Comparative assessment of nanomaterial definitions and safety evaluation considerations.

Nanomaterials continue to bring promising advances to science and technology. In concert have come calls for increased regulatory oversight to ensure their appropriate identification and evaluation, which has led to extensive discussions about nanomaterial definitions. Numerous nanomaterial definitions have been proposed by government, industry, and standards organizations. We conducted a comprehensive comparative assessment of existing nanomaterial definitions put forward by governments to highlight their similarities and differences. We found that the size limits used in different definitions were inconsistent, as were considerations of other elements, including agglomerates and aggregates, distributional thresholds, novel properties, and solubility. Other important differences included consideration of number size distributions versus weight distributions and natural versus intentionally-manufactured materials. Overall, the definitions we compared were not in alignment, which may lead to inconsistent identification and evaluation of nanomaterials and could have adverse impacts on commerce and public perceptions of nanotechnology. We recommend a set of considerations that future discussions of nanomaterial definitions should consider for describing materials and assessing their potential for health and environmental impacts using risk-based approaches within existing assessment frameworks. Our intent is to initiate a dialogue aimed at achieving greater clarity in identifying those nanomaterials that may require additional evaluation, not to propose a formal definition.

Workshop report: strategies for setting occupational exposure limits for engineered nanomaterials.

Occupational exposure limits (OELs) are important tools for managing worker exposures to chemicals; however, hazard data for many engineered nanomaterials (ENMs) are insufficient for deriving OELs by traditional methods. Technical challenges and questions about how best to measure worker exposures to ENMs also pose barriers to implementing OELs. New varieties of ENMs are being developed and introduced into commerce at a rapid pace, further compounding the issue of OEL development for ENMs. A Workshop on Strategies for Setting Occupational Exposure Limits for Engineered Nanomaterials, held in September 2012, provided an opportunity for occupational health experts from various stakeholder groups to discuss possible alternative approaches for setting OELs for ENMs and issues related to their implementation. This report summarizes the workshop proceedings and findings, identifies areas for additional research, and suggests potential avenues for further progress on this important topic.

Activation of mouse and human peroxisome proliferator-activated receptors (PPARs) by phthalate monoesters.

Administration of phthalates is known to cause toxicity and liver cancer in rodents through the activation of peroxisome proliferator-activated receptors (PPARs), and the monoesters appear to be the active metabolites that function as ligands of PPARs. There is evidence that PPARs exhibit significant species differences in response to ligand activation. In this study, the activation of mouse and human PPARalpha, PPARbeta, and PPARgamma by a broad class of phthalate monoesters was investigated using a trans-activation assay, functional analysis of PPARalpha target gene expression, and a PPARgamma-mediated differentiation assay. These studies demonstrated a range in the ability of various phthalate monoesters to activate PPARalpha, with the mouse PPARalpha generally being activated at lower concentrations and exhibiting a greater response than human PPARalpha. Similarly, a range in the trans-activation of mouse PPARbeta by phthalate monoesters was also observed, but this effect was not found with human PPARbeta. A number of phthalate monoesters activated both mouse and human PPARgamma, with similar sensitivity being exhibited by both receptors. These studies show that the potency and efficacy of phthalate monoesters for the activation of PPARalpha and PPARgamma increase with increasing side-chain length. These studies also show that mouse PPARