ENVIRONMENTAL RESEARCH54, 52--73 (1991)

Tumorigenicity of Fine Man-Made Fibers after Intratracheal Administrations to Hamsters SHU1CHI ADACHI, K A Z U O TAKEMOTO, AND K J K U Z I K I M U R A *

Department of Public Health, Saitama Medical School, Morohongo 38, Moroyarna, lruma, Saitama 350-04, Japan; and *Institute of Science for Labour, Sugao 1544, Miyamae-ku, Kawasaki 213, Japan Received December 14, 1989 Six types of man-made fibers were administered intratracheally (2.0 mg/animal each a week, for 5 weeks; total 10 mg/animal) to female Syrian hamsters that were observed histologically for 2 years after administration. The fibers were rock wool [average diameter (D) = 6.1 ~xm, average length (L) = 296 fxm], fiberglass (D = 0.65 p,m, L = 16.8 I~m), potassium titanate fiber (D = 0.36 Ixm, L = 7.17 txm), calcium sulfate fiber (D = 1.0 ~m, L = 17.8 Ixm), basic magnesium sulfate fiber (D = 0.45 txm, L = 22.4 txm), and metaphosphate fiber (D = 2.38 jxm, L = 64.1 txm). Tumors were observed in hamsters that had received basic magnesium sulfate fiber (9/20), metaphosphate fiber (6/20), calcium sulfate fiber (3/20), and fiberglass (2/20) but not in the control, rock wool, or potassium titanate fiber groups. The primary sites of the tumors were not only in the pleural cavity but also in the intracelial organs, kidney, adrenal gland, bladder, and uterus. Only a few of the tumors were identified as mesotheliomas by histological examination. In addition to neoplastic lesions, fibrosis, pleural thickening, and chronic inflammatory changes in the lungs were observed in the hamsters, but these changes appeared too mild to foster a pneumoconiosis such as asbestosis. © 1991 Academic Press, Inc.

INTRODUCTION It has been well recognized that asbestos is a carcinogenic substance according to many epidemiological studies and animal experiments. Because of this, the demand for asbestos has decreased and the use of asbestos has been prohibited in several countries. Consequently, not only the production of man-made fibers but also the development of new fibers are increasing. However, there is a limited knowledge of the carcinogenicity and other biological effects of these asbestos substitutes. The asbestos substitutes include several different types of man-made fibers, including amorphous silicate fibers (fiberglass, rock wool, slag wool), monocrystalline fibers (whiskers), and other miscellaneous fibers (carbon fiber, silicon carbide, etc). These classifications are based on chemical compositions but not on size or physical properties. For instance, there are wide ranges for the diameter and length of fiberglass and rock wool; these dimensions are variable by changing or modifying the manufacturing process. Monocrystalline whiskers, which are very fine and have a small variance of dimensions, are added to polymer resins in order to give smoothness, hardness, and precision. In a number of epidemiological and experimental studies it has been suggested that the carcinogenesis and other negative health effects caused by exposure to man-made fibers (almost all the studies were on fiberglass and rock wool) are not 52 0013-9351/91 $3.00 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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as severe (or are nonexistent) as those caused by asbestos. (Environmental Health Criteria, 1986). Therefore, there are no specific regulations for the production or the use of man-made fibers at present. However, it is suspected that the diameter and length of any fibers might be one of the most important factors of carcinogenicity. This hypothesis was suggested by Stanton et al. (1977) and Pott (1978) from their experimental studies, in which the asbestos and man-made fibers that have different dimensions were injected to the pleural or peritoneal cavities of rats. The results showed the significance between the incidence of tumor and the dimension of the fiber. This hypothesis might give attention to the use of fine man-made fibers as asbestos substitutes but there is a serious lack of information on the health effects of man-made fibers. To obtain more information on the biological reactions caused by the man-made fibers, such as fibrogenic reaction and pleural thickening, an intrapleural and intraperitoneal injection experiment is insufficient. These routes of exposure would increase the localization of fibers at the injection site and this is not the case in occupational or general environmental exposure. In order to evaluate the biological effects of inhaled man-made fibers, especially crystalline whiskers, six types of fine fibers were administered intratracheally to female Syrian hamsters and histological examinations were performed for 2 years after the administration. MATERIALS AND METHODS Fibers and Their Preparation The fiber samples were two types of amorphous silicate, three types of monocrystalline whiskers, and one polymer of metaphosphate. Scanning electron micrographs of these fibers are shown in Figs. 1-6. The amorphous silicates were the rock wool (specific gravity (sp grav) was not available) used as a spraying material for construction and fiberglass (sp grav 2.6 g/cm 3) used as a filter for quantitative chemical analysis. The elemental composition of the rock wool was mainly silica and calcium. A small amount of magnesium, aluminum, potassium, titanium, and manganese were also detected by an energy dispersion X-ray microanalyzer fitted with the scanning electron microscope (SEM-EDX). Monocrystalline whiskers were composed of potassium titanate fiber (KzTi6Ol3, sp grav 3.3 g/cm3), calcium sulfate fiber ( C a S O 4 , sp grav 2.96 g/cm3), and basic magnesium sulfate fiber ( M g S O 4 • 5MgO • 8 H 2 0 , sp grav 2.3 g/cm3). Metaphosphate fiber (sp grav 2.86 g/cm 3) is expressed in the chemical form of 2CaO • Na20 • 3 P 2 0 5 . Distributions of the diameter and length of these fibers were determined by using scanning electron microscopy and are described in Tables 1-6. Each 500 mg of fiber was suspended in 50 ml of sterilized saline with 0.25 g of sodium carboxymethylcellulose to delay fiber sedimentation. These suspensions were sonicated before administration. Experimental Animals One hundred forty female Syrian hamsters, weighing 80 g, were purchased from the Shizuoka Experimental Animal Cooperative (Shizuoka, Japan). Commercial

