Metabolite profiles of rats in repeated dose toxicological studies after oral and inhalative exposure.

The MetaMap(®)-Tox database contains plasma-metabolome and toxicity data of rats obtained from oral administration of 550 reference compounds following a standardized adapted OECD 407 protocol. Here, metabolic profiles for aniline (A), chloroform (CL), ethylbenzene (EB), 2-methoxyethanol (ME), N,N-dimethylformamide (DMF) and tetrahydrofurane (THF), dosed inhalatively for six hours/day, five days a week for 4 weeks were compared to oral dosing performed daily for 4 weeks. To investigate if the oral and inhalative metabolome would be comparable statistical analyses were performed. Best correlations for metabolome changes via both routes of exposure were observed for toxicants that induced profound metabolome changes. e.g. CL and ME. Liver and testes were correctly identified as target organs. In contrast, route of exposure dependent differences in metabolic profiles were noted for low profile strength e.g. female rats dosed inhalatively with A or THF. Taken together, the current investigations demonstrate that plasma metabolome changes are generally comparable for systemic effects after oral and inhalation exposure. Differences may result from kinetics and first pass effects. For compounds inducing only weak changes, the differences between both routes of exposure are visible in the metabolome.

Prevalidation of the ex-vivo model PCLS for prediction of respiratory toxicity.

In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23 h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration-response curves were analyzed. Log10[IC50 (µM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC50 by IC50) were developed and showed promising results.

Short term inhalation toxicity of a liquid aerosol of glutaraldehyde-coated CdS/Cd(OH)2 core shell quantum dots in rats.

Quantum dots exhibit extraordinary optical and mechanical properties, and the number of their applications is increasing. In order to investigate a possible effect of coating on the inhalation toxicity of previously tested non-coated CdS/Cd(OH)2 quantum dots and translocation of these very small particles from the lungs, rats were exposed to coated quantum dots or CdCl2 aerosol (since Cd(2+) was present as impurity), 6h/d for 5 consecutive days. Cd content was determined in organs and excreta after the end of exposure and three weeks thereafter. Toxicity was determined by examination of broncho-alveolar lavage fluid and microscopic evaluation of the entire respiratory tract. There was no evidence for translocation of particles from the respiratory tract. Evidence of a minimal inflammatory process was observed by examination of broncho-alveolar lavage fluid. Microscopically, minimal to mild epithelial alteration was seen in the larynx. The effects observed with coated quantum dots, non-coated quantum dots and CdCl2 were comparable, indicating that quantum dots elicited no significant effects beyond the toxicity of the Cd(2+) ion itself. Compared to other compounds with larger particle size tested at similarly low concentrations, quantum dots caused much less pronounced toxicological effects. Therefore, the present data show that small particle sizes with corresponding high surfaces are not the only factor triggering the toxic response or translocation.

Short term inhalation toxicity of a liquid aerosol of CdS/Cd(OH)2 core shell quantum dots in male Wistar rats.

Colloidal quantum dots (QD) show great promise as fluorescent markers. The QD used in this study were obtained in aqueous medium rather than the widely used colloidal QD. Both methodologies used for the production of QD are associated with the presence of heavy metals such as cadmium (Cd). Here we investigate the short-term inhalation toxicity of water-soluble core-shell CdS/Cd(OH)2 QD. Male Wistar rats were head-nose exposed for 6 h/day on 5 days at the technically maximum concentration (0.52 mg Cd/m³). Histological examination was performed directly after the last exposure. Additional rats were used for Cd organ burden determinations. Clinical parameters in blood, bronchoalveolar lavage fluid and lung tissue were determined 3 days after the last exposure. To analyze the reversibility or progression of effects, the examinations were performed again after a recovery period of 3 weeks. The results of the study indicate that CdS/Cd(OH)2 QD caused local neutrophil inflammation in the lungs that partially regressed after the 3-week recovery period. There was no evidence that QD were translocated to the central nervous system nor that a systemic acute phase response occurred.

Generation and characterization of test atmospheres with nanomaterials.

