Biopersistences of man-made vitreous fibers and crocidolite fibers in rat lungs following short-term exposures.

Biopersistence of commercial man-made vitreous fibers (MMVF) and crocidolite were studied in Fischer 344 rats. MMVF used were size-selected to be rat-respirable, and rats were exposed nose-only 6 h/day for 5 days to gravimetric concentrations (30 mg/m3) of two fiber glass compositions--a rockwool, and a slagwool--or to 10 mg/m3 of long-fibered crocidolite, or to filtered air. Animals were sacrificed at 1 hr, 1, 5, 31, 90, 180, 270, 365, and 545 days after exposure stopped. Fibers were recovered from digested lung tissue to determine changes in concentrations (fibers/mg dry lung) and fiber retentions (expressed as percent of day 1 retention [PR]) for selected dimension categories. One-day average concentrations of lung-retained MMVF and crocidolite fibers, of diameter > or = 0.5 micron or > 20 microns in length, were nearly equal, permitting direct comparisons between MMVF and crocidolite. At 270 days average PR for MMVF > or = 0.5 micron in diameter were from 3 to 6 +/- 2% and 27 +/- 9% for crocidolite. For fibers > 20 microns, PR were 1 to 4 +/- 4% for MMVF and 37 +/- 20% for crocidolite. At 545 days, MMVF > 20 microns in length were at background level while concentration of crocidolite fibers > 20 microns in length remained at 2000 +/- 400 f/mg DL (dry lung), or 38 +/- 9% of day-1 retention. These results suggest strongly that MMVF dissolved or fractured in vivo whereas crocidolite fibers did not change.


Introduction
Studies of animal exposure to airborne fibrous materials (e.g., crocidolite fibers) have shown that certain factors largely control tissue response in terms of fibrogenesis or malignant transformation. First, fibers must be respirable, i.e., short and thin enough to reach the distal parts of the pulmonary region (1,2). Second, durable fibers of specific diameters and lengths ("Stanton" fibers) retained in the pulmonary region are needed to produce an adverse tissue response. Those fibers must be "biopersistent", so that specific dimensions are maintained at the cellular level for long enough (e.g., years) to attain the cumulative dose necessary to induce pathogenic effects (3,4). Moreover, the durable fibers should be longer than 17 pm to have the greatest effect, because such fibers can-not be phagocytized by the alveolar macrophages and would therefore be the most biopersistent . Focus on the fate of these fibers in the lung would also add to our understanding of in vivo dissolution.
This study compared the biopersistences in rat lung of respirable fractions of some commercial MMVF, with the biopersistence of respirable crocidolite fibers under the same conditions. Parameters studied included changes in concentrations (f/mg dry lung tissue) and dimensions (percent of day 1 lung retention, for selected lengths and diameters) of fibers EStock

Materials and Methods
Commercial fibers were Fiber 10, a glasswool (Manville Corp.), Fiber 11, a glasswool (Certainteed Corp.), Fiber 21, a rockwool (Roxul International), and Fiber 22, a slagwool (USG Interiors). Table 1 presents the chemical compositions of these bulk fibers. From each MMVF bulk product, the fraction selected had fibers of average dimensions of approximately 1 pm diameter and 15 pm length. Fibers of such dimensions are found in workplace aerosols. The fibers obtained accounted for 0.25 to 5 wt-% of the bulk product, depending on the initial diameter distribution of the MMVF. Crocidolite [obtained from the National Institute of Environmental Health Sciences (NIEHS)], also was size-selected to obtain a fraction of an average length of 7 pm.
Groups of Fischer 344 rats were exposed "nose-only" for 6 hr/day for 5 days to a gravimetric concentration of 30 mg/m3 for each MMVF. A group of rats was exposed to 10 mg/m3 of crocidolite fibers, and another group breathed filtered air. Fiber aerosols were lofted using a generation system designed not to break, grind, or contaminate the fibers. Aerosol concentrations for fiber mass were monitored by gravimetric sampling. Fiber concentration levels (fibers/cm3) were determined by phase contrast microscopy (x 400 magnification) for total and WHO fiber numbers (8), and by optical and scanning electron microscopy (SEM) for length Table 2. Fiber concentration (f/mg DLx1000) and percent of day 1 retention for crocidolite in rat lungs from 1  Subgroups of nine rats for each fiber type and nine controls were sacrificed 1 hr, 1, 5, 31, 90, 180, 270, 365, and 545 days after fiber inhalation had stopped. Lungs were removed, the infracardiac lobes separated, and the remaining lung tissues preserved for future reference and study. Lobes from five rats from each exposed group and from the controls were dehydrated and dried to constant weight to obtain the initial dry weight of tissue for determination of fiber concentrations. Tissues were low temperature ashed, the fibers were recov-ered, and numbers of fibers, and their diameters and lengths were determined using SEM and optical microscopy. Concentrations of fibers per mg of dry lung tissue* (f/mg DL) were calculated, as were concentrations of fibers and the percentages of day 1 retention (or PR) for selected diameter and length categories for each sacrifice period.

