Bisphenol A is a high production volume chemical used extensively in the manufacture of poly­carbonate plastics, epoxy resins, and thermal printer paper with a high potential for occupational and post-production dermal exposure. Bisphenol A-containing plastics were commonly used in food packaging, resulting in significant public exposure through leaching into foodstuff. The public is also at risk of dermal exposure due to environmental contamination. Due to public health con­cerns regarding the potential for endocrine disrupting effects, efforts have been applied to replace bisphenol A with safer alternatives. Bisphenol A has been shown to cause skin sensitization in humans; however, there is a paucity of information available on the sensitizing potential of the struc­tural analogues which are increasingly being employed as substitutes. We utilized new approach methodologies (NAMs) addressing key events 1-3 of the adverse outcome pathway for skin sen­sitization to assess the potential of bisphenol A substitutes to induce dermal sensitization. Defined approaches (DA) were applied to further classify and categorize potency according to OECD TG 497. The NAMs and DAs confirmed that bisphenol A was a skin sensitizer in potency category UN GHS 1B. Bisphenol B, AP, and E were also classified as UN GHS 1B sensitizers, and bis­phenol AF as UN GHS 1A/1B depending on the DA, while 2,4-bisphenol S and F were borderline sensitizers, and bisphenol S was classified as a non-sensitizer. These data provide evidence of skin sensitization hazard for the bisphenol structural analogues tested, except for bisphenol S, suggesting that they present risks for dermal allergy.

Exposure to environmental agents has been linked to inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD) in humans. In these studies, we describe a modification of an experimental model used historically in the pharmaceutical industry to help identify therapies for the treatment of IBD to facilitate its use for identification of environmental agents that have the potential to accelerate, exacerbate, and/or impair recovery from IBD. In this model, female C57BL/6 mice were exposed to low levels of dextran sodium sulfate (DSS) for 7 consecutive days in drinking water to allow for a modest level of colon inflammation and pathology as measured by a battery of clinical, pathological, toxicological endpoints (water consumption, body weights, colon length, body temperature, stool consistency, and hematochezia), and cytokine/chemokine production in the serum and colon. Treatment with DSS for 7 d showed a clear dose-response with 1% DSS producing minimal changes in the colon and 3% DSS inducing severe damage with IBD. A concentration of 2% DSS in drinking water for 7 d was selected for investigating disease recovery and exacerbation by an environmental agent as it induced mild colon inflammation that showed nearly complete resolution within 21 d following cessation of DSS exposure. Cytokine and chemokine profiles showed a Type 1 predominant immune response in the colon and serum that is consistent with inflammation observed in human IBD. The model was used to determine the impact of administration of a high salt diet (HSD) on DSS IBD progression, severity, and recovery. While administration of HSD by itself had no effect on indicators of colon damage or inflammation, co-administration of HSD with DSS, produced marked exacerbation and persistence of disease supporting the potential of the model for identifying environmental agents that can affect IBD.

Immunotoxicity assessment is nearing a crossroads predicated on mounting pressure for reduction/replacement of animals in toxicology. This has fueled the development of alternative New Approach Methodologies (NAMs) for hazard identification. This work details a comprehensive human whole blood NAM battery for immunotoxicity testing. The test system is a closed tube culture containing whole blood diluted 1:3 in culture media with or without an immune stimulant, anti-CD3/CD28 or viral peptide pool. Model immunotoxicants dexamethasone (DEX; 0 - 1.94 μM) and benzo(a)pyrene [B(a)P; 0-6.3 μM], were added to the test system for 24 hours. Immune cells were identified and counted by flow cytometric immunophenotyping and assessed for natural killer (NK) cell activity and T cell activation. Supernatants were interrogated for proinflammatory cytokine concentrations. In vitro treatment with DEX resulted in concentration-dependent suppression of cytokine production, NK cell activity, and T cell activation induced by anti-CD3/CD28, as well as viral-induced cytokine production. B(a)P caused suppression of cytokine production and a nonsignificant reduction in T cell activation but did not impact NK cell activity, however, immunosuppression by B(a)P only occurred following metabolic activation by S9 fraction enzymes. Advantages of this NAM battery include assessment of adaptive immunity and direct translation of immunotoxicity to viral host resistance. These results provide evidence of in vitro immunotoxicity that reflect known outcomes from in vivo studies. This multi-endpoint human whole blood NAM battery should be useful for screening compounds for immunotoxicity hazard identification without reliance on animal systems for increased translatability to humans.

