Eosinophilic globules (syn. hyaline inclusions) and protein crystalloids in the nasal mucosa of rats treated with synthetic amorphous silica, an unspecific chitinase-like-protein-positive background lesion.

In a 13-week inhalation toxicity study with three recovery periods (3, 6, and 12 months), Crl: WI rats were allocated to nine groups, each containing 25 animals per sex. Eight groups were treated daily by inhalation with the test items at concentrations of 0.5, 1.0, 2.5, or 5.0 mg/m3 (SAS 1 groups 2, 3, 4, or 5, respectively; SAS 2 groups 6, 7, 8, or 9, respectively). Controls (group 1) were treated with air only. In nasal cavities, the major lesions consisted of increased eosinophilic globules and chitinase-3-like-protein-positive crystalloids* in the nasal mucosa, mainly in nasal cavity levels 2-4 up to week 26 of recovery without any further injury in olfactory mucosa, mainly in SAS 1-treated animals. Eosinophilic globules in the rodent nasal cavity are common and increase with age; they represent a particular finding of the rodent nasal mucosa. The relevance of chitinase-3-like protein (Ym1 + Ym2) expression in the rodent nasal mucosa is unknown but is normal in control animals. Both findings developed without any indicator for inflammatory processes. The increase of these unspecific background findings is considered an indicator of minor irritative effects. Due to the clear lack of nasal tissue injury or concurrent changes (degeneration, necrosis, inflammatory infiltrate, dysplasia, and/or neoplasia) following repeated inhalation exposure to SAS, it is deemed that the eosinophilic globules (hyaline inclusions) combined with the formation of eosinophilic protein crystalloids in this study represent an adaptive response.

Enhanced study design for acute inhalation studies with hydrophobic surface-treated particles to determine toxicological effects including suffocation.

High concentrations of low-density particles may cause effects in acute inhalation toxicity studies which can be easily underestimated or misinterpreted following strictly the OECD TG 436, i.e., limited parameters as mortality and gross lesions will be evaluated only. Seven particle types (synthetic amorphous silica (SAS) HMDZ-SAS, silica gel, pyrogenic SAS, and precipitated SAS, calcium carbonate, aluminum oxide pyrogenic alumina, organic red pigment) were chosen at the highest technically feasible concentration of approximately 500 mg/m3 for acute inhalation studies with an expanded endpoint setup. Therefore additional parameters and a thorough histopathological evaluation of an extensive set of organs, including the respiratory tract emphasizing the nasal cavities were added. Six Crl:WI rats per study were exposed for four hours from which three animals were sacrificed after 24 hours and three animals after 14 days. HMDZ-SAS caused early death in all animals due to blockage of the nasal passages caused by its hydrophobicity. For all other Si-containing compounds, histology revealed minor inflammatory and reactive lesions in lungs after 24 hours that were still present after 14 days, except in silica gel-treated animals. After 14 days, for pyrogenic SAS, precipitated SAS, and pyrogenic alumina, granulomas formed in the BALT and lung-associated lymph nodes. In contrast, the calcium carbonate induced almost no findings, and the red pigment (also tested for the additional dose of 1000 mg/m3) stuck partially to the nasal mucosa without causing pathological damage and partly entered the lungs without showing any adverse effects. The results of the present study highlight the advantage of improving the rather simple study design of acute inhalation studies by implementing an extended study design.

Particle aerosol generation and potential altering in airflow used for acute/repeated inhalation toxicity testing.

Reproducible aerosol generation in combination with stable aerosol properties are essential prerequisites for compliant performance of acute or repeated inhalation toxicity tests of particulate materials according to OECD TG 403, 436, 412, or 413. A frequent problem of powder aerosol generation is the formation of coarse agglomerates with low shear resistance, which are beyond the tolerable size range but not detected by the prescribed aerodynamic measurement techniques by cascade impactor as the measurement conditions cause a disintegration into smaller fragments. But such agglomerates are observed during the transport to the inhalation chambers. These effects particularly apply to high mass concentrations and low-density powders, i.e., pyrogenic oxides. This study describes the transport influence in the airflow on the change of powder aerosols and on their respirability. A simplified short tube set-up was developed for the aerosol transport pre-tests, which allows the determination of the optimal aerosol formation conditions for the inhalation tests. The particles were measured with low shear using laser diffraction measurement or optical particle counters. The calculation of the aerodynamic particle sizes prescribed in the guidelines requires knowledge of the effective particle density of the porous aerosol particles. A practicable method for determining these is presented and described. In the outlook, first low concentration measurements show that clear agglomeration effects can also occur at particle concentrations around 20 mg/m³.

Regenerative and progressing lesions in lungs and lung-associated lymph nodes from fourteen 90-day inhalation studies with chemically different particulate materials.

Rat lungs and lung-associated lymph nodes from 14 inhalation studies with chemically different particulate materials were histopathologically re-evaluated, and the bronchoalveolar lavage fluid (BALF) data and lung burden analyses were compared. All investigated substances caused similar lesions. For most substances, 1 mg/m3 of respirable particulate matter was established as the borderline for adverse morphological changes after the 90-day exposure period, confirmed by the increase in polymorphonuclear neutrophils in BALF. Possible reversibility was demonstrated when recovery groups are included in the study especially allowing the differentiation between regeneration or progressing of inflammatory changes during the recovery period. It was concluded, that the major driver of toxicity is not an intrinsic chemical property of the particle but a particle effect. Concerning classification for specific target organ toxicant (STOT) repeated exposure (RE), this paper highlights that merely comparing the lowest concentration, at which adverse effects were observed, with the Classification Labelling and Packaging (CLP) regulation (EC) no. 1272/2008 guidance values is inappropriate and might lead to a STOT classification under CLP for a large part of the substances discussed in this paper, on the basis of typically mild to moderate findings in rat lung and lung-associated lymph nodes on day 1 after exposure. An in-depth evaluation of the pathologic findings is required and an expert judgement has to be included in the decision on classification and labeling, evaluating the type and severity of effects and comparing these with the classification criteria.

