Five-day inhalation toxicity study of three types of synthetic amorphous silicas in Wistar rats and post-exposure evaluations for up to 3 months

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Abstract

Evidence suggests that short-term animal exposures to synthetic amorphous silicas (SAS) and crystalline silica can provide comparable prediction of toxicity to those of 90-day studies, therefore providing the opportunity to screen these types of substances using short-term rather than 90-day studies. To investigate this hypothesis, the inhalation toxicity of three SAS, precipitated silica Zeosil 45, silica gel Syloid 74, and pyrogenic silica Cab-O-Sil M5 was studied in Wistar rats. Rats were exposed nose-only to concentrations of 1, 5 or 25 mg/m3 of one of the SAS 6 h a day for five consecutive days. Positive controls were exposed to 25 mg/m3 crystalline silica (quartz dust), negative controls to clean air. Animals were necropsied the day after the last exposure or 1 or 3 months later. All exposures were tolerated without serious clinical effects, changes in body weight or food intake. Differences in the effects associated with exposure to the three types of SAS were limited and almost exclusively confined to the 1-day post-exposure time point. Silicon levels in tracheobronchial lymph nodes were below the detection limit in all groups at all time points. Silicon was found in the lungs of all high concentration SAS groups 1-day post-exposure, and was cleared 3 months later. Exposure to all three SAS at 25 mg/m3 induced elevations in biomarkers of cytotoxicity in bronchoalveolar lavage fluid (BALf), increases in lung and tracheobronchial lymph node weight and histopathological lung changes 1-day post-exposure. Exposure to all three SAS at 5 mg/m3 induced histopathological changes and changes in BALf only. With all three SAS these effects were transient and, with the exception of slight histopathological lung changes at the higher exposure levels, were reversible during the 3-month recovery period. No adverse changes were observed in animals exposed to any of the SAS at 1 mg/m3. In contrast, with quartz-exposed animals the presence of silicon in the lungs was persistent and toxicological effects differed from those seen with SAS both with regard to the type and severity as well as in the time-response profile. In quartz-exposed animals silicon in the tracheobronchial lymph nodes was below the detection limit but silicon was found in the lungs at comparable levels 0-, 1- and 3-months post-exposure. One-day post-exposure to quartz, elevations in biomarkers of cytotoxicity in BALf, increases in lung and tracheobronchial lymph node weight and histopathological lung changes were minimal. These effects were present at 1-month post-exposure and progressively more severe at 3-months post-exposure. Overall, the results of the current study are similar to those of other published studies that had a 90-day exposure period and both types of studies indicate that the lack of lung clearance is a key factor in the development of silicosis.

Introduction

Silicas are some of the most abundant compounds found naturally in the earth’s crust and can be divided into crystalline or non-crystalline (amorphous) silicas, all having the same basic molecular formula (almost 100% SiO2). There are at least three polymorphs of crystalline silica including quartz, cristobalite, and tridymite (Warheit, 2001; see also Fig. 1).

Amorphous silicas are divided into naturally occurring amorphous silicas and synthetic forms. The naturally occurring amorphous silicas such as diatomaceous earth usually contain significant amounts of crystalline silica, sometimes up to 60 wt%. Certain industrial processes produce silica fume and fused silica as by-products. These materials often contain a number of impurities including crystalline silica and should not be confused with the commercial product known as fumed silica (ECETOC, 2006; see also Fig. 1). Synthetic amorphous silicas (SAS) are intentionally manufactured amorphous silicas that do not contain measurable levels of crystalline silica and as such are not associated with the negative health impacts attributed to crystalline silica or the naturally occurring amorphous silicas (Merget et al., 2002). The synthetic forms may be classified as (1) wet process manufactured silica and (2) pyrogenic (thermal or fumed) silica. These two types of SAS can be further modified by surface treatments (Merget et al., 2002; Fig. 1).

SAS are used in many materials such as synthetic resins, plastics, lacquers, vinyl coatings, varnishes, adhesives, paints, printing inks, silicone rubber, fillers in the rubber industry, tyre compounds, insulation material, liquid systems in coatings, as free-flow and anti-caking agents in powder materials, as tooth paste additives, pharmaceuticals, cosmetics, as liquid carriers particularly in the manufacture of agrochemicals and animal feed, and foods, resulting in widespread exposure to these substances (Reuzel et al., 1991, Merget et al., 2002).

