Subchronic 13-Week Inhalation Exposure of Rats to Multiwalled Carbon Nanotubes: Toxic Effects Are Determined by Density of Agglomerate Structures, Not Fibrillar Structures

Abstract

Wistar rats were nose-only exposed to multiwalled carbon nanotubes (MWCNT, Baytubes) in a subchronic 13-week inhalation study. The focus of study was on respiratory tract and systemic toxicity, including analysis of MWCNT biokinetics in the lungs and lung-associated lymph nodes (LALNs). The time course and concentration dependence of pulmonary effects were examined by bronchoalveolar lavage (BAL) and histopathology up to 6 months postexposure. Particular emphasis was directed to the comparative characterization of MWCNT structures prior to and after micronization and dry powder dispersion into inhalation chambers. These determinations were complemented by additional analyses in digested BAL cells. Animals were exposed on 6 h/day, 5 days per week for 13 consecutive weeks to 0, 0.1, 0.4, 1.5, and 6 mg/m(3). The subchronic exposure to respirable solid aerosols of MWCNT was tolerated without effects suggestive of systemic toxicity. Kinetic analyses demonstrated a markedly delayed clearance of MWCNT from lungs at overload conditions. Translocation into LALNs occurred at 1.5 and 6 mg/m(3) and required at least 13 weeks of study to become detectable. At these exposure levels, the lung and LALN weights were significantly increased. Sustained elevations in BAL polymorphonuclear neutrophils and soluble collagen occurred at these concentrations with borderline effects at 0.4 mg/m(3). Histopathology revealed principal exposure-related lesions at 0.4 mg/m(3) and above in the upper respiratory tract (goblet cell hyper- and/or metaplasia, eosinophilic globules, and focal turbinate remodeling) and the lower respiratory tract (inflammatory changes in the bronchioloalveolar region and increased interstitial collagen staining). Granulomatous changes and a time-dependent increase of a bronchioloalveolar hyperplasia occurred at 6 mg/m(3). All end points examined were unremarkable at 0.1 mg/m(3) (no-observed-adverse-effect-level). In summary, this study demonstrates that the induced pathological changes are consistent with overload-related phenomena. Hence, the etiopathological sequence of inflammatory events caused by this type of MWCNT appears to be related to the high displacement volume of the low-density MWCNT assemblage structure rather than to any yet ill-defined intrinsic toxic property. Thus, the hypothesis of study is verified, namely, common denominators between carbon black and MWCNT do exist.

Long‐term retention of pristine multi‐walled carbon nanotubes in rat lungs after intratracheal instillation

“…Most of the previous studies analyzed the organ distributions of MWCNTs indirectly, for example by analyzing catalytic metal (Pauluhun 2010) or functionalized MWCNTs (Deng et al 2007, Liu et al 2007). Pauluhun (2010) estimated pulmonary MWCNT burdens in rats for 26 weeks after a 13‐week inhalation exposure by analyzing the catalytic Co. Catalytic metal can distribute throughout the body (Huang et al ., 2010).…”

“…Previous inhalation exposure tests using MWCNTs have also low clearance rates. After inhalation exposure to tangled MWCNTs (6.0 mg m −3 ), pulmonary retention at 26 weeks was >90% of that observed 1 day after exposure [0.1 mg m −3 , half‐time 5 month; 0.4–6.0 mg m −3 , half‐time 10 to > 13 months (Pauluhun, 2010)]. After inhalation exposure and intratracheal instillation to original and functionalized MWCNTs, no clearance was observed within 21 days (Silva et al ., 2014).…”

“…As overload occurred after administration of the exceeding threshold volume of MWCNTs (Pauluhn, 2010), we discuss the volume of MWCNTs employed in the present study. Assuming the density of MWCNT in the present study was a true density of 1.72 g cm −3 because the suspension was well dispersed, pulmonary MWCNTs was estimated to be only 0.12 μ l g −1 per lung, which is within the range of minimal lung overload for tangled MWCNTs [0.1–0.3 μ l g −1 per lung (0.1 mg m −3 ); Pauluhn, 2010]. In the present study, however, only 0.12 μ l g −1 per lung of MWCNTs induced a complete delay in clearance.…”

“…In the present study, however, only 0.12 μ l g −1 per lung of MWCNTs induced a complete delay in clearance. In Pauluhn (2010), complete lung overload occurred at a much higher volume of tangled MWCNTs [1.2–12 μ l g −1 per lung (1.5–6.0 mg m −3 )]. There are two possible explanations for this difference between Pauluhn (2010) and the present study.…”

“…In Pauluhn (2010), complete lung overload occurred at a much higher volume of tangled MWCNTs [1.2–12 μ l g −1 per lung (1.5–6.0 mg m −3 )]. There are two possible explanations for this difference between Pauluhn (2010) and the present study. One possibility relates to the severity of cytotoxicity, as the toxicity of well‐dispersed rigid MWCNTs could be more severe than that of tangled MWCNTs.…”