Immunotoxicity Testing Research Articles

Regulatory view on the relevance of the TDAR in immunotoxicity testing

Regulatory view on the relevance of the TDAR in immunotoxicity testing

Use of IL-18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens

Assessment of allergenic potential of chemicals is performed using animal models, such as the murine local lymph node assay, which does not distinguish between respiratory and contact allergens. Progress in understanding the mechanisms of skin sensitization, provides us with the opportunity to develop in vitro tests as an alternative to in vivo sensitization testing. The aim of the present study was to evaluate the possibility to use intracellular interleukin-18 (IL-18) production to assess in vitro the contact sensitization potential of low molecular weight chemicals. The human keratinocyte cell line NCTC2455 was used. Cells were exposed to contact allergens (cinnamaldehyde, dinitrochlorobenzene, glyoxal, isoeugenol, p-phenylediamine, resorcinol, tetramethylthiuram disulfide, 2-mercaptobenzothiazole, 4-nitrobenzylbromide), to proaptens (cinnamyl alcohol, eugenol), to respiratory allergens (diphenylmethane diisocyanate, trimellitic anhydride, ammonium hexachloroplatinate) and to irritants (sodium lauryl sulphate, salicylic acid, phenol). Cell associated IL-18 were evaluated 24 later. At not cytotoxic concentrations (cell viability higher of 75%, as assessed by MTT reduction assay), all contact sensitizers, including proaptens, induced a dose-related increase in IL-18, whereas both irritants and respiratory failed. Similar results were also obtained using primary human keratinocytes. Results were reproducible, and the method could be transferred to another laboratory, suggesting the potential use of the test in immunotoxicity testing strategies. Overall, results obtained indicated that cell-associated IL-18 may provide an in vitro tool for identification and discrimination of contact versus respiratory allergens and/or irritants.

Patterns of Immunotoxicity Associated with Chronic as Compared with Acute Exposure to Chemical or Physical Stressors and their Relevance with Regard to the Role of Stress and with Regard to Immunotoxicity Testing

Previous studies have demonstrated that the stress response induced by some drugs and chemicals contributes in a predictable way to alteration of particular immunological parameters in mice. It has not been determined if mice can become tolerant or habituated with regard to the stress response and consequent immunological effects. Addressing this issue was the purpose of the present study. Mice were dosed daily for 28 days with atrazine, ethanol, propanil, or subjected to restraint, which are known to induce neuroendocrine stress responses and thereby to alter several immunological parameters. On day 29, a blood sample was taken and the spleen was removed for analysis of cellular phenotypes, differential cell counts (for blood), and natural killer (NK) cell activity. Corticosterone concentration at various times after dosing (or restraint) was also measured. Comparison of these results with results from previous studies with a single acute exposure revealed that the corticosterone response was almost completely absent in mice treated with ethanol, reduced in mice treated with restraint and propanil, and for atrazine the response was the same as noted for acute exposure. In most cases, the changes in immunological parameters were consistent with expectations based on these corticosterone responses. However, in a few cases (e.g., NK cell activity), it was clear that there were effects not mediated by stress. These results indicate that the nature of the stressor determines whether mice become tolerant with regard to the stress response and consequent immunological effects. This finding has practical implications for safety testing in mice.

Summary of a workshop on nonclinical and clinical immunotoxicity assessment of immunomodulatory drugs

The number of anti-inflammatory and immunomodulatory drugs being developed in the pharmaceutical industry has increased considerably in the past decade. This increase in research and development has been paralleled by questions from both regulatory agencies and industry on how best to assess decreased host resistance to infections or adverse immunostimulation caused by immunomodulatory agents such as anti-cytokine antibodies (e.g., the tumor necrosis factor-α inhibitors), anti-adhesion molecule antibodies (e.g., anti-α-4 integrin inhibitors) and immunostimulatory molecules (e.g., anti-CD28 antibodies). Although several methods have been developed for nonclinical assessment of immunotoxicity, highly publicized adverse events have brought to light significant gaps in the application of nonclinical immunotoxicity testing in assessing potential risk in humans. Confounding this problem is inconsistent application of immunotoxicology methods for risk assessment within the scientific community, limited understanding of appropriate immunotoxicity testing strategy for immunomodulators and inconsistent testing requests by regulatory agencies. To address these concerns, The Immunotoxicology Technical Committee (ITC) of the International Life Science Institute (ILSI) Health and Environmental Sciences Institute (HESI) organized a workshop on Immunomodulators and Clinical Immunotoxicology in May 2007. The Workshop was convened to identify key gaps in nonclinical and clinical immunotoxicity testing of anti-inflammatory and immunomodulatory agents and to begin to develop consistent approaches for immunotoxicity testing and risk assessment. This paper summarizes the outcome of the HESI ITC Immunomodulators and Clinical Immunotoxicology Workshop. Topics not discussed at the Workshop were outside the scope of this report. Although more work is needed to develop consistent approaches for immunotoxicity assessment of immunomodulators, this Workshop provided the foundation for future discussion.