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ADACHI, TAKEMOTO, AND KIMURA

Fie. la. Scanning electron micrograph (SEM) of rock wool, showing smooth surface.

diet CE-2 (Japan Clea) and tap w a t e r were administered ad libitum. The animal r o o m was kept at 25 -+ I°C and 50 +- 5% (RH), and fresh filtered air was used. H a m s t e r s were kept for 1 w e e k before the first administration and then were divided into seven groups.

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Fl6. lb. SEM-EDX spectrum of the rock wool.

Intratracheal Administration Fiber suspension (0.2 ml) was carefully injected intratracheally with 0.3 ml of air under diethyl ether anesthesia once a week for 5 weeks. Uniformity of the injected fiber in each lung lobe was confirmed by preliminary study using macroscopically and light microscopically visible potassium titanate fiber. The total dose was 10.0 mg/animal.

Histological Examinations Two years after the first administration, all hamsters were sacrificed and routine autopsies were performed. Only moribund hamsters were killed before this time and all organs were fixed in 10% formalin. Formalin-fixed organs were prepared for histopathological examinations using common methods. In addition to the hematoxylin eosin stain, special stains [periodic acid-Schiff (PAS), Azan, Mallory, Elastica van Gieson, and others] were used. Alcian blue stain with and without hyaluronidase was used for all tumors. RESULTS

There were no deaths after the administrations of fibers due to acute inflammation or other acute changes. Throughout this experimental period, there was no significant difference in mortality and body weight between groups.

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ADACH|, TAKEMOTO, AND KIMURA

FIG. 2. SEM of fiberglass, showing thin fibers with diameters less than 0.5 ixm.

Observed tumors are summarized in Table 7. Tumors were not found in hamsters that received rock wool, potassium titanate fiber, or nothing (control group). The numbers of tumor-bearing hamsters were two in the fiberglass group, three in the calcium sulfate fiber group, nine in the basic magnesium sulfate fiber group, and five in the metaphosphate fiber group. The sites or primary sites of these

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FIo. 3. SEM of potassium titanate fiber. These fibers have similar diameters and lengths.

tumors were the lung, pericardium, rib, kidney, urinary bladder, adrenal gland, ovary, uterus, and a part of peritoneum (the exact origin could not be identified). Tumors in the pleural cavity included a squamous cell carcinoma of the lung with slight keratinization in a hamster exposed to fiberglass (Fig. 7), two mesotheliomas (sarcomatous type, both showed the presence of hyaluronic acid by the

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ADACHI, TAKEMOTO, AND KIMURA

FIG. 4. SEM of calcium sulfate fiber, showing polyfilamentous structure, longitudinal splitting, and adhesion of fine fiber.

histochemical observations using hyaluronidase) originating from the pericardium (Figs. 8a, 8b, 9, 10a, 10b), one mesothelioma that metastasized to the kidney and other tissues, and nodular tumors (pleomorphic tumor, large and epithelial structure, PAS-positive) on the pleural surface of the lung (Fig. 11) in a hamster

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FIG. 5. SEM of basic magnesium sulfate fiber, showing split fine fibers and polyfilamentous structure.

exposed to basic magnesium sulfate fiber. Other tumors included a heart tumor (occupied left and right ventricles; tumor cells had round and polygonal structures and prominent nucleoli; mitosis were encountered), a rib tumor (unspecified cell types) in hamsters exposed to calcium sulfate fiber (Fig. 12, 13), and a pleural

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ADACHI, TAKEMOTO, AND KIMURA

FIG. 6. SEM of metaphosphate fiber, showing adhesion of pulp-like fibrils.

mesothelioma (sarcomatous type) invading the sternum (Fig. 14a, 14b) in hamsters exposed to metaphosphate fiber. For tumors other than those found in the pulmonary region, two kidney tumors (an anaplastic malignant tumor and a malignant histocytoma), three adrena! tumors (a neuroblastoma, a cortical adenoma, and an A&B cell tumor), a leiomyoma in the urinary bladder, and a leiomyosarcoma in the uterus were observed in

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Tumorigenicity of fine man-made fibers after intratracheal administrations to hamsters.

Six types of man-made fibers were administered intratracheally (2.0 mg/animal each a week, for 5 weeks; total 10 mg/animal) to female Syrian hamsters ...
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