Results
Geometric mean diameters and lengths of lofted fibers and fibers recovered from lungs are shown in Figures 1 and 2. The average diameters and lengths of crocidolite fibers recovered from the rat lungs at various retention times differed little, whereas the MMVF showed a decrease in both geo-  metric mean diameter and length with time retained in the animals' lungs. Fibers recovered from the 1-hr sacrifice represent the deposition in both the conducting airway and alveolar regions of the lung, since particle clearance from the conducting airways is assumed to be complete within 24 hr after stopping exposure (9). Results were reported as average f/mg DL for selected dimension categories (Tables  2,3) and as PR for each sacrifice period (Tables 2,4).
For crocidolite fibers .5 pm in length, the average fiber concentrations from the day 1 sacrifice were 168,000 ± 47,000 and for 31, 100,000 ± 31,000 f/mg DL. The average decreased to 58,000 ± 12,000 at day 365 sacrifice and 56,000 ± 6000 at day 545 (Table 2).
Long crocidolite fiber retention tended to be prolonged compared to retention of crocidolite particles (i.e., <5 pm long) as indicated by higher retention values for the longer fibers beginning after 31 days.
Percent retentions of fibers .10 pm did not differ significantly from the PRs of fibers .5 pm. PRs of fibers >20 pm in length tended to be higher than those for fibers .5 pm at 365 and 545 days. For crocidolite fibers >20 pm in length, average concentrations were roughly equal between days 1 and 31 (first day, 5300; 31st day, 4000 f/mg DL), decreased to 1100 ± 800 f/mg DL at 180 days, and tended to remain constant thereafter (2000 ± 1000 f/mg DL, Table 2). Table 3 presents average concentrations of MMVF retained from day 1 to day 545. For MMVF .5 pm in length, the average fiber concentrations from day 1 sacrifices ranged from 11,500 f/mg DL to 37,600 f/mg DL. PRs of MMVF >5 pm at day 31 ranged from 42 ± 3% to 87 ± 19% and were about equal by day 180 (Table 4). *Reporting results as fibers per milligram of dry lung facilitates comparison with fiber data obtained from analyses of human lung tissue (e.g., ref. 17), but does not account for increase in lung mass with age. For example, from age 6 weeks to 78 weeks, the average rat lung mass nearly doubles. Reporting results as fibers per lung or fibers per lobe would increase the PR values given in Tables 2 and 4, especially for crocidolite since its fibers cleared from the lung at a much lower rate than did MMVF. For crocidolite fibers longer than 20 pm, for example, PRs would be 100% (day 1); 96% (day 31); 58% (day 90); 22% (day 180); 53% (day 270); 59% (day 545). In contrast, for fibers longer than 20 pm of MMVF 22, PRs would be 100% (day 1); 15% (day 31); 1% (day 90); 4% (day 180); 1% (day 270); 3% (day 365); not detected (day 545). A more complete presentation of these observations will be included in a forthcoming publication.
Volume 102, Supplement 5, October 1994 days, the PRs for MMVF > 20 pm in length were near background level. Discussion Movement of a particle in the pulmonary region is principally mediated by the alveolar macrophage that engulfs the particle and migrates either towards the bronchial lumen or into subpleural, paraseptal, perivascular, or peribronchial locations or into bronchial lymphatics and onward to lymphoid nodules (7,9). Clearance by this mechanism is slow, taking months or longer. The average diameter of an alveolar macrophage is 7.5 pm in rats and 8.5 pm in humans (11). Macrophage-mediated mechanical clearance is less effective for fibers .10 pm in length than for fibers between 5 pm and 10 pm in length (7,12). Analyses of anthophyllite fibers in lung tissues from Finnish workers suggested that the critical length for mechanical clearance of a fiber from the lung is 17 pm (13). Hence, macrophage clearance is most probably ineffective for fibers > 20 pm in length, both in rat and human lungs, and study of this fraction should permit more direct observation of in vivo fiber dissolution. In this study approximately equal concentrations of MMVF and crocidolite fibers longer than 20 pm were retained in the pulmonary region of lungs of animals one day after exposure had ceased (Tables 2,3) but after 180 days 90 to 100% of the long MMVF had been cleared, while no significant differences in percent clearance (PC) values were observed for long crocidolite fibers from 31 days on, and at 545 days, the PC for these fibers was about 60%*.
Furthermore, results from the 1-day sacrifices indicated that roughly the same concentrations of MMVF and crocidolite fibers .0.5 pm in diameter (Tables 2,3) were retained in the pulmonary regions of exposed animals. Within 180 days, approximately 90 to 95% of MMVF .0.5 pm in diameter had cleared from the lungs, and by 270 days this had increased to approximately 95 to 99%. In contrast, no significant differences were observed in the clearance of crocidolite fibers .0.5 pm in diameter from day 90 on, with values remaining approximately 70 to 75%.
Comparison of MMVF clearances is difficult because the day 1 deposition for each fiber type differed in size distribution. However, clearance of MMVF 21 >20 pm appeared to be slower up to 90 days compared to the other MMVFs. A previous study suggested that rockwool with an iron content similar to that of MMVF 21 appeared to be more persistent than glass fibers in rat lungs (12). Further study of the MMVF size distributions is needed to better understand these observations.
Lifetime animal inhalation studies of MMVF21 and 22 and crocidolite fibers were completed at Research and Consulting Company (RCC), Geneva in 1993 (14). Sixteen of 106 (14.1%) crocidoliteexposed animals had primary lung neoplasms compared to 2 of 126 (1.6%; p<0.01) unexposed control animals. One mesothelioma was observed in a rat from the crocidolite group. Fibrosis was observed in the crocidolite exposure group in all lungs examined after 3 months of exposure.

Environmental Health Perspectives
Retentions at day 545 approached background levels observed in lung tissues from unexposed animals ( Table 2).
Retention of MMVF particles <5 pm in length generally exceeded the corresponding PR's of MMVF .5 pm long.
Likewise, fibers <0.5 pm in diameter had consistently higher PRs than fibers >0.5 pm in diameter at each corresponding sacrifice point (Table 4).
Day 1 concentrations of fibers >20 pm in length ranged from 1500 ± 1700 f/mg DL (Fiber 10) to 4700 ± 600 f/mg DL (Fiber 11, Table 3). The PRs for Fiber 21 were higher than the average PR for the other MMVF both at 31 and 90 days; but by day 180 the PR had decreased to values close to those of the other MMVF. At 545