Testing Human Biologicals in Animal Host Resistance Models

The purpose of immunotoxicity testing is to obtain data that is meaningful for safety assessment. Host resistance assays are the best measure of a toxicant's effect on the overall ability to mount an effective immune response and protect the host from infectious disease. An outline is presented for immunotoxicological evaluation using host resistance assays. The influenza virus host resistance model is useful to evaluate the overall health of the immune system and is one of the most thoroughly characterized host resistance models. Viral clearance requires all aspects of the immune system to work together and is the ultimate measure of the health of the immune system in this model. Mechanistic immune functions may be included while measuring viral clearance and include: cytokines, macrophage activity, natural killer (NK) cell activity, cytotoxic T-lymphocyte (CTL) activity, and influenza-specific IgM and IgG. Measurement of these immunological functions provides an evaluation of innate immunity (macrophage or NK activity), an evaluation of cell-mediated immunity (CMI) (CTL activity), and an evaluation of humoral-mediated immunity (HMI) (influenza-specific IgM or IgG). Measurement of influenza-specific IgM or IgG also provides a measurement of T-dependent antibody response (TDAR) since influenza is a T-dependent antigen. There are several targeted host resistance models that may be used to answer specific questions. Should a defect in neutrophil and/or macrophage function be suspected, Streptococcus pneumoniae, Pseudomonas aeruginosa, or Listeria monocytogenes host resistance models are useful. Anti-inflammatory pharmaceuticals or therapeutics for rheumatoid arthritis or Crohn's disease that target TNFα may also be evaluated for immunotoxicity using the S. pneumoniae intranasal host resistance assay. Marginal zone B (MZB) cells are required for production of antibody to T-independent antigens such as the polysaccharide capsule of the encapsulated bacteria that are so prominent in causing blood-borne infections and pneumonia. Intravenous infection with Streptococcus pneumoniae, an encapsulated bacterium, results in a blood-borne infection that requires MZB cells for clearance. The systemic S. pneumoniae host resistance assay evaluates whether a therapeutic test article exerts immunotoxicity on MZB cells and measures the T-independent antibody response (TIAR). Suppression of CMI or in some cases HMI may result in reactivation of latent virus that may result in a fatal disease such as progressive multifocal leukoencephalopathy (PML). The murine cytomegalovirus (MCMV) reactivation model may be used to evaluate a pharmaceutical agent to determine if suppression of CMI or HMI results in reactivation of latent virus. Candida albicans is another host resistance model to test potential immunotoxicity. Host resistance assays have been the ultimate measure of immunotoxicity testing for environmental chemicals and pharmaceutical small molecules. Human biologicals are now an important component of the drug development armamentarium for biotech and pharmaceutical companies. Many human biologicals are fusions of IgG, and/or target immune mediators, immunological receptors, adhesion molecules, and/or are indicated for diseases that have immune components. It is therefore necessary to thoroughly evaluate human biological therapeutics for immunotoxicity. Numerous biologicals that are pharmacologically active in rodents can be evaluated using well-characterized rodent host resistance assays. However, biologicals not active in rodents may use surrogate biologicals for testing in rodent host resistance assays, or may use host resistance assays in genetically engineered mice that mimic the effect of the human biological pharmacological agent.

Alternative approaches to immunotoxicity and allergy testing

Alternative approaches to immunotoxicity and allergy testing

Can flow cytometry play a part in cell based high‐content screening?

