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The European Society of Toxicologic Pathology (ESTP) organized a panel of 24 international experts from many fields of toxicologic clinical pathology (e.g., industry, academia, and regulatory) that came together in 2021 to align the use of terminology to convey the importance of clinical pathology findings in preclinical toxicity studies. An additional goal consisted of how to identify important findings in standard and nonstandard clinical pathology associated endpoints. This manuscript summarizes the information and opinions discussed and shared at the ninth ESTP International Expert Workshop, April 5 to 6, 2022. In addition to terminology usage, the workshop considered topics related to the identification and conveyance of the importance of test item-related findings. These topics included sources of variability, comparators, statistics, reporting, correlations to other study data, nonstandard biomarkers, indirect/secondary findings, and an overall weight-of-evidence approach. [ABSTRACT FROM AUTHOR]

Disruption of the immune system during embryonic brain development by environmental chemicals was proposed as a possible cause of neurodevelopmental disorders. We previously found adverse effects of di-n-octyltin dichloride (DOTC) on maternal and developing immune systems of rats in an extended one-generation reproductive toxicity study according to the OECD 443 test guideline. We hypothesize that the DOTC-induced changes in the immune system can affect neurodevelopment. Therefore, we used in-vivo MRI and PET imaging and genomics, in addition to behavioral testing and neuropathology as proposed in OECD test guideline 443, to investigate the effect of DOTC on structural and functional brain development. Male rats were exposed to DOTC (0, 3, 10, or 30 mg/kg of diet) from 2 weeks prior to mating of the F0-generation until sacrifice of F1-animals. The brains of rats, exposed to DOTC showed a transiently enlarged volume of specific brain regions (MRI), altered specific gravity, and transient hyper-metabolism ([18F]FDG PET). The alterations in brain development concurred with hyper-responsiveness in auditory startle response and slight hyperactivity in young adult animals. Genomics identified altered transcription of key regulators involved in neurodevelopment and neural function (e.g. Nrgrn, Shank3, Igf1r, Cck, Apba2, Foxp2); and regulators involved in cell size, cell proliferation, and organ development, especially immune system development and functioning (e.g. LOC679869, Itga11, Arhgap5, Cd47, Dlg1, Gas6, Cml5, Mef2c). The results suggest the involvement of immunotoxicity in the impairment of the nervous system by DOTC and support the hypothesis of a close connection between the immune and nervous systems in brain development. [ABSTRACT FROM AUTHOR]

The evaluation of changes in the immune system serves to determine the efficacy and potential immunotoxicologic effects of new products under development. Toxicologic pathologists play critical roles in identifying immune system changes that drive the immunosafety determination. Standard pathology evaluations of therapies and chemicals remain similar; however, biopharmaceutical therapies have moved from simply affecting the immune system to being specifically developed to modify the immune system, which can impact interpretation. Recent explosive growth in immunomodulatory therapies presents a challenge to the toxicologic pathologist, toxicologist, and regulatory reviewer in terms of evaluating the clinical relevance and potential adversity of immune system changes. Beyond the recognition of such changes, there is an increasing expectation to evaluate, describe, and interpret how therapies affect complex immune system pathways for both immunomodulatory therapies and non-immunomodulatory drugs with off-target immunotoxic effects. In this opinion piece, considerations regarding immune system evaluation, the current landscape of immunomodulatory therapies, a brief description of immunotoxicologic (and immunopathologic) endpoints, the importance of integrating such immunosafety data, and relevance to adversity determination are discussed. Importantly, we describe how the current paradigm of determining adversity for immune system changes may be challenging or insufficient and propose a harmonized and flexible approach for assessing adversity. [ABSTRACT FROM AUTHOR]

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative changes in rats and mice. The purpose of this publication is to provide a standardized nomenclature for classifying changes observed in the hematolymphoid organs, including the bone marrow, thymus, spleen, lymph nodes, mucosa-associated lymphoid tissues, and other lymphoid tissues (serosa-associated lymphoid clusters and tertiary lymphoid structures) with color photomicrographs illustrating examples of the lesions. Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. The nomenclature for these organs is divided into 3 terminologies: descriptive, conventional, and enhanced. Three terms are listed for each diagnosis. The rationale for this approach and guidance for its application to toxicologic pathology are described in detail below. [ABSTRACT FROM AUTHOR]