Cytokines are low molecular weight extracellular glycoproteins and polypeptides synthesized primarily by T cells, neutrophils, and macrophages although they can be produced by nearly all nucleated cells. While cytokines have been evaluated in disease states and correlations of abnormal profiles have been established, cytokine cutoff values as biomarkers of disease have been well studied. Monoclonal antibodies targeting the cytokines and their receptors provide potential for therapeutic intervention. Cytokine assessments also provide valuable information on immune activation and mechanisms in the course of nonclinical and clinical studies. Cytokine release assays (CRAs) have been developed and are routinely used in the preclinical safety assessment of new therapeutic monoclonal antibodies and therapies for hazard identification of CRS and its associated pathologies. Despite these challenges, cytokine assessments provide valuable information on immune status, disease pathogenesis, therapeutic effects, dose and dose escalation, and potential toxicities during the course of nonclinical and clinical studies.

Abstract Proactive identification of chemicals with skin sensitizing properties is a key toxicological endpoint within chemical safety assessment, as required by legislation for registration of chemicals. In order to meet demands of increased animal welfare and facilitate increased testing efficiency also in nonregulatory settings, considerable efforts have been made to develop nonanimal approaches to replace current animal testing. Genomic Allergen Rapid Detection (GARD™) is a state-of-the-art technology platform, the most advanced application of which is the assay for assessment of skin sensitizing chemicals, GARD™skin. The methodology is based on a dendritic cell (DC)-like cell line, thus mimicking the mechanistic events leading to initiation and modulation of downstream immunological responses. Induced transcriptional changes are measured following exposure to test chemicals, providing a detailed evaluation of cell activation. These changes are associated with the immunological decision-making role of DCs in vivo and include among other phenotypic modifications, up-regulation of co-stimulatory molecules, induction of cellular and oxidative stress pathways and xenobiotic responses, and provide a holistic readout of substance-induced DC activation. Here, results from an inter-laboratory ring trial of GARD™skin, conducted in compliance with OECD guidance documents and comprising a blinded chemical test set of 28 chemicals, are summarized. The assay was found to be transferable to naïve laboratories, with an inter-laboratory reproducibility of 92.0%. The within-laboratory reproducibility ranged between 82.1% and 88.9%, whereas the cumulative predictive accuracy across the 3 laboratories was 93.8%. It was concluded that GARD™skin is a robust and reliable method for the identification of skin sensitizing chemicals and suitable for stand-alone use or as a constituent of integrated testing. These data form the basis for the regulatory validation of GARD™skin.

The goal of immunotoxicity testing is to obtain data useful for immunotoxicity safety assessment. Guidance in the performance of immunotoxicity safety evaluations is provided in documents from the US EPA for chemicals and the ICH S8 document for pharmaceuticals. The ICH S8 document outlines a tiered approach that includes (1) standard toxicity studies with associated hematology, immune system organ weights, and histopathology data; (2) functional assays, such as cytotoxic T lymphocyte (CTL) assays, natural killer (NK) cell assays, respiratory burst, phagocytosis, and T-cell-dependent antibody response (TDAR) assays; and (3) host resistance assays. Host resistance assays are considered the gold standard in immunotoxicity testing and provide a critical overview of the extent to which innate, adaptive, and homeostatic regulatory immune functions are integrated to protect the host. Both comprehensive and targeted host resistance assays are available, each with distinct advantages. This chapter serves to provide a general overview of the various assays that may be used, as well as a summary of procedures.

Metabolic Syndrome and Associated Diseases: From the Bench to the Clinic, a Society of Toxicology Contemporary Concepts in Toxicology (CCT) workshop was held on March 11, 2017. The meeting was convened to raise awareness of metabolic syndrome and its associated diseases and serve as a melting pot with scientists of multiple disciplines (eg, toxicologists, clinicians, regulators) so as to spur research and understanding of this condition. The criteria for metabolic syndrome include obesity, dyslipidemia (low high-density lipoprotein and/or elevated triglycerides), elevated blood pressure, and alterations in glucose metabolism. It can lead to a greater potential of type 2 diabetes, lipid disorders, cardiovascular disease, hepatic steatosis, and other circulatory disorders. Although there are no approved drugs specifically for this syndrome, many drugs target diseases associated with this syndrome thus potentially increasing the likelihood of drug-drug interactions. There is currently significant research focusing on understanding the key pathways that control metabolism, which would be likely targets of risk factors (eg, exposure to xenobiotics, genetics) and lifestyle factors (eg, microbiome, nutrition, and exercise) that contribute to metabolic syndrome. Understanding these pathways could also lead to the development of pharmaceutical interventions. As individuals with metabolic syndrome have signs similar to that of toxic responses (eg, oxidative stress and inflammation) and organ dysfunction, these alterations should be taken into account in drug development. With the increasing frequency of metabolic syndrome in the general population, the idea of a "normal" individual may need to be redefined. This paper reports on the substance and outcomes of this workshop.

