Considerations for the Identification and Conveyance of Clinical Pathology Findings in Preclinical Toxicity Studies: Results From the 9th ESTP International Expert Workshop.

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.

Perinatal exposure to the immune-suppressant di-n-octyltin dichloride affects brain development in rats.

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.

Toxicologic Pathology Forum: Considerations Regarding Determination of Adversity for Immunopathology Findings in Nonclinical Toxicology Studies with Immune-Modulating Therapeutics.

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.

Nanomaterials and the Serosal Immune System in the Thoracic and Peritoneal Cavities.

The thoracic and peritoneal cavities are lined by serous membranes and are home of the serosal immune system. This immune system fuses innate and adaptive immunity, to maintain local homeostasis and repair local tissue damage, and to cooperate closely with the mucosal immune system. Innate lymphoid cells (ILCs) are found abundantly in the thoracic and peritoneal cavities, and they are crucial in first defense against pathogenic viruses and bacteria. Nanomaterials (NMs) can enter the cavities intentionally for medical purposes, or unintentionally following environmental exposure; subsequent serosal inflammation and cancer (mesothelioma) has gained significant interest. However, reports on adverse effects of NM on ILCs and other components of the serosal immune system are scarce or even lacking. As ILCs are crucial in the first defense against pathogenic viruses and bacteria, it is possible that serosal exposure to NM may lead to a reduced resistance against pathogens. Additionally, affected serosal lymphoid tissues and cells may disturb adipose tissue homeostasis. This review aims to provide insight into key effects of NM on the serosal immune system.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Hematolymphoid System.

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.

A practical approach to assess inhalation toxicity of metal oxide nanoparticles in vitro.

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 , Mn2 O3 , CuO, ZnO, Co3 O4 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 Mn2 O3 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 Mn2 O3 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.

Inhaled multiwalled carbon nanotubes modulate the immune response of trimellitic anhydride-induced chemical respiratory allergy in brown Norway rats.

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/m(3)) or a high (22 mg/m(3)) 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.

Potentially increased sensitivity of pregnant and lactating female rats to immunotoxic agents.

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.

Nasal passages of Göttingen minipigs from the neonatal period to young adult.

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.

Pre-exposure to sulfur dioxide attenuates most allergic reactions upon trimellitic anhydride challenge in sensitized Brown Norway rats.

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 (SO(2)) 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 SO(2), and challenged by inhalation to a distinctly irritating or minimally irritating concentration of TMA after the (last) SO(2) exposure. Repeated exposure to SO(2) 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 SO(2) exposure. Repeated pre-exposure to SO(2) reduced the number of TMA-induced apnoeas, in an SO(2) exposure duration-dependent manner. This effect of SO(2) on TMA-induced functional allergic reactions (apnoeas) was distinct only when the TMA challenge concentration was not too irritating itself. Repeated pre-exposure to SO(2) reduced TMA-induced laryngeal ulceration, goblet-cell hyperplasia, and inflammation in the lungs in most animals, regardless of the TMA challenge concentration. The SO(2)-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, SO(2) appeared to facilitate TMA-induced irritation, probably due to incomplete protection. Overall, SO(2) exposure of TMA-sensitized rats reduced TMA-related allergic respiratory responses in most animals.

The contact allergen dinitrochlorobenzene (DNCB) and respiratory allergy in the Th2-prone Brown Norway rat.

All LMW respiratory allergens known to date can also induce skin allergy in test animals. The question here was if in turn skin allergens can induce allergy in the respiratory tract. Respiratory allergy was tested in Th2-prone Brown Norway (BN) rats by dermal sensitization with the contact allergen dinitrochlorobenzene (DNCB; 1%, day 0; 0.5%, day 7) and a head/nose-only inhalation challenge of 27mg/m3 of DNCB (15 min, day 21), using a protocol that successfully identified chemical respiratory allergens. Skin allergy to DNCB was examined in BN rats and Th1-prone Wistar rats in a local lymph node assay followed by a topical patch challenge of 0.1% DNCB. Sensitization of BN rats via the skin induced DNCB-specific IgG in serum, but not in all animals, and an increased number of CD4+ cells in the lung parenchyma. Subsequent inhalation challenge with DNCB did not provoke apneas or allergic inflammation (signs of respiratory allergy) in the BN rats. However, microarray analysis of mRNA isolated from the lung revealed upregulation of the genes for Ccl2 (MCP-1), Ccl4 (MIP-1beta), Ccl7 and Ccl17. Skin challenge induced considerably less skin irritation and allergic dermatitis in the BN rat than in the Wistar rat. In conclusion, the Th2-prone BN rat appeared less sensitive to DNCB than the Wistar rat; nevertheless, DNCB induced allergic inflammation in the skin of BN rats but even a relatively high challenge concentration did not induce allergy in the respiratory tract, although genes associated with allergy were upregulated in lung tissue.

Molecular characterization of trimellitic anhydride-induced respiratory allergy in Brown Norway rats.

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/m(3) 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.

Preexposure to amorphous silica particles attenuates but also enhances allergic reactions in trimellitic anhydride-sensitized brown Norway rats.

Irritant-induced inflammation of the airways may aggravate respiratory allergy induced by chemical respiratory allergens. Therefore, the effect of airway irritation by synthetic amorphous silica (SAS) on respiratory allergy to trimellitic anhydride (TMA) was studied. Brown Norway (BN) rats were topically sensitized on day 0 and on day 7, subsequently exposed for 6 h/day for 6 days to 27 mg/m(3) SAS, and challenged by inhalation to a minimally irritating concentration of 12 mg/m(3) TMA, 24 h after the last SAS exposure. An additional group was exposed to SAS before a second challenge to TMA. Control groups were treated with vehicle, and/or did not receive SAS exposure. Breathing parameters, cellular and biochemical changes in bronchoalveolar lavage (BAL) fluid, and histopathological airway changes 24 h after challenge were the main parameters studied. Exposure to SAS alone resulted in transient changes in breathing parameters during exposure, and in nasal and alveolar inflammation with neutrophils and macrophages. Exposure to SAS before a single TMA challenge resulted in a slightly irregular breathing pattern during TMA challenge. SAS also diminished the effect of TMA on tidal volume, laryngeal ulceration, laryngeal inflammation, and the number of BAL (lung) eosinophils in most animals, but aggravated laryngeal squamous metaplasia and inflammation in a single animal. The pulmonary eosinophilic infiltrate and edema induced by a second TMA challenge was diminished by the preceding SAS exposure, but the number of lymphocytes in BAL was increased. Thus, a respiratory particulate irritant like SAS can reduce as well as aggravate certain aspects of TMA-induced respiratory allergy.

Setting an indoor air exposure limit for formaldehyde: factors of concern.

The paper aims to evaluate the indoor air limit of 1 microg/m(3) (0.8 ppb) formaldehyde as advised by the European Commission [the INDEX project; Kotzias, D., Koistinen, K., Kephalopoulos, S., Schlitt, C., Carrer, P., Maroni, M., Jantunen, M., Cochet, C., Kirchner, S., Lindvall, T., McLaughlin, J., Mølhave, L., de Oliveira Fernandes, E., Seifert, B., 2005. Critical appraisal of the setting and implementation of indoor exposure limits in the EU. European Commission, Institute for Health and Consumer Protection, Physical and Chemical Exposure Unit, Ispra, Italy, pp. 1-50]. The limit has been based on a nose and throat irritation threshold of 0.1mg/m(3) (0.08 ppm; LOAEL), a NOAEL of 0.03 mg/m(3) (0.025 ppm) and an assessment factor of 30, including a factor of 3 for the higher sensitivity of children. Nose and throat irritation, at concentrations below which hyperplasia/metaplasia occurs, are most likely the manifestation of trigeminal nerve stimulation (sensory irritation). The threshold for sensory irritation in human volunteers is 1 ppm, much higher than the 0.1mg/m(3) indicated above. Eye irritation is the most sensitive effect reported in human volunteers but has been mentioned only occasionally in the studies used by the European Commission. Moreover, sensory irritation is a local reaction that requires a low assessment factor, if any. It is difficult to judge the sensitivity for sensory irritation in children because of the potential confounding factors in the evaluated studies. It is concluded that an indoor air level of 0.1 ppm (0.12 mg/m(3)) formaldehyde, as indicated by Appel et al. (2006) [Appel, K.E., Bernauer, U., Herbst, U., Madle, S., Schulte, A., Richter-Reichhelm, H.B., Gundert-Remy, U. 2006. Kann für Formaldehyd eine "sichere" Konzentration abgeleitet werden?--Analyse der Daten zur krebserzeugenden Wirkung (Can a "safe" concentration be established for formaldehyde?--Analysis of carcinogenicity data)? Umweltmed. Forsch. Prax. 11, 347-361], can be considered a safe and appropriate level.

