INVESTIGATION OF THE DURABILITY OF CELLULOSE FIBRES IN RAT LUNGS

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1 Pergamon Ann. occup. Hyg., Vol. 41, Supplement 1, pp , Published by Elsevier Science Ltd on behalf of BOHS Printed in Great Britain. All rights reserved /97 $ Inhaled Particles VUI PII: S (96) INVESTIGATION OF THE DURABILITY OF CELLULOSE FIBRES IN RAT LUNGS H. Muhle, H. Ernst and B. Bellmann Fraunhofer Institute of Toxicology and Aerosol Research, Nikolai-Fuchs-Str. 1, D Hannover, Germany INTRODUCTION Inhalable cellulose fibres can be released in production processes in the paper industry or during spraying of specially prepared chips of recycled newspapers which are used for thermal insulation of walls in houses. In the latter process, fibre concentrations may amount up to 50 x 10 6 fibres m~ 3 in building sites (Tiesler and Schnittger, 1992). For the definition of a "critical" fibre the same criteria as for inorganic fibres were used (fibre length > 5 um, fibre diameter < 3 urn, aspect ratio >3:1). Milton et al. (1990) instilled cellulose dust intratracheally in hamsters by a single dose of 0.75 mg/100 g body weight. Hamsters were killed after 8 weeks. Lungs of animals showed a significant number of granulomata and thickened interalveolar septae. In a 28-day inhalation study with cellulose in rats, Davis (1993) reported the appearance of alveolitis and granulomata. The test material originated from thermal insulation products. It is an open question, whether cellulose fibres can accumulate in lungs or whether these fibres will show a degradation under physiological conditions. The goal of this investigation was to analyse quantitatively the biodurability of cellulose fibres in lungs of rats. MATERIALS AND METHODS Chemical pure microcrystalline wood cellulose fibres (type Avicel PH 105 Serva, Heidelberg, Germany) and the commercially available product Isofloc originating from recycled newspaper were classified to obtain a respirable fraction of these materials, Isofloc is used as a thermal insulating product and was treated with 12% borax and 8% boric acid. Airborne fibres were sized in a high-volume heavy-gain impaction sampler (Bellmann and Muhle, 1994). The obtained rat-inhalable fractions were named Cellulose-F (reference substance) and. The size distribution was determined by electron microscopic investigations. For the median fibre length was 7.6 um, the median fibre diameter was 0.50 um. For Cellulose-F the corresponding values were 4.2 and 0.87 um, respectively (see Table 1)- Test materials were suspended in saline and 2 mg per animal was instilled intratracheally into 10-weeks-old female Wistar rats (30 animals per group). This 184

2 Durability of cellulose fibres in rat lungs 185 Table 1. Percentiles of the frequency distribution of the materials length and diametei of fibres in the sized Material 10% < Fibre length [urn] 50% < 90% < 99% < 10% < Fibre diameter [urn] 50% < 90% < 99% < Cellulose-F amount of mass leads to dust overload conditions in lungs. Macrophage-mediated particle clearance is reduced at this lung burden (Muhle et al., 1990). A third group of 30 animals was treated by saline only. At 2 days, 1, 3, 6 and 12 months after treatment at each date six animals per group were sacrificed and lungs were isolated. Tissues of four lungs per group were digested by 26% hypochlorite for 4 h at 4 C on a roller. The suspension was centrifuged for 25 min at g. The pellet was resuspended in water and filtered on a nuclepore filter. The treatment of test materials by hypochlorite under these conditions did not change the size distribution of the fibres. The wood Cellulose-F was analysed by a scanning electron microscope. Fibres originating from recycled newspapers were thinner and, therefore, investigated in a transmission electron microscope. For further details of methods of fibre analysis see Bellmann and Muhle (1994). Two lungs were taken for histopathological examination after H and E stain. For calculation of clearance kinetics a regression analysis of logarithm of number or mass of fibres per lung of individual animals versus time after instillation was done (Muhle et al., 1994). RESULTS Lung weight Lung weight was significantly increased in the -treated group up to half a year after treatment. In the Cellulose-F-exposed group only a mild increase of lung weight was observed. After one year no statistically significant differences were seen in both treatment groups. Fibre retention One year after treatment of the experimental animals cellulose fibres of both materials were present in lungs. Fibre number of the reference material Cellulose-F was almost unchanged in the lungs (see Fig. 1). The calculated half-time of the fibre clearance was in the range of 1000 days (see Table 2). This means that this fibre type shows a considerable biopersistence. Up to half a year after treatment for fibres a splitting into thinner fibrils were observed (see Fig. 2). After one year the evaluation by transmission electron microscopy with regard to fibre number and fibre diameter was difficult for because branching of fibrils and a low contrast in the electron microscope; however, the presence of these fibres could be demonstrated unequivocally. Because of the splitting of fibres no half-time can be calculated for the fibre number. On the basis of fibre mass which was calculated from the fibre size distribution and fibre numbers a half-time of 72 days was obtained for the first half year (see Table 2).

