 books relating to pharmaceuticals, healthcare, and life sciences, as well as over 170 peer-reviewed papers and some 500 technical articles. Sandle has presented at over 200 events and he currently works at Bio Products Laboratory Ltd. (BPL), and he is a visiting professor at the University of Manchester and University College London, as well as a consultant to the pharmaceutical industry. Visit his microbiology website at