In parallel to gene therapy and cellular therapy it is clear that the more traditional pharmacological approaches still have a major role to play in the treatment of most diseases. Two drug-discovery strategies are widely used in the pharmaceutical industry: the so-called rational drug design and the most recent screening-based strategy. Rational drug design requires usually an excellent knowledge of the biomolecular target: molecular mechanisms of action, three-dimensional structure, etc. It is scientifically sound and often efficient but rather time consuming. To cope with an exponentially increasing number of potentially interesting targets derived from genomic and proteomic studies, the industry has developed the screening-based approach since the 1990s. It has proven to be very efficient to afford original hit compounds that may open the way to drug development. During the last decades

The immunosuppressive effects of 10 mg/kg cyclophosphamide in Wistar rats

The purpose of this study is to identify the immunosuppressive effects of cyclophosphamide (a known immunosuppressant) in male Wistar rats which were administered orally for 30 days repeatedly, and to find a proper dose of cyclophosphamide at which the immunosuppressive effects is identified. The dose of cyclophosphamide is 2, 5, 10 mg/kg bw, respectively. The results showed that 10 mg/kg cyclophosphamide treatment induced decreases in body weight, body weight gain, relative weight of spleen and thymus, antibody plaque-forming cells, delayed-type hypersensitivity reaction, natural killer cell activity, lipopolysaccharide-induced B-cell proliferation, and Concanavalin A-induced T-cell proliferation. The histopathology observation on spleen and thymus revealed as decreased cellularity. But, no significant clinical symptoms of toxicities and stress were observed and no rats died. These results indicated that the immunosuppressive effects of 10 mg/kg cyclophosphamide dosed for 30 days orally were identified in male Wistar rats. Based on the results, it is considered that the dose of 10 mg/kg of cyclophosphamide orally for 30 days in male Wistar rats could be used as the dose of positive control group for chemicals immunotoxicity assessment tests. This study also proved that the testing procedures and the experimental techniques established in our laboratory for immunotoxicity tests in male Wistar rats are reliable and feasible.

Immunotoxicity Testing in Non-Rodent Species

Evaluation of the immunotoxicity potential of some pharmaceuticals, including immunomodulatory chemicals and biologics, cannot be limited to testing in rodents. Thus, immune function tests have also been applied in studies with non-human primates and more recently dogs that assess various components of the immune system. These assays include TDAR responses with various immunogens, lymphocyte phenotyping, natural-killer cell activity, delayed-type hypersensitivity, and macrophage function assays. Approaches for incorporating immune function testing in non-rodent species, results from these tests, their interpretation and limitations with respect to drug safety assessment will be reviewed.

Immunotoxicity Evaluation by Immune Function Tests: Focus on the T-Dependent Antibody Response (TDAR) [Overview of a Workshop Session at the 45th Annual Meeting of the Society of Toxicology (SOT) March 5–9, 2006 San Diego, CA

Increased expectations from a number of regulatory agencies, e.g., Environmental Protection Agency (EPA), Food and Drug Administration (FDA), European Medicines Agency (EMEA), and the Ministry of Health, Labour and Welfare (MHLW) of Japan, call for the evaluation of potential adverse effects on the immune system. As recently summarized in the ICH S8 guideline, the T-cell-dependent antibody response (TDAR) has been identified in a regulatory context as a main functional test of immunotoxicity. While the characterization of immunotoxic potential is pertinent to both the chemical and pharmaceutical industries, the use of immunotoxicology data for hazard identification and/or risk assessment in each case is different. Therefore, multiple approaches to immunotoxicity testing have evolved. The assays that evaluate TDAR function include both well-established tests, e.g., anti-sheep red blood cell plaque-forming cell (PFC) assay, and newer models, e.g., anti-keyhole limpet hemocyanin (KLH) antibody ELISA. These tests vary in the study design, antigen application and analytical methods. However, they all evaluate the same endpoint—a competent immune (e.g., antibody) response to an antigen. Numerous issues have been identified in the application of TDAR tests, including high animal to animal variability; differences in antigen source and potency; a lack of established “normal range” of the immune response and uncertainty about the degree of inhibition of the TDAR to be considered toxicologically important. As such, the need for a forum to discuss these issues was recognized by the immunotoxicology community, and was addressed at the 2006 Society of Toxicology (SOT) Workshop. A series of papers will summarize that forum with the ultimate objectives being to build a consensus among immunotoxicologists on the implications of these factors on using TDAR results in hazard identification and/or risk assessment, and to establish a criteria to classify compounds as immunotoxicants.

