Bruce K. Zoitos

IN VITRO MEASUREMENT OF FIBER DISSOLUTION RATE RELEVANT TO BIOPERSISTENCE AT NEUTRAL pH: AN INTERLABORATORY ROUND ROBIN

Measurements are presented of the dissolution rates in neutral pH simulated lung (SLF) of several man-made vitreous fibers (MMVF) and crocidolite asbestos that were recently in chronic rodent inhalation studies. The measured dissolution rate depended strongly on the fiber composition. The MMVF tested dissolved from 30 times to nearly 1000 times faster than the crocidolite asbestos. Measurements were made in flow-through equipment in four different laboratories in North America and Europe. The standard deviations of the measured values for each fiber were typically between 30 and 50% of average value. It is believed that in order to be relevant to the dissolution of long fibers the extracellular fluid of the lung, the in vitro measurement must be performed at a rate high enough that corrosion products do not accumulate in sufficient concentration affect the dissolution rate.

Measurement of in- vitro fibre dissolution rate at acidic pH

A low biopersistence of man-made vitreous fibres (MMVFs) has often been related to a high invitro dissolution rate at near-neutral pH. For some fibre types, however, a low in-vivo biopersistence cannot be explained by the in-vitro dissolution rate in near-neutral physiological fluids. It has been suggested, that the high in-vitro dissolution rate of these fibres at the acidic pH which is found inside the phagolysosomes of the alveolar macrophages could be the reason for the fast in-vivo clearance of such fibres. The aim of this study was, through interlaboratory comparison of in-vitro dissolution measurements at acidic pH, to investigate the causes of variations and to identify key parameters for a measurement method similar to the method used at near neutral pH. Results of in-vitro measurements of different fibre types at acidic pH are presented, and the influence of different test parameters described. The measurements and calculations at acidic pH can be made similar to what is done for measurements at near-neutral conditions. Since incongruent dissolution is often very pronounced, it is relevant to calculate two dissolution rates, one for the network represented by silicon and one for the leaching elements. In order to obtain comparable results it is necessary to use a liquid which is well-defined with respect to type and concentration of buffer system, and to compare only values obtained under similar experimental conditions. The measured dissolution rates differ substantially with different liquids and different experimental conditions. However, the ranking of the different tested fibres with respect to the dissolution rates remains the same as long as they are tested under identical conditions.

Lack of marked cyto- and genotoxicity of cristobalite in devitrified (heated) alkaline earth silicate wools in short-term assays with cultured primary rat alveolar macrophages

Abstract Alkaline earth silicate (AES) wools are low-biopersistence high-temperature insulation wools. Following prolonged periods at high temperatures they may devitrify, producing crystalline silica (CS) polymorphs, including cristobalite, classified as carcinogenic to humans. Here we investigated the cytotoxic and genotoxic significance of cristobalite present in heated AES wools. Primary rat alveolar macrophages were incubated in vitro for 2 h with 200 µg/cm2 unheated/heated calcium magnesium silicate wools (CMS1, CMS2, CMS3; heat-treated for 1 week at, or 4 weeks 150 °C below, their respective classification temperatures) or magnesium silicate wool (MS; heated for 24 h at 1260 °C). Types and quantities of CS formed, and fiber size distribution and shape were determined by X-ray diffraction and electron microscopy. Lactate dehydrogenase release and alkaline and hOGG1-modified comet assays were used, ± aluminum lactate (known to quench CS effects), for cytotoxicity/genotoxicity screening. Cristobalite content of wools increased with heating temperature and duration, paralleled by decreases in fiber length and changes in fiber shape. No marked cytotoxicity, and nearly no (CMS) or only slight (MS) DNA-strand break induction was observed, compared to the CS-negative control Al2O3, whereas DQ12 as CS-positive control was highly active. Some samples induced slight oxidative DNA damage, but no biological endpoint significantly correlated with free CS, quartz, or cristobalite. In conclusion, heating of AES wools mediates changes in CS content and fiber length/shape. While changes in fiber morphology can impact biological activity, cristobalite content appears minor or of no relevance to the intrinsic toxicity of heated AES wools in short-term assays with rat alveolar macrophages.

Horizontal diffusion elutriation: a new size-separation technique for preparation of rodent-respirable fibers for animal testing.

Short-and long-term animal experiments are used to examine the toxicology and biopersistence of various types of fibers. In order to ensure an adequate exposure dose for testing, modern experimental protocols specify that the exposure aerosol (in an inhalation test) or the fibers (in an intratracheal instillation [IT] test) must contain at least a minimum concentration of long (> 20 μm) rodent-respirable fibers. As produced and handled, most fibers contain a distribution of diameters and lengths, only some of which are both long and rodent-respirable. Therefore, it is necessary to size-separate the fibers to enrich the proportion of long, rodent-respirable fibers in the material to be tested. This article presents a new and relatively simple method for size separation that avoids some of the difficulties associated with other methods. The method, termed horizontal diffusion elutriation (HDE), is illustrated by size-separating refractory ceramic fiber (RCF) and four polycrystalline alumina (PCA) fibers.