To ensure the product safety of nanomaterials, BASF has initiated an extensive program to study the potential inhalation toxicity of nanosize particles. As preparation work for upcoming inhalation studies, the following manufactured nanomaterials have been evaluated for their behavior in an exposure system designed for inhalation toxicity studies: titanium dioxide, carbon black, Aerosil R104, Aerosil R106, aluminum oxide, copper(II) oxide, amorphous silicon dioxide, zinc oxide, and zirconium(IV) oxide. As the physicochemical properties and the complex nature of ultrafine aerosols may substantially influence the toxic potential, the particle size, specific surface area, zeta potential, and morphology of each of the materials were determined. Aerosols of each material were generated using a dry powder aerosol generator and by nebulization of particle suspensions. The mass concentration of the particles in the inhalation atmosphere was determined gravimetrically and the particle size was determined using a cascade impactor, an optical particle counter, and a scanning mobility particle sizer. The dispersion techniques used generated fine aerosols with particle size distributions in the respiratory range. However, as a result of the significant agglomeration of nanoparticles in the test materials evaluated, no more than a few mass percent of the materials were present as single nanoparticles (i.e., < 100 nm). Considering the number, a greater percentage of nanoparticles was present. Based on the obtained results and experience with the equipment, a technical setup for inhalation studies with nanomaterials is proposed. Furthermore, a stepwise testing approach is recommended that also could reduce the number of animals used in testing.

Determination of pulmonary irritant threshold concentrations of hexamethylene-1,6-diisocyanate (HDI) prepolymers by bronchoalveolar lavage in acute rat inhalation studies according to TRGS 430.

Pulmonary irritant threshold concentrations of two hexamethylene-1,6-diisocyanate (HDI)-based prepolymers (I: polymeric emulsfier modified and II: oligomeric allophanate modified) were determined in acute inhalation studies according to TRGS 430 (Dangerous Substances Technical Rule, isocyanates, Germany), based on benchmark extrapolation of bronchoalveolar lavage fluid (BALF) total protein. It was also investigated if the method is robust enough to be transferred to an independent laboratory. Five male Wistar rats per group were exposed nose-only to the test substances as liquid aerosols to concentrations of 0, 0.5, 3, 15 mg/m(3) for both test substances with an additional test group at 50 mg/m(3) for test substance I. The duration of the exposure was 6h, followed by serial sacrifices 1 day, 3 days and 7 days post exposure. BALF was analyzed for biochemical and cytological markers indicative for injury of the bronchoalveolar region. The exposure of rats to test substance I and II caused dose depended lung irritation with BALF total protein concentration being the most sensitive indicator of pulmonary effects. The extrapolated no observed adverse effect level of test substance I was 1.1 mg/m(3) and that of test substance II 2.3 mg/m(3). The acute pulmonary irritant threshold concentrations were found to be similar to those reported by [Pauluhn, J., 2004. Pulmonary irritant potency of polyisocyanate aerosols in rats: comparative assessment of irritant threshold concentrations by bronchoalveolar lavage. J. Appl. Toxicol. 24, 231-247] for HDI-homopolymers and other HDI-based polyisocyanates, and were at least 30 times higher than the MAK (occupational exposure limit) value for the HDI monomer (0.035 mg/m(3)). Thus the EBW (exposure assessment value) for these two HDI-based prepolymers can be established at 10x MAK, i.e. at 0.35 mg/m(3).

Investigations on the subchronic toxicity of 2-methoxypropanol-1(acetate) in rats.

Wistar rats were exposed to 2-methoxypropylacetate-1 (2-MPAc-1) vapours in concentrations of 0, 110, 560 and 2800 ppm for (equiv. to 0; 0.6; 3.0 and 14.9 mg/L) for 4 weeks in chambers (6 hours/day; 5 days/week; five male and five female animals per group). The top concentration was equivalent to a 95% vapour saturation at 20 degrees C and the animals reacted to this with a moderate respiratory irritation during the 6 hours exposure times; at 560 ppm these effects were only slight. The top dose was also associated with a significantly reduced body weight development and some hematologic and biochemical alterations of little specificity. The most prominent effect was thymic atrophy. No effects were noted on the testes or on the cellularity in blood or bone marrow. 560 ppm were without systemic effects. Furthermore, 2-methoxypropanol-1 (2-MP-1), 2-MPAc-1 and 2-ethoxyethanol (EE) were administered in parallel by gavage to groups of five male Wistar rats daily for 10 days at near equimolar dose levels (1800, 2600 and 1800 mg/kg per day, respectively). At the end of the administration period the testes were investigated. There was a pronounced testicular atrophy in animals exposed to EE, whereas no adverse effects were observed with 2-MP-1 and 2-MPAc-1. The results of these studies indicate that 2-MP-1 and 2-MPAc-1 which previously had been shown to exert pronounced prenatal toxicity in rabbits and weak prenatal effects in rats are devoid of other forms of systemic toxicity in rats that are typically observed with ethoxyethanol and methoxyethanol.