Methodologies for developmental immunotoxicity (DIT) testing

Developmental immunotoxicity has gained increasing recognition as a significant factor influencing the risk of later life disease. Based on the data collected thus far on different chemicals and drugs, the developing immune system can be significantly more sensitive than the adult immune system to xenobiotic-induced insult. There are distinct differences between the immune system surrounding birth and that in the mature adult as well as differences in the nature of immunotoxic changes based on age. Immunosuppresssion is not the only concern. Immunotoxic changes that increase the risk for allergic or autoimmune responses should also be considered. Therefore, one should not assume that immunotoxicity assays validated for adult exposure assessment are inherently the most predictive for developmental immunotoxicology (DIT) evaluation. Many of those adult-based protocols were developed solely to detect immunosuppression, whereas DIT concerns include shifts in immune balance. For this reason, it is useful to examine the various immune endpoints that have been employed in recent perinatal immunotoxicity studies, compare those against routine adult immunotoxicity evaluation protocols, and consider the options that are available for effective DIT testing. The results published on several chemicals and drugs in recent years suggest that functional tests are a front-line priority for perinatal immunotoxicity detection and that a combination of at least two functional tests (such as a multi-isotype T-dependent antibody response (TDARs), and a cell-mediated immune response assay such as the delayed-type hypersensitivity assay and/or T cell or NK cytotoxicity assays) should be paired with immune cell populations and histopathological analysis. Cytokine production measurements offer outstanding promise and may eventually be able to be substituted for other more laborious procedures. However, multi-cytokine analysis needs to be standardized in terms of optimum source for analysis and protocol.

Use of SRBC antibody responses for immunotoxicity testing

The production of antigen-specific antibodies represents a major defense mechanism of humoral immune responses and involves the cooperation and interaction of several immune cell types: antigen presenting cells, T helper cells, and B cells. Thus, there are several cells or cell products (e.g., interleukins) that may be altered following xenobiotic exposure, making assays that evaluate the production of antigen specific antibody a relatively comprehensive and sensitive assessment of immune function. Data suggest that the primary antibody response to SRBC may be one of the most sensitive endpoints available to assess chemical-induced alterations to the immune system. As a result, this endpoint has become the cornerstone of several recently established guidelines for assessing the potential immunotoxicity of xenobiotics. Five types of antibody may be produced in a humoral immune response (i.e., IgGs of various subtypes, IgM, IgD, IgA, or IgE). For immunotoxicity assessment, the focus has primarily been on assays that assess production of IgM antibodies. Although a number of assays have been developed to evaluate antibody production, the antibody forming cell (AFC) assay and enzyme-linked immunosorbent assay (ELISA) are the two most frequently employed to evaluate the potential immunotoxicity of a xenobiotic. In this manuscript, background information, as well as the pros and cons of each of these assays are discussed and detailed methods on conducting each assay are provided.

Influenza virus host resistance model

Host resistance (HR) models are used to evaluate the effect of a test article on clearance of an infectious microorganism in order to assess total immunocompetence. HR models serve as biomarkers of net immunological health or immunological well-being. Immunotoxicity can result either in an impaired clearance of an infectious agent, increased susceptibility to an opportunistic microorganism, prevention of immunization, or exacerbation of latent viral infections. The purpose of immunotoxicity testing is to obtain data that is meaningful for safety assessment, and for immunosuppression the major objective is to determine the significance with respect to increased susceptibility to infectious disease. Host resistance models provide the only sure method of examining the influence of test articles on the functional integrity of the immune system and its ability to eliminate pathogenic microorganisms and tumor cells. They provide the means to directly assess the functional reserve of the immune system. Clearance of influenza virus requires an intact and functional immune system that incorporates a cascade of immune responses. Mechanistic studies can be included in the influenza virus host resistance model by measuring the effect of a test article on innate immunity (cytokine and interferon production, macrophage function, and natural killer (NK) cell function) and acquired or adaptive immunity (cytotoxic T lymphocyte (CTL) activity as well as influenza-specific IgM and/or IgG antibody).