Evaluation of cell-mediated immunity (CMI) is a significant component in any assessment designed to predict the full range of potential immunotoxic risk underlying health risks. Among measures of CMI, the cytotoxic T Lymphocyte (CTL) response is recognized as perhaps the most relevant functional measure that reflects cell-mediated acquired immune defense against viral infections and cancer. The CTL response against T-dependent antigens requires the cooperation of at least three different major categories of immune cells. These include professional antigen presenting cells (e.g., dendritic cells), CD4+ T helper lymphocytes, and CD8+ T effector lymphocytes. It is also among the few functional responses dependent on and, hence, capable of evaluating effective antigen presentation via both class I and class II molecules of the major histocompatibility complex (MHC). For this reason the CTL assay is an excellent candidate for evaluation of potential immunotoxicity. This chapter provides an example of a mouse CTL assay against influenza virus that has been utilized for this purpose.

The purpose of immunotoxicity testing is to obtain data that is meaningful for safety assessment and a number of approaches are available to achieve this goal. Regardless of the approach, it is important that the testing evaluate the three arms of the immune system and include assessments of innate immunity, CMI, and HMI. When host resistance is used for immunotoxicity testing, immune suppression caused by a test compound or chemical may be reflected as impaired clearance of the infectious agent, increased sensitivity to opportunistic infections, prevention of immunization, or exacerbation of latent infections. In this immunotoxicity testing paradigm, the host resistance assay allows for an assessment of total functional immunocompetence and serves as a biomarker of net immunological health and well-being. It is difficult to assess the biological relevance of an ‘x’ percent change in a particular immune parameter, such as reduction in thymus weight or reduction of NK activity, unless this change is evaluated in terms of overall biological impact. Host resistance assays, with the available redundancy inherent in the immune system, allow such an evaluation. Comprehensive host resistance assays are used to evaluate the overall health of the immune system, i.e., how the numerous components of the functional immune system work together to clear an infection, while targeted host resistance assays are available to evaluate specific immunotoxicity questions. The choice of which host resistance assay to employ is dependent on the question being asked, and on the context of the question being posed (immunotoxicity or efficacy).

4-Methylcyclohexanemethanol (MCHM) is a flotation reagent used in fine coal beneficiation. On January 9, 2014, crude MCHM, a mixture containing predominantly MCHM, was inadvertently released into the Elk River, a municipal water source that serves about 300,000 people in the Charleston, WV area, resulting in temporary contamination of 15 percent of the state's tap water and causing significant dermal exposure. The current studies were undertaken to determine whether crude MCHM or MCHM has the potential to produce dermal irritancy and/or sensitization. BALB/c female mice were treated daily for 3 consecutive days by direct epicutaneous application of 25 μL of various concentrations of crude MCHM or MCHM to the dorsum of each ear. A mouse ear-swelling test was used to determine irritancy potential and was undertaken in combination with the standardized Local Lymph Node Assay (LLNA) to determine skin sensitizing potential. MCHM was found to produce skin irritation at concentrations above 20% and did not produce sensitization. Crude MCHM also produced irritation, although weaker, and in addition was found to be a weak to moderate skin sensitizer. The results are discussed in terms of potential human health hazard.

Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.