Issues in the inhalation toxicity testing and hazard assessment for low density particulate materials such as synthetic amorphous silica (SAS).

Inhalation toxicity testing of particulate materials is mandated for classification. According to CLP, particulate materials should be tested as marketed and many particulate materials are marketed as non-respirable particles. However, OECD TG 413 requires exposure to particle sizes that are respirable and reach the alveoli. The requirement for exposure of rats to respirable particles is thus in contrast to CLP and requires the application of high shear forces. The exposure to artificially small particles causes a number of issues that hamper the interpretation of the results of the testing. These issues are aerosol altering in the exposure system, assessment of the adversity of the inflammatory lung responses, inclusion of recovery groups, and extrapolation of the results to humans exposed under occupational condition. In addition, effects of many particulate materials after testing according to OECD 413 are not intrinsic properties, but a general reaction of the lung to the deposited material, show very similar NOAECs for chemical diverse materials, and often are completely reversible.

Physical Obstruction of Nasal Cavities With Subsequent Asphyxia, Causes Lethality of Rats in an Acute Inhalation Study With Hydrophobic HMDZ Surface-Treated Synthetic Amorphous Silica (SAS).

The aim of the present study was to understand the mechanism of lethality associated with high dose inhalation of a low-density hydrophobic surface-treated SAS observed in some acute inhalation studies. It was demonstrated that physical obstruction of the upper respiratory tract (nasal cavities) caused the effects observed. Hydrophobic surface-treated SAS was inhaled (flow-past, nose-only) by six Wistar rats (three males and three females) in an acute toxicity study at a concentration of ~500 mg/m3 for an intended 4-hr exposure. Under the conditions of the test set-up, the concentration applied was found to be the highest that can be delivered to the test animal port without significant alteration of the aerosol size distribution over time. None of the test- material-exposed animals survived the planned observation time of 4 h; three animals died between 2 34 h after starting exposure and cessation of exposure at 3 14 h, two died after transfer to their cages and the remaining animal was sacrificed due to its poor condition and welfare considerations. Histology accomplished by energy dispersive X-ray (EDX) analysis demonstrated that test material particles agglomerated and formed a gel-like substrate that ultimately blocked the upper respiratory airways, which proved fatal for the rat as an obligatory nose breather. This observation is in line with the findings reported by Hofmann et al. showing a correlation between lethality and hydrophobicity determined by contact angle measurement. The aerosol characterizations associated with this study are provided in detail by Wessely et al.

Influence of the H1 Antihistamine Mepyramine on the Antibacterial Effect of Florfenicol in Pigs.

The effect of florfenicol against Escherichia coli (E. coli) was investigated in vivo to confirm results of an in vitro study of Bruer et al. (2019), which has shown positive effects of various antibacterial agents in combination with the antihistamine mepyramine (MEP). Therefore, pigs were treated in three different settings: An untreated control group, 10 mg/kg florfenicol (FFC) and 10 mg/kg FFC in combination with 20 mg/kg MEP. E. coli were isolated from faecal samples and analyzed in growth quantity and resistance to FFC. The FFC medication induced an increased number of resistant E. coli strains isolated from faecal samples. The number of colonies detected after cultivation of animal samples treated with 10 mg/kg FFC was higher than the number of colonies after treatment with 10 mg/kg FFC in combination with of FFC and MEP. Furthermore, the effect of both compounds was examined on bacterial susceptibility of Pasteurella multocida in vitro, where the combination of FFC with MEP resulted in a diminished minimum inhibitory concentration. We confirmed the development of bacterial resistance in the intestine as non-target tissue caused by the use of the antibacterial agent florfenicol. Moreover, the combination of FFC with an antihistamine like MEP offers a possibility to enhance the efficacy of an antibacterial treatment and modifies the effect on gut microbiota.

Histamine 2 Receptor Agonism and Histamine 4 Receptor Antagonism Ameliorate Inflammation in a Model of Psoriasis.

Psoriasis is a chronic inflammatory skin disorder characterized by hyperproliferative keratinocytes and immune cell infiltration into the skin, often accompanied by itch. Histamine, acting via histamine 1-4 receptors, is known to modulate immune responses in the skin and to induce itch. The aim of this study was to test the role of histamine 2 receptors and histamine 4 receptors in the imiquimod-induced psoriasis-like skin inflammation model. BALB/c mice were treated intraperitoneally with amthamine (histamine 2 receptor agonist), JNJ-39758979 (histamine 4 receptor antagonist), a combination of both, or vehicle twice daily in a preventive manner. Imiquimod was applied once daily onto the back skin for 10 consecutive days. Stimulation of histamine 2 receptors and blockade of histamine 4 receptors ameliorated imiquimod-induced skin inflammation. The combination of amthamine and JNJ-39758979 reduced skin inflammation even more pronounced, diminished epidermal hyperproliferation, and inhibited spontaneous scratching behaviour. A combination of histamine 2 receptor agonist and histamine 4 receptor antagonists could represent a new strategy for the treatment of psoriasis.