Immunotoxic effects of chemicals: A matrix for occupational and environmental epidemiological studies

Many biological and chemical agents have the capacity to alter the way the immune system functions in human and animals. This study evaluates the immunotoxicity of 20 substances used widely in work environments. A systematic literature search on the immunotoxicity of 20 chemicals was performed. The first step was to review literature on immunotoxicity testing and testing schemes adopted for establishing immunotoxicity in humans. The second step consisted of providing a documentation on immunotoxicity of substances that are widely used in work environment, by building tables for each chemical of interest (benzene, trichloroethylene, PAHs, crystalline silica, diesel exhausts, welding fumes, asbestos, styrene, formaldehyde, toluene, vinyl chloride monomer, tetrachloroethylene, chlorophenols, 1,3-butadiene, mineral oils, P-dichlorobenzene, dichloromethane, xylene, 1,1,1-trichloroethane, ethylene oxide). The third step was the classification of substances; an index (strong, intermediate, weak, nil) was assigned on the basis of the evidence of toxicity and type of immunotoxic effects (immunosuppression, autoimmunity, hypersensitivity) on the basis of the immune responses. Finally substances were assigned a score of immunotoxic power. Tables have been produced that include information for the 20 substances of interest, based on 227 animal studies and 94 human studies. Each substance was assigned an index of immunotoxic evidence, a score of immunotoxic power and type of immunotoxic effect. This matrix can represent a tool to identify chemicals with similar properties concerning the toxicity for the immune system, and to interpret epidemiological studies on immune-related diseases.

Developmental immunotoxicity testing and protection of children's health.

Prenatal and postnatal exposure to persistent organochlorine pollutants, such as polychlorinated biphenyls (PCBs), has been implicated as a possible cause of impaired immune function in children. For example, studies have shown that children who have been exposed to PCBs and related compounds from their mothers' contaminated diets have reduced concentrations of immunoglobulins and increased frequency of childhood infections [1–3]. And a new study by Heilmann et al. published in PLoS Medicine shows that children exposed to PCBs have reduced antibody responses to childhood vaccinations, adding to the growing evidence that PCBs are associated with developmental immunotoxicity (DIT) [4]. The study provides a stark example of the heightened immune vulnerabilities that exist during early life and that must be adequately protected from environmental insult if we are to minimize health risks to children.

Immunotoxicogenomics: The Potential of Genomics Technology in the Immunotoxicity Risk Assessment Process

Evaluation of xenobiotic-induced changes in gene expression as a method to identify and classify potential toxicants is being pursued by industry and regulatory agencies worldwide. A workshop was held at the Research Triangle Park campus of the Environmental Protection Agency to discuss the current state-of-the-science of "immunotoxicogenomics" and to explore the potential role of genomics techniques for immunotoxicity testing. The genesis of the workshop was the current lack of widely accepted triggering criteria for Tier 1 immunotoxicity testing in the context of routine toxicity testing data, the realization that traditional screening methods would require an inordinate number of animals and are inadequate to handle the number of chemicals that may need to be screened (e.g., high production volume compounds) and the absence of an organized effort to address the state-of-the-science of toxicogenomics in the identification of immunotoxic compounds. The major focus of the meeting was on the theoretical and practical utility of genomics techniques to (1) replace or supplement current immunotoxicity screening procedures, (2) provide insight into potential modes or mechanisms of action, and (3) provide data suitable for immunotoxicity hazard identification or risk assessment. The latter goal is of considerable interest to a variety of stakeholders as a means to reduce animal use and to decrease the cost of conducting and interpreting standard toxicity tests. A number of data gaps were identified that included a lack of dose response and kinetic data for known immunotoxic compounds and a general lack of data correlating genomic alterations to functional changes observed in vivo. Participants concluded that a genomics approach to screen chemicals for immunotoxic potential or to generate data useful to risk assessors holds promise but that routine use of these methods is years in the future. However, recent progress in molecular immunology has made mode and mechanism of action studies much more practical. Furthermore, a variety of published immunotoxicity studies suggest that microarray analysis is already a practical means to explore pathway-level changes that lead to altered immune function. To help move the science of immunotoxicogenomics forward, a partnership of industry, academia, and government was suggested to address data gaps, validation, quality assurance, and protocol development.