Exposure of humans to metal oxide nanoparticles (NPs) occurs mainly via air, and inhaled metal oxide NPs may generate inflammation. The aim of this study was to investigate the proinflammatory potential of six metal oxide NPs (CeO2, Mn2O3, CuO, ZnO, Co3O4 and WO3; 27-108 μg ml−1) using human primary 3-dimensional airway epithelium (MucilAir™) and dendritic cell (DC) models. Metal oxide NPs were mainly aggregated/agglomerated in the cell media, as determined by dynamic light scattering, scanning electron microscopy and differential centrifugal sedimentation. WO3 and ZnO were highly soluble, both with and without respiratory mucus. Proinflammatory signalling by the epithelium was evaluated after a 24 hour exposure by increased interleukin-6 and -8 and monocyte chemoattractant protein 1 cytokine release, which occurred only for CuO. Moreover, maturation of immature human DCs, which play a key role in the lung immune system, were evaluated by expression of surface markers HLA-DR, CD80, CD83 and CD86 after a 48 hour exposure. Only Mn2O3 consistently upregulated DC maturation markers. Furthermore, by addition of medium from metal oxide NP-exposed 3-dimensional airway cultures to metal oxide NP-exposed DC cultures, the interplay between lung epithelium and DCs was studied. Such an interplay was again only observed for Mn2O3 and in one of five DC donors. Our results show that, even when using dosages that represent very high in vivo exposure levels, up to 27 hours of constant human airway exposure, metal oxide NPs cause minimal proinflammatory effects and that epithelial cells not necessarily interfere with DC maturation upon metal oxide NP exposure. The present approach exemplifies a relevant translation towards human safety assessment. [ABSTRACT FROM AUTHOR]

The interaction between exposure to nanomaterials and existing inflammatory conditions has not been fully established. Multiwalled carbon nanotubes (MWCNT; Nanocyl NC 7000 CAS no. 7782-42-5; count median diameter in atmosphere 61 ± 5 nm) were tested by inhalation in high Immunoglobulin E (IgE)-responding Brown Norway (BN) rats with trimellitic anhydride (TMA)-induced respiratory allergy. The rats were exposed 2 days/week over a 3.5-week period to a low (11 mg/m3) or a high (22 mg/m3) concentration of MWCNT. Nonallergic animals exposed to MWCNT and unexposed allergic and nonallergic rats served as controls. At the end of the exposure period, the allergic animals were rechallenged with TMA. Histopathological examination of the respiratory tract showed agglomerated/aggregated MWCNT in the lungs and in the lung-draining lymph nodes. Frustrated phagocytosis was observed as incomplete uptake of MWCNT by the alveolar macrophages and clustering of cells around MWCNT. Large MWCNT agglomerates/aggregates were found in granulomas in the allergic rats, suggesting decreased macrophage clearance in allergic rats. In allergic rats, MWCNT exposure decreased serum IgE levels and the number of lymphocytes in bronchoalveolar lavage. In conclusion, MWCNT did not aggravate the acute allergic reaction but modulated the allergy-associated immune response. [ABSTRACT FROM PUBLISHER]

Immunogenicity is a major issue of concern for monoclonal antibodies used in human diseases and is by default mainly determined in non-human primates (NHP), as target molecules are considered most similar in NHP compared to human. In this manuscript the predictive value of immunogenicity testing in minipigs for human safety is evaluated, as the immune system of the pig is functionally similar to that in other mammalian species. Adalimumab and infliximab (both monoclonal antibodies blocking TNFα) were used as model substances. Female Göttingen minipigs (4/group) were treated every other week with low (0.1 mg/kg), mid (1.0 mg/kg), or high dose (5 mg/kg) adalimumab or 5 mg/kg infliximab subcutaneous (SC) over a period of 8 weeks. After first and last dosing, pharmacokinetic analysis was performed. Anti-drug antibodies (ADAs) were measured on several time points. Furthermore, hematology, clinical chemistry, body weight, clinical signs, and histopathology of several organs were evaluated. No signs of toxicity of the treatments were observed in the limited organs and tissues collected. Eleven out of 12 minipigs treated with adalimumab elicited a detectable ADA response. Induction of ADA was correlated with decreased plasma levels of adalimumab. Infliximab clearance was comparable after first and last dose. Therefore, the presence of ADA directed to infliximab was considered highly unlikely. It was concluded that the minipig and NHP showed comparable suitability for immunogenicity prediction in humans. More studies with other biopharmaceutical products are needed to strengthen the status of the minipig as an alternative model for immunotoxicity testing including immunogenicity. [ABSTRACT FROM AUTHOR]

The use of recombinant human proteins for the treatment of several diseases has increased considerably during the last decades. A major safety and efficacy issue of biopharmaceuticals is their potential immunogenicity. To prevent immunogenicity, biotechnology-derived proteins are engineered to be as human-like as possible. Immunogenicity is mainly determined in non-human primates (NHP), as they are considered to be the best predictive animal species for human safety, based on their close relatedness to man. As minipigs are increasingly used in the safety evaluation of (bio)pharmaceuticals, the predictive value of the minipig in immunogenicity testing was evaluated in this study, using anakinra as a model compound. Animals were treated subcutaneously with either placebo, low- (0.5 mg/kg), or high-dose (5 mg/kg) anakinra daily on 29 consecutive days. After the first and last dose, the pharmacokinetic (PK) profile of anakinra was evaluated. Antibodies directed to anakinra were measured on several time points during the treatment period. Furthermore, hematology, clinical chemistry, body weight, clinical signs, and histopathology of several organs were evaluated. No signs of toxicity were observed upon treatment with anakinra. PK parameters were comparable with those found in human and NHP studies performed with anakinra. All animals developed anti-anakinra antibodies. The results obtained in minipigs were comparable to those observed in monkeys. For anakinra, the predictive value of the minipig for immunogenicity testing was found to be comparable to that seen in NHP. However, more studies evaluating additional biopharmaceutical products are needed to support the use of the minipig as an alternative model for (immuno)toxicity testing, including immunogenicity. [ABSTRACT FROM AUTHOR]

Histopathological examination of the nasal passages requires a standardized approach for recording lesion distribution patterns. Nasal diagrams provide guidance to map the lesions. Information on lesions exists for rodents, dogs, and monkeys, which all have been used in inhalation studies. Recently, minipigs have garnered interest as an inhalation model because minipigs resemble humans in many features of anatomy, physiology, and biochemistry and may be a good alternative to monkeys and dogs. The present work explored the microanatomy and histology of the nasal passages of Göttingen minipigs from postnatal day 1 until 6 months of age. Six nasal levels were selected, which allow examination of the squamous, transitional (nonciliated) and ciliated respiratory, and olfactory epithelia; the nasopharynx; and relevant structures such as the vomeronasal organ, olfactory bulb, and nasal/nasopharynx-associated lymphoid tissue. [ABSTRACT FROM PUBLISHER]

Characteristic susceptibility to environmental and pharmaceutical exposure may occur during periods in life of marked histophysiological changes of the immune system. Perinatal development is such a period; pregnancy followed by lactation is potentially another one. Here, we explored the influence of pregnancy and lactation on the model immunotoxic compound di-n-octyltin dichloride (DOTC) in rats using clinical and histopathological parameters. Female rats were exposed to 0, 3, 10, or 30 mg DOTC/kg feed during pregnancy and up to 20 (at weaning) or 56 days after delivery. Age-matched nonmated females were exposed during the same time periods. DOTC at the level of 10 and 30 mg/kg decreased thymus weight and affected thymus morphology in the lactating rats. In addition, DOTC decreased the numbers of neutrophils in the lactating rats. These effects were no longer apparent at day 56 despite continuous exposure to DOTC. This explorative study indicates that the innate and adaptive immune system may be especially sensitive to immunotoxicants during pregnancy and lactation. [ABSTRACT FROM PUBLISHER]