Supported by several epidemiological studies and a large number of animal studies, certain polyfluorinated alkyl acids are believed to be immunotoxic, affecting particularly humoral immunity. Our aim was to investigate the relationship between the antibody response following vaccination with an inactivated trivalent influenza vaccine and circulating levels of perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS). The study population consisted of 411 adults living in the mid-Ohio region of Ohio and West Virginia where public drinking water had been inadvertently contaminated with PFOA. They participated in a larger cross-sectional study in 2005/2006 and were followed up in 2010, by which time serum levels of PFOA had been substantially reduced but were still well above those found in the general population. Hemagglutination inhibition tests were conducted on serum samples collected preinfluenza vaccination and 21 ± 3 days postvaccination in 2010. Serum samples were also analyzed for PFOA and PFOS concentrations (median: 31.5 and 9.2 ng/ml, respectively). Questionnaires were conducted regarding the occurrence and frequency of recent (during the last 12 months) respiratory infections. Our findings indicated that elevated PFOA serum concentrations are associated with reduced antibody titer rise, particularly to A/H3N2 influenza virus, and an increased risk of not attaining the antibody threshold considered to offer long-term protection. Although the direct relationship between weakened antibody response and clinical risk of influenza is not clear, we did not find evidence for an association between self-reported colds or influenza and PFOA levels nor between PFOS serum concentrations and any of the endpoints examined.

Murine gammaherpesvirus-68 (MHV-68), a natural pathogen of mice, is being evaluated as a model of Epstein Barr Virus (EBV) infection for use in investigation of the effects of immunomodulatory therapy on herpesvirus pathogenesis in humans. Immunosuppressive agents are used for treatment of a variety of autoimmune diseases as well as for prevention of tissue rejection after organ transplantation and can result in recrudescence of latent herpesvirus infections. Prior to examination of MHV-68 as a suitable model for EBV, better characterization of the MHV-68 model was desirable. Characterization of the MHV-68 model involved development of assays for detecting virus and for demonstration of safety when present in murine colonies. Limited information is available in the literature regarding MHV-68 transmission, although recent reports indicate the virus is not horizontally spread in research facilities. To further determine transmission potential, immunocompetent and immunodeficient mice were infected with MHV-68 and co-habitated with naïve animals. Molecular pathology assays were developed to characterize the MHV-68 model and to determine viral transmission. Horizontal transmission of virus was not observed from infected animals to naïve cagemates after fluorescence microscopy assays and quantitative PCR (qPCR). Serologic analysis complemented these studies and was used as a method of monitoring infection amongst murine colonies. Overall, these findings demonstrate that MHV-68 infection can be controlled and monitored in murine research facilities, and the potential for unintentional infection is low.

Background. Dimethylfumarate (DMF) was the cause of a major outbreak of allergic contact dermatitis as a consequence of its use as an antifungal agent in leather products, particularly in furniture, with what became known as toxic sofa dermatitis'. Objectives. To determine whether the frequency and severity of reactions to DMF arose as a function of its intrinsic potency and/or the nature and extent of exposure. Methods. The intrinsic potency of DMF was measured with the standard local lymph node assay (LLNA), with determination of an EC3 value, which is the threshold in the LLNA and serves as an indicator of relative skin-sensitizing potency in humans. Results. The EC3 value for DMF was 0.35% when tested in dimethylformamide as a vehicle, indicating that DMF is a strong, but not an extreme, skin sensitizer in this mouse model. Conclusions. DMF appears to have a sensitizing potency in the mouse that is very similar to that of formaldehyde, which is also a strong human skin sensitizer. However, the frequency and intensity of allergic contact dermatitis reactions to DMF suggest that it was the prolonged, repeated and occlusive exposure to this chemical over large skin areas, combined with the strong sensitizing potency, that generated the perfect storm' conditions that caused the DMF epidemic.

OBJECTIVE:This study determined the influence of a 2-component polyethylene glycol surgical sealant (Coseal) as an adhesion prevention device on sepsis-related mortality and/or systemic bacterial translocation to the spleen.STUDY DESIGN:A bacterial inoculum and telemetry probe were implanted in 50 treated and 49 untreated rats. Telemetry probes monitored core-body temperature to determine time of death. Spleens were collected on day 3 for quantitative bacteriology of Escherichia coli and Bacteroides fragilis.RESULTS:Median survival time and mortality of treated rats (37.0 hours, 54.0%) were noninferior to untreated rats (47.5 hours, 55.1%). Median E coli titers in treated rats (2.24 log colony forming units/spleen) were significantly less than untreated rats (4.32 log colony forming units/spleen). B fragilis titers were not different.CONCLUSION:This study demonstrates intraperitoneal administration of a 2-component polyethylene glycol surgical sealant as an adhesion prevention device does not alter time to death or sepsis-related mortality and/or systemic bacterial translocation to the spleen.