Regulatory aspects of immunotoxicology

Since years, differences among the regulatory requirements on preclinical immunotoxicity testing for pharmaceuticals in the EU, Japan and US indicated a need for an internationally accepted approach. Requests for immunotoxicity investigations are also addressed by guidelines in non-drug areas. While some contain more detailed information in their requirements, other regulations comprise only vague descriptions for consideration of (non-intended) effects on the immune effects. Since 2002, the International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use put effort in the development of a harmonised approach for testing of immunosuppression and immunoenhancement. Consensus on the ICH S8 guideline on immunotoxicity testing for pharmaceuticals was achieved which now can be implemented into national regulations. The new concept contains in-depth testing, e.g., by functional tests in a concern/weight of evidence approach if the standard toxicity studies or other causes of concern give evidence of an immunotoxic potential or when the target populations are specifically vulnerable. It is expected that the progress on immunotoxicity testing reached by the ICH process will also have an impact on other regulatory areas. Additionally, the regulatory differences in testing requirements on immunotoxicity in other pharmaceutical areas including biotechnology-derived drugs, medicinal products and vaccines and in non-drug areas consisting of chemicals, agrochemicals or food additives are briefly highlighted.

Evaluation of serum immunoglobulins among individuals living near six Superfund sites.

Residents living in communities near Superfund sites have expressed concern that releases from these facilities affect their health, including adverse effects on their immune systems. We used data from six cross-sectional studies to evaluate whether people who live near several Superfund sites are more likely to have individual immunoglobulin test results (IgA, IgG, and IgM) below or above the reference range than those who live in comparison areas with no Superfund site. Study participants consisted of target-area residents who lived close to a Superfund site and comparison-area residents who were not located near any Superfund or hazardous waste sites. A consistent modeling strategy was used across studies to assess the magnitude of the relationship between area of residence and immunoglobulin test results, adjusting for potential confounders and effect modifiers. In all study areas, the results suggest that people who live near a Superfund site may have been more likely to have IgA test results above the reference range than comparison areas residents regardless of modeling strategy employed. The effect measures were larger for residents who lived in communities near military bases with groundwater contamination. For all analyses the wide confidence intervals reflect uncertainty in the magnitude of these effects. To adequately address the question of whether the immune system is affected by low-level exposures to hazardous substances, we recommend that more functional immunotoxicity tests be conducted in human populations where individual exposure information is available or when it can be reasonably estimated from environmental exposure measurements.

The Comparative Immunotoxicity of Five Selected Compounds Following Developmental or Adult Exposure

It is well established that human diseases associated with abnormal immune function, including some common infectious diseases and asthma, are considerably more prevalent at younger ages. Although not established absolutely, it is generally believed that development constitutes a period of increased immune system susceptibility to xenobiotics, since adverse effects may occur at lower doses and/or immunomodulation may be more persistent, thus increasing the relative risk of xenobiotic exposure to the immunologically immature organism. To address this issue, a brief overview of immune maturation in humans is provided to demonstrate that functional immaturity alone predisposes the young to infection. Age-dependent differences in the immunotoxic effects of five diverse compounds, diethylstilbestrol (DES), diazepam (DZP), lead (Pb), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and tributyltin oxide (TBTO), which have undergone adult and developmental immunotoxicity testing in rodents, are then reviewed, as are human data when available. For all five chemicals, the developing immune system was found to be at greater risk than that of the adult, either because lower doses produced immunotoxicity, adverse effects were more persistent, or both.

Flow cytometric analysis and optimisation for measuring phagocytosis in three Australian freshwater fish

The phagocytic activity of fish immunocytes has been measured by a wide range of methods, and has been used as a bio-indicator to assess the immunotoxicity of environmental pollutants and the efficiency of immunostimulants used in aquaculture. This study demonstrates the utilisation of a flow cytometric technique for measuring phagocytosis as an alternative to manual evaluations by light microscopy. Optimal conditions for the phagocytosis of latex beads were ascertained, including incubation period, cell:bead ratio and media components, for head kidney cells isolated from three native Australian fish that inhabit the Murray–Darling basin, i.e. silver perch ( Bidyanus bidyanus), golden perch ( Macquaria ambigua) and crimson-spotted rainbowfish ( Melanotaenia fluviatilis). Thus, standardised protocols have now been established for future use in the immunotoxicity testing of xenobiotics in native Australian freshwater fish.