Irritant-induced inflammation of the airways may aggravate respiratory allergy induced by chemical respiratory allergens. Therefore, it was studied whether airway irritation by sulfur dioxide (SO2) would enhance respiratory allergic reactions to trimellitic anhydride (TMA), using a rat model. Brown Norway (BN) rats were topically sensitized, subsequently exposed for a single time or repeatedly to 300 ppm SO2, and challenged by inhalation to a distinctly irritating or minimally irritating concentration of TMA after the (last) SO2 exposure. Repeated exposure to SO2 alone reduced breathing frequency during exposure, and caused epithelial alterations including hyperplasia and squamous metaplasia, and infiltration of polymorphonuclear inflammatory cells into nasal tissues, larynx, trachea, and bronchi/bronchioli. Histopathological changes were less prominent after 1 day of SO2 exposure. Repeated pre-exposure to SO2 reduced the number of TMA-induced apnoeas, in an SO2 exposure duration-dependent manner. This effect of SO2 on TMA-induced functional allergic reactions (apnoeas) was distinct only when the TMA challenge concentration was not too irritating itself. Repeated pre-exposure to SO2 reduced TMA-induced laryngeal ulceration, goblet-cell hyperplasia, and inflammation in the lungs in most animals, regardless of the TMA challenge concentration. The SO2-induced replacement of normal respiratory epithelium by less sensitive, squamous epithelium may offer an explanation for the, unexpected, reduced allergic manifestation. However in a few animals, SO2 appeared to facilitate TMA-induced irritation, probably due to incomplete protection. Overall, SO2 exposure of TMA-sensitized rats reduced TMA-related allergic respiratory responses in most animals. [ABSTRACT FROM AUTHOR]

To contribute to the hazard identification of low molecular weight (LMW) respiratory allergens, respiratory allergy induced by trimellitic anhydride (TMA) was characterized by whole genome analysis of lung tissue and blood proteomics in Brown Norway rats. Dermal sensitization (50% and 25% w/v) with TMA and an inhalation challenge of 15 mg/m3 TMA-induced apneas, laryngeal inflammation, increased numbers of eosinophils, neutrophils and macrophages in bronchoalveolar lavage (BAL), and increased immunoglobulin E levels in serum and lung tissue. Whole genome analysis of lung, sampled 24 hours after challenge, showed expression changes of not only genes belonging to several Gene Ontology groups with up-regulation of inflammatory-associated genes and those associated with lung remodeling but also genes involved in downsizing these processes. Blood proteomics reflected activation of inflammation-inhibiting pathways. Unsensitized animals challenged with TMA exhibited also an increased number of macrophages in BAL, but gene expression in the above-mentioned gene pathways was unchanged or down-regulated. The authors conclude that parameters for lung remodeling can be a valuable tool in hazard identification of LMW respiratory allergens. [ABSTRACT FROM AUTHOR]

The effects of cyclosporin A (CY) and cyclophosphamide (CPS) on Peyer's patches (PP) were studied in Wistar rats, exposed in utero and neonatally or during adult age. In one study, pregnant dams received 5 or 15 mg/kg bw/day CY from gestation day 6 to day 21 of lactation. In two other studies, animals were exposed at young adult age: female rats received orally 5 or 20 mg/kg/day CY or 5 or 10 mg/kg bw CPS for 4 weeks; males received orally 5 mg/kg bw CPS for 4 weeks, or a single iv injection of 50 mg/kg bw CPS. Upon in utero and neonatal exposure, the numbers of grossly observed PP were increased in male pups from the high-dose CY dams at 70 days of age. Exposure to high-dose CY at adult age only tended to decrease the numbers of PP; germinal center development was reduced in the PP from the middle segment of the small intestines, as examined microscopically. Exposure to both doses CPS at adult age reduced the numbers of PP and reduced germinal centre development and the number of lymphocytes in all compartments of PP. It was concluded that the effects of CPS and CY could be established by counting the number of grossly visible PP and by microscopic observation of PP, provided that regional differences of PP were taken into account. Moreover, the type of effects of an immunotoxic agent may vary with age of exposure. [ABSTRACT FROM AUTHOR]

Mucosa-associated lymphoid tissue (MALT) is a generalized term incorporating a disseminated collection of lymphoid tissues in multiple sites throughout the body. MALT sites that have been/are primarily studied include bronchus-associated lymphoid tissue (BALT), gut-associated lymphoid tissue (GALT), and nasopharynx-associated lymphoid tissue (NALT). Since MALT sites are often under-sampled in conventional toxicity studies, MALT lesions have not been extensively documented in these lymphoid effector sites. Lesions of the nasopharyngeal lymphoid tissue and Peyer's patches include degeneration, inflammation, and both primary and metastatic neoplasia. [ABSTRACT FROM AUTHOR]

