Multi-walled carbon nanotubes (Baytubes®): Approach for derivation of occupational exposure limit

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Abstract

Carbon nanotubes come in a variety of types, but one of the most common forms is multi-walled carbon nanotubes (MWCNT). This paper focuses on the dose–response and time course of pulmonary toxicity of Baytubes®, a more flexible MWCNT type with the tendency to form assemblages of nanotubes. This MWCNT has been examined in previous single and repeated exposure 13-week rat inhalation studies. Kinetic endpoints and the potential to translocate to extrapulmonary organs have been examined during postexposure periods of 3 and 6 months, respectively. The focus of both studies was to compare dosimetric endpoints and the time course of pulmonary inflammation characterized by repeated bronchoalveolar lavage and histopathology during the respective follow-up periods. To better understand the etiopathology of pulmonary inflammation and time-related lung remodeling, two metrics of retained lung dose were compared. The first used the mass metric based on the exposure concentration obtained by filter analyses and aerodynamic particle size of airborne MWCNT. The second was based on calculated volumetric lung burdens of retained MWCNT. Kinetic analyses of lung burdens support the conclusion that Baytubes®, in principal, act like poorly soluble agglomerated carbonaceous particulates. However, the difference in pulmonary toxic potency (mass-based) appears to be associated with the low-density (≈0.1–0.3 g/m3) of the MWCNT assemblages. Of note is that assemblages of MWCNT were found predominantly both in the exposure atmosphere and in digested alveolar macrophages. Isolated fibers were not observed in exposure atmospheres or biological specimens. All findings support the conclusion that the low specific density of microstructures was conducive to attaining the volumetric lung overload-related inflammatory response conditions earlier than conventional particles. Evidence of extrapulmonary translocation or toxicity was not found in any study. Thus, pulmonary overload is believed to trigger the cascade of events leading to a stasis of clearance and consequently increased MWCNT doses high enough to trigger sustained pulmonary inflammation. This mechanism served as conceptual basis for the calculation of the human equivalent concentration. Accordingly, multiple interspecies adjustments were necessary which included species-specific differences in alveolar deposition, differences in ventilation, and the time-dependent particle accumulation accounting for the known species-specific differences in particle clearance half-times in rats and humans. Based on this rationale and the NOAEL (no-observed adverse effect level) from the 13-week subchronic inhalation study on rats, an occupational exposure limit (OEL) of 0.05 mg Baytubes/m3 (time weighted average) is considered to be reasonably protective to prevent lung injury to occur in the workplace environment.

Introduction

It has been suggested that currently about thousands of different types of engineered carbon nanotubes (CNT) have to be considered due to different starting materials, production processes, catalysts, surface functionalization, and resultant microstructures. The same type of diversity applies to many other types of engineered nanomaterials (Savolainen, 2009). In addition to deliberate functionalization also inadvertent functionalization of CNT may arise from “accidental” modification during synthesis, processing and/or by the means to make this material amenable to toxicity testing (Muller et al., 2008, Salvador-Morales et al., 2008). This may lead to differences in protein binding properties and biological response. This diversity precludes both to find an ad hoc unifying denominator of the metric of dose, biocompatibility and critical toxicity as well as risk assessment of all types of all CNT. The association between the physical–chemical properties and the target organ dose–effect relationships are an issue awaiting resolution for engineered nanomaterials (Savolainen, 2009). Along with these concerns and the risks exposure to nanomaterials may pose to workers, issues regarding the most appropriate unifying metric of dose are still unresolved (Maynard, 2007).

Yet, no single particle characteristic as a hallmark indicator directing fate and pulmonary toxicity has been identified (Madl and Pinkerton, 2009). Emerging views suggest that the assemblage displacement volume of multi-walled CNT (MWCNT), which is critical to the impairment of alveolar macrophage-mediated clearance and elicitation of pulmonary inflammation, may dictate the fate and pulmonary response to this type of structures. Nonetheless, in the inhalation studies referred to the mass metric was used to allow a straightforward comparison of external exposure concentrations and retained cumulative MWCNT dose.

In vivo studies of MWCNT are very limited, and the results of these studies are not consistent. Reasons of this inconsistency appear to be associated to the methodological variables used to make MWCNT amenable to testing, often lacking characterization of the MWCNT actually administered to experimental animals, and the non-adherence to internationally recognized OECD inhalation testing guidelines (OECD, 2009). Thus, in regard to MWCNT, many experimental variables (test substance related, method of substance pre-conditioning and administration related, and test design related) exist. A site-by-site comparison with other types of MWCNT would have required comparable testing approaches and methods to characterize the extent of external and internal exposure. Due to the absence of such studies, the focus of this treatise is on Baytubes only.