3 186 H. Muhle et al. Cellulose-F Number of all fibres. *--- * Number of WHO fibres *--x -* Calculated mass of particles i i i Days after Instillation Fig. 1. Retention of microcrystalline wood cellulose fibres and of their calculated particle mass in rat lungs. Histopathology Lungs treated with Cellulose-F and showed almost identical changes. At 2 and 30 days after treatment mild multifocal granulomatous inflammation was observed. Cellulose fibres were phagocytised by alveolar macrophages. After 3 and 6 months fibre associated granulomata, slight interstitial fibrosis, alveolar histiocytosis, alveolar lipoproteinosis and alveolar cell hyperplasias were found. After one year the severity of lesions reported at earlier sacrifice dates increased. 400 DISCUSSION The objective of this paper was to investigate the biodur ability of the cellulose containing dust. As this material originates from recycled newspaper dust it contains also other constituents of wood like lignin. Therefore, as a reference Table 2. Calculated half-times of the clearance with 95% confidence limits Group Half-times in days calculated on the basis of Number of WHO Number of fibres fibres Mass of fibres Cellulose-F 564 (274-oo) 1046 (351-oo) 72(53-113) 1000 (676-oo)

4 Durability of cellulose fibres in rat lungs o o Number of all fibres * * * Number of WHO fibres -*--x * Calculated mass of particles Days after Instillation Fig. 2. Retention of fibres from recycled newspaper and of their calculated particle mass in rat lungs. material chemical pure microcrystalline wood cellulose was used. After sizing these materials rat-inhalable fractions were obtained. The lung burden which was given to the animals was in the range of "dust overload" of lungs (Muhle et al., 1990). The reason for dose selection was to prevent to a great extent the macrophage-mediated fibre clearance. This type of clearance physically removes fibres from lungs. This could possibly interfere with the kinetics of the degradation of the cellulose fibres. Retention data showed that in rat lungs fibres of microcystalline wood cellulose are quite persistent. Fibres originating from recycled newspapers splitted in lungs. Obviously no significant enzymatic digestion of cellulose fibres takes place in lungs. It is concluded that cellulose fibres show a higher biodurability in lungs than ceramic fibres tested by the same protocol (Bellmann and Muhle, submitted). Therefore, cellulose fibres have the potential to accumulate in lungs. Histopathological results and published data show inflammatory reactions and fibrotic lesions in rat lungs. It is recommended that a long-term inhalation study is performed to investigate chronic effects. Acknowledgements This project was funded by: Hauptverband der gewerblichen Berufsgenossenschaften, Arbeitsgemeinschaft der Bau-Berufsgenossenschaften and Okologische Bautechnik Hirschhagen GmbH.

5 188 H. Muhle et al. REFERENCES Bellmann, B. and Muhle, H. Biopersistence of various types of mineral fibres in the rat lung after intratracheal application (submitted). Bellmann, B. and Muhle, H. (1994) Investigation of the biodurability of wollastonite and xonotlite. Environ. Health Perspect. 102 (Suppl. 5) Davis, J. M. G. (1993) The need for standardising testing procedures for all products capable of liberating respirable fibres: the example of materials based on cellulose. Br. J. ind. Med. 50, Milton, D. K., Godleski, J. L., Feldman, H. A. and Greaves, I. A. (1990) Toxicity of intratracheally instilled cotton dust, cellulose, and endotoxin. Am. Rev. Respir. Dis. 142, Muhle, H., Bellmann, B., Creutzenberg, O., Heinrich, U. and Mermelstein, R. (1990) Dust overloading of lungs: investigations of various materials, species differences and irreversibility of effects. /. Aerosol Med. 3, S111-S128. Muhle, H., Bellmann, B. and Pott, F. (1994) Comparative investigations of the biodurability of mineral fibres in the rat lung. Environ. Health Perspect. 102 (Suppl. 5) Tiesler, H. and Schnittger, J. (1992) Untersuchungen zur Belastung durch faserformige Staube bei der Verarbeitung von Cellulose-Dammstoffen. Zbl. Arbeitsmed. 42,