The present study investigated whether airway responses of sensitized rats to trimellitic an, hydride (TMA) were concentration dependent and whether these were related to irritation by TMA. Groups of BN and Wistar rats were sensitized by two dermal applications of TMA (50% w/v, followed by 25% w/v in vehicle). Controls received vehicle (acetone-olive oil 4:1, v/v). All animals were challenged 3 wk after the first sensitization by inhalation of one of a range of concentrations of TMA (0.2-61 mg/m³ for BN rats, 15-250 mg/m³ for Wistar rats). Breathing pattern, breathing frequency, and tidal volume were measured before, during, and after challenge to assess allergic and irritative airway responses. One day after challenge, nonspecific airway responsiveness to a range of concentrations of methacholine was measured. At necropsy on the same day, blood was withdrawn for measuring total serum immunoglobulin E (IgE) and organs were weighed. Larynx, trachea and lungs were examined histopathologically. In BN rats, TMA sensitization elevated total IgE levels; subsequent inhalation challenge with 2 mg/m³ of TMA and higher caused laryngeal inflammation with squamous epithelial metaplasia, and pulmonary hemorrhages. Concentration-related decreases in breathing frequency and alterations in breathing pattern, which differed from the irritation-induced pattern, were also observed at these levels. Inhalation challenge with TMA concentrations of 12 mg/m³ and higher increased lung weight. Increased nonspecific airway responsiveness was observed at the 2 next higher tested concentrations of 46 and 61 mg/m³. In unsensitized BN rats, only laryngeal squamous metaplasia was observed, albeit at higher challenge concentrations of TMA, and decreased breathing frequency, a typical breathing pattern characteristic of irritation. Identically sensitized Wistar rats showed airway inflammation and pulmonary hemorrhages upon challenge with TMA, but no functional changes, even at distinctly irritating concentrations of TMA up to 250 mg/m³. In conclusion, TMA challenge of sensitized BN rats caused challenge concentration-related allergic airway inflammation, asthmalike changes in breathing pattern, and increased nonspecific airway responsiveness. The lowest no-observed-effect level (NOEL) based on the most sensitive endpoint investigated was 0.2 mg/m³, a value that is well below the irritation concentration. The presence of a NOEL in the sensitized BN rat suggests that assessment of safe human exposure levels is feasible. [ABSTRACT FROM AUTHOR]

The mucosal membranes form a weak mechanical barrier, but they are provided with an extensive specific and non-specific defence system. Antigenic stimulation of the mucosal immune system of the oronasal passages induces specific, local immune responses, and activates immune components of mucosae elsewhere as well as the systemic immune system. Nasal lymphocytes are disseminated diffusely in the mucosa or are organised in structures at the entrance of the nasopharynx (nasal-associated lymphoid tissues, NALT). Nasal lymphatics, and possibly NALT, play an important role in drainage of brain fluid, especially in small animals. Little is known about toxicity to the NALT, despite its central role in mucosal immunity. Its strategic position in the nasal passages suggests that it comes easily into contact with inhaled nasal toxicants. Therefore, we recommend to include histopathological examination of NALT in standard guideline-driven inhalation toxicity studies. [Copyright &y& Elsevier]

Several acid anhydrides are known for their sensitizing and irritative properties. Since both irritation and respiratory allergy can cause changes of lung function, proper testing of allergen-dependent effects on the respiratory tract requires knowledge of the respiratory irritant effects. To study the latter effects, groups of female Brown Norway (BN) and Wistar rats were exposed for 30 min to a range of concentrations (10 to 300 mg/m[sup 3]) of the well-known respiratory allergen trimellitic anhydride (TMA). Breathing pattern and frequency were monitored before, during, and after exposure. Animals were necropsied and lung weights were determined 1 day after exposure. In BN rats, changes in breathing pattern were seen at levels of 29 mg/m[sup 3] and higher and decreases in frequency at 60 mg/m[sup 3] and higher, whereas in Wistar rats changes in both pattern and frequency (increases followed by decreases) were seen at levels of 34 mg/m[sup 3] and higher. Changes in breathing pattern consisted of a spiked form instead of a wave form of the respiratory cycle, with a pause between breaths at the end of expiration. The length of the pause increased with increasing concentrations of TMA while the duration of the respiratory cycle decreased slightly, implying that breathing frequency was mainly determined by the magnitude of the increase in pause. These reversible changes in breathing pattern and frequency were considered to be suggestive of lower airway irritation, rather than upper airway irritation. No concentration-related changes in lung weights were observed. The highest level at which no acute airway irritation as based on both breathing pattern and frequency was observed in both rat strains was 14 mg/m[sup 3]. [ABSTRACT FROM AUTHOR]