Five-day inhalation toxicity study of three types of synthetic amorphous silicas in Wistar rats and post-exposure evaluations for up to 3 months.

Evidence suggests that short-term animal exposures to synthetic amorphous silicas (SAS) and crystalline silica can provide comparable prediction of toxicity to those of 90-day studies, therefore providing the opportunity to screen these types of substances using short-term rather than 90-day studies. To investigate this hypothesis, the inhalation toxicity of three SAS, precipitated silica Zeosil 45, silica gel Syloid 74, and pyrogenic silica Cab-O-Sil M5 was studied in Wistar rats. Rats were exposed nose-only to concentrations of 1, 5 or 25mg/m(3) of one of the SAS 6h a day for five consecutive days. Positive controls were exposed to 25mg/m(3) crystalline silica (quartz dust), negative controls to clean air. Animals were necropsied the day after the last exposure or 1 or 3 months later. All exposures were tolerated without serious clinical effects, changes in body weight or food intake. Differences in the effects associated with exposure to the three types of SAS were limited and almost exclusively confined to the 1-day post-exposure time point. Silicon levels in tracheobronchial lymph nodes were below the detection limit in all groups at all time points. Silicon was found in the lungs of all high concentration SAS groups 1-day post-exposure, and was cleared 3 months later. Exposure to all three SAS at 25mg/m(3) induced elevations in biomarkers of cytotoxicity in bronchoalveolar lavage fluid (BALf), increases in lung and tracheobronchial lymph node weight and histopathological lung changes 1-day post-exposure. Exposure to all three SAS at 5mg/m(3) induced histopathological changes and changes in BALf only. With all three SAS these effects were transient and, with the exception of slight histopathological lung changes at the higher exposure levels, were reversible during the 3-month recovery period. No adverse changes were observed in animals exposed to any of the SAS at 1mg/m(3). In contrast, with quartz-exposed animals the presence of silicon in the lungs was persistent and toxicological effects differed from those seen with SAS both with regard to the type and severity as well as in the time-response profile. In quartz-exposed animals silicon in the tracheobronchial lymph nodes was below the detection limit but silicon was found in the lungs at comparable levels 0-, 1- and 3-months post-exposure. One-day post-exposure to quartz, elevations in biomarkers of cytotoxicity in BALf, increases in lung and tracheobronchial lymph node weight and histopathological lung changes were minimal. These effects were present at 1-month post-exposure and progressively more severe at 3-months post-exposure. Overall, the results of the current study are similar to those of other published studies that had a 90-day exposure period and both types of studies indicate that the lack of lung clearance is a key factor in the development of silicosis.

Development of immune organs and functioning in humans and test animals: Implications for immune intervention studies.

A healthy immune status is mostly determined during early life stages and many immune-related diseases may find their origin in utero and the first years of life. Therefore, immune health optimization may be most effective during early life. This review is an inventory of immune organ maturation events in relation to developmental timeframes in minipig, rat, mouse and human. It is concluded that time windows of immune organ development in rodents can be translated to human, but minipig reflects the human timeframes better; however the lack of prenatal maternal-fetal immune interaction in minipig may cause less responsiveness to prenatal intervention. It is too early to conclude which immune parameters are most appropriate, because there are not enough comparative immune parameters. Filling these gaps will increase the predictability of results observed in experimental animals, and guide future intervention studies by assessing relevant parameters in the right corresponding developmental time frames.