Occupational exposure to dust of crystalline silica (quartz or cristobalite) has been shown to induce silicosis, a chronic lung disease characterised by granulomas and severe fibrosis in the lungs (Reiser and Last, 1979, Weill et al., 1994, Merget et al., 2002). In addition, occupational exposure to quartz is associated with an increased risk for pulmonary diseases such as chronic bronchitis, chronic obstructive pulmonary disease (COPD), and lung cancer (McDonald, 1996, IARC, 1997, Warheit, 2001, Merget et al., 2002). In contrast, apart from two publications of case reports which included exposure to naturally occurring amorphous silica, and therefore, co-exposure to crystalline silica (Mohrmann and Kann, 1985, Philippou et al., 1992), SAS have not been shown to induce fibrosis in workers with high occupational exposure (McLaughlin et al., 1997, Merget et al., 2002). It has been determined that there is inadequate evidence for the carcinogenicity of SAS in both humans and experimental animals (IARC, 1997). Sufficient data to assess the risk of chronic bronchitis, COPD and emphysema following occupational exposure to SAS are not available (Merget et al., 2002).

Experiments using rats have supported the association of quartz exposure with the induction of lung tumours and silicotic nodules (Reiser et al., 1982, Reiser et al., 1983, Groth et al., 1986, Holland et al., 1986, Reuzel et al., 1991, Barbaro et al., 2002), but this was not found in hamsters (Renne et al., 1985). In contrast, the evidence of SAS-induced fibrogenicity in animal studies is limited. Studies in rats during the past two decades (Groth et al., 1981, Hemenway et al., 1986, Lee and Kelly, 1992, Lee and Kelly, 1993, Reuzel et al., 1991, Warheit et al., 1991, Warheit et al., 1995, Johnston et al., 2000) have consistently demonstrated largely (often completely) reversible inflammation, granuloma formation and focal emphysema, with no progressive fibrosis. In a 3-month exposure study with a recovery period up to 12 months, previously performed in our Institute, the toxicity of three SAS (Aerosil 200, a pyrogenic SAS; Aerosil R974, a surface treated pyrogenic SAS; and Sipernat 22S, a precipitated SAS) were compared to quartz (Reuzel et al., 1991). All test materials induced increases in lung weights and inflammation in the lungs, however, only quartz exposure resulted in the development of progressive lesions post-exposure, including silicotic nodules. In contrast, markers of inflammation in rats exposed to SAS were partly or completely reversible.

Evidence suggests that short-term animal exposures to SAS and crystalline silica (Warheit et al., 1995) can provide comparable prediction of toxicity to those of 90-day studies (Reuzel et al., 1991, Johnston et al., 2000), therefore providing the opportunity to screen these types of substances using short-term rather than 90-day studies. To investigate this hypothesis a 5-day inhalation toxicity assessment in rats was conducted. The effects of three SAS at concentrations of 1, 5 and 25 mg/m3 were compared to those of quartz at 25 mg/m3. The SAS were precipitated silica Zeosil 45 and silica gel Syloid 74 (both wet process silica), and pyrogenic silica Cab-O-Sil M5 (thermal or fumed silica). The short-term exposure period (5 days) was followed by post-exposure observation periods of 1 and 3 months.

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Animals and maintenance

Male and female, young adult, Wistar (Crl:(WI)WU BR) rats were purchased from Charles River Deutschland (Sulzfeld, Germany). The animals were acclimatised for at least 5 days before the start of the study. They were kept under conventional laboratory conditions in suspended, stainless steel cages fitted with wire mesh floor and front, two, three or five rats per cage, and received powdered RM3 rodent diet and unfluoridated tap water ad libitum. The animal rooms were ventilated with about 10 air

Exposure concentrations and particle size distributions

Mean actual concentrations of SAS and quartz were close to target concentrations (Table 2). The mass median aerodynamic diameters were approximately 2–3 μm with mean geometric standard deviations (gsd) of approximately 1.5–2, indicating that particles were highly respirable under these test conditions.

Clinical signs, body weights, and food intake

No treatment-related clinical signs were observed except for a slightly decreased breathing frequency visually observed during exposure to Zeosil 45 and quartz. It was transient in animals exposed

SAS exposures

None of the groups exhibited treatment-related gross lesions at necropsy. Microscopic examination revealed SAS-related changes in the lungs and tracheobronchial lymph nodes. With all three SAS exposures, most histopathological changes were observed at 25 mg/m3 and to a lesser degree at 5 mg/m3, and none at 1 mg/m3. Directly after the exposure period, the histopathological changes in the lungs that were observed consisted of increased intra-alveolar accumulation of macrophages (Syloid 74 and

Discussion

In the present inhalation toxicity study with various SAS, daily exposure for five consecutive days resulted in treatment-related adverse effects in the respiratory tract. Several of the changes were found to be similar for all SAS but some limited differences were also observed. The pyrogenic silica Cab-O-Sil M5 induced the most pronounced increases in markers of lung inflammation but these were almost completely reversible within the 3-month recovery period. Silica gel Syloid 74 induced the

Acknowledgements

These studies were performed at the request of and were sponsored by CEFIC-ASASP, Brussels, Belgium. The authors wish to thank Gerard Roverts, Frank Hendriksma and Joost Bruijntjes for technical assistance, and Dr. B. Hendrickx (Rhodia, France) for providing valuable information.

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