The studies described in this paper were designed to be compliant with current OECD testing standards and Good Laboratory Practice (GLP) regulations. The objective of this paper is to derive an occupational exposure limit (OEL) for Baytubes®, a thin-walled type of MWCNT, based on previous single and repeated inhalation exposure studies on rats (Ellinger-Ziegelbauer and Pauluhn, 2009, Pauluhn, 2010) and taking into account the wealth of information generated for uniform poorly soluble particles of higher-than-unit density. Little disagreement exists that OELs provide health and safety professionals with an important tool for protecting worker health. However, despite the general agreement on the need for OELs, significant disparities can exist in the specific OEL values established by the different methods used to derive them (Haber and Maier, 2002). For complex, essentially insoluble particle-like agglomerate structures of carbon nanotubes, this derivation is further complicated by multiple factors. These include the diverse views on the most critical physical/physicochemical property determining the biopersistence of deposited nanostructures in the lung, the ensuing sustained pulmonary inflammation, and possible chronic sequelae thereof under conditions causing lung particle overload in experimental models (commonly the rat) (Brown et al., 2005, Donaldson et al., 2008, Elder et al., 2005, Green et al., 2007, Madl and Pinkerton, 2009, Oberdörster, 1995, Oberdörster et al., 2007). Prevailing experimental evidence obtained in the most sensitive bioassay (rat) with granular biopersistent particles demonstrated that the prevention of overload-like conditions may also prevent from secondary long-term effects to occur (ILSI, 2000). Opposite to humans, rats have been shown to develop preferentially intraluminal and to a lesser extent interstitial inflammatory changes in the lung (Green et al., 2007). Accordingly, emphasis was directed towards the analysis of the most sensitive endpoint to define the threshold of intraluminal pulmonary inflammation characterized by bronchoalveolar lavage (BAL) in subchronically (13 weeks) exposed rats. This study duration provides the minimum database for the derivation of a DFG-MAK (Deutsche Forschungsgemein schaft-Maximale Arbeitsplatz Konzentration) value in the Work Area (DFG, 2009).

Along with the objective to derive an OEL for Baytubes, attempts were made to identify common mechanistic denominators between higher-than-unit density, uniform biopersistent particles compared to lower-than-unit density MWCNT microstructures. Based on the results of these studies, it appears as if that the critical toxicity of this type of MWCNT arises from the collective behavior of the inhaled microstructures (assemblages) of nanotubes rather than the individual tube structure per se. Typically, following dispersion into inhalation chambers, in subchronically exposed rats assemblages of MWCNT were found both in exposure atmospheres as well as in lavaged lung cells (Pauluhn, 2010).

Section snippets

Experimental variables

It is important to keep in mind that the model system or testing regimen themselves may influence measured responses irrespective of the nanomaterial investigated. At present, there is no one characteristic across different studies that seem to correlate with the inflammatory and fibrotic responses that are being observed. For instance, intratracheal instillation studies generally show similar trends of toxicity as inhalation studies, and vary in timing and severity depending on the

Microstructure size, surface area, agglomeration, and deagglomeration

The stability of MWCNT microstructures is determined by the sum of the van der Waals attractive and electrical double layer repulsive forces. The Derjaguin, Landau, Verwey and Overbeek (DLVO) theory (Derjaguin and Landau, 1941) proposes that the energy barrier resulting from the repulsive force prevents two particles approaching one another and adhering together. If the ζ-potential is reduced, e.g. by high ionic strength of the medium, particle adhesion is reversed. The erosion of

Regulatory toxicity studies

So far, internationally harmonized toxicity testing procedures for nanostructures are not available. Therefore, Baytubes® was examined using the regulatory base set for the hazard identification of new substances by classical toxicity testing (such as relevant to REACH and OECD considerations). The acute oral and dermal toxicity studies in rats, according to the test guidelines OECD#423 and OECD#402, did not reveal any specific toxicity (LD50-oral > 5000 mg/kg bw; LD50-dermal > 2000 mg/kg bw.) There

Derivation of OEL

Suffice it to say, for hazard identification purposes, the respective OECD (2009) testing guidelines for subchronic inhalation toxicity mandate that the material tested in inhalation chambers as solid aerosol is of adequate respirability to rats. Technical manipulations could make the test substance somewhat different to that produced and thus potentially inhaled by the occupationally exposed workers. Accordingly, hazards identified in such rat bioassays need to be put into a real world human

Conclusions

In regard to MWCNT, numerous material-related and mode-of-testing-related variables have to be accounted for. Among others; these include the nanotube diameter and length, surface functionalization, and the various types of adhesion forces acting between tube surfaces which, eventually, are decisive whether MWCNT are present as fibers or assemblages. These variables may determine whether MWCNT are present as isolated fibers or as self-assembled, intertwined, coil-like microstructures

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