Integrated analysis of toxicity data of two pharmaceutical immunosuppressants and two environmental pollutants with immunomodulating properties to improve the understanding of side effects-A toxicopathologist׳s view.

Data in a toxicity test are evaluated generally per parameter. Information on the response per animal in addition to per parameter can improve the evaluation of the results. The results from the six studies in rats, described in the paper by Kemmerling, J., Fehlert, E., Rühl-Fehlert, C., Kuper, C.F., Stropp, G., Vogels, J., Krul, C., Vohr, H.-W., 2015. The transferability from rat subacute 4-week oral toxicity study to translational research exemplified by two pharmaceutical immunosuppressants and two environmental pollutants with immunomodulating properties (In this issue), have been subjected to principal component analysis (PCA) and principal component discriminant analysis (PC-DA). The two pharmaceuticals azathioprine (AZA) and cyclosporine A (CSA) and the two environmental pollutants hexachlorobenzene (HCB) and benzo(a)pyrene (BaP) all modulate the immune system, albeit that their mode of immunomodulation is quite diverse. PCA illustrated the similarities between the two independent studies with AZA (AZA1 and AZA2) and CSA (CSA1 and CSA2). The PC-DA on data of the AZA2 study did not increase substantially the information on dose levels. In general, the no-effect levels were lower upon single parameter analysis than indicated by the distances between the dose groups in the PCA. This was mostly due to the expert judgment in the single parameter evaluation, which took into account outstanding pathology in only one or two animals. The PCA plots did not reveal sex-related differences in sensitivity, but the key pathology for males and females differed. The observed variability in some of the control groups was largely a peripheral blood effect. Most importantly, PCA analysis identified several animals outside the 95% confidence limit indicating high-responders; also low-to-non-responders were identified. The key pathology enhanced the understanding of the response of the animals to the four model compounds.

The transferability from rat subacute 4-week oral toxicity study to translational research exemplified by two pharmaceutical immunosuppressants and two environmental pollutants with immunomodulating properties.

Exposure to chemicals may have an influence on the immune system. Often, this is an unwanted effect but in some pharmaceuticals, it is the intended mechanism of action. Immune function tests and in depth histopathological investigations of immune organs were integrated in rodent toxicity studies performed according to an extended OECD test guideline 407 protocol. Exemplified by two immunosuppressive drugs, azathioprine and cyclosporine A, and two environmental chemicals, hexachlorobenzene and benzo[a]pyrene, results of subacute rat studies were compared to knowledge in other species particular in humans. Although immune function has a high concordance in mammalian species, regarding the transferability from rodents to humans various factors have to be taken into account. In rats, sensitivity seems to depend on factors such as strain, sex, stress levels as well as metabolism. The two immunosuppressive drugs showed a high similarity of effects in animals and humans as the immune system was the most sensitive target in both. Hexachlorobenzene gave an inconsistent pattern of effects when considering the immune system of different species. In some species pronounced inflammation was observed, whereas in primates liver toxicity seemed more obvious. Generally, the immune system was not the most sensitive target in hexachlorobenzene-treatment. Immune function tests in rats gave evidence of a reaction to systemic inflammation rather than a direct impact on immune cells. Data from humans are likewise equivocal. In the case of benzo[a]pyrene, the immune system was the most sensitive target in rats. In the in vitro plaque forming cell assay (Mishell-Dutton culture) a direct comparison of cells from different species including rat and human was possible and showed similar reactions. The doses in the rat study had, however, no realistic relation to human exposure, which occurs exclusively in mixtures and in a much lower range. In summary, a case by case approach is necessary when testing immunotoxicity. Improvements for the translation from animals to humans related to immune cells can be expected from in vitro tests which offer direct comparison with reactions of human immune cells. This may lead to a better understanding of results and variations seen in animal studies.

Toxicity to nasal-associated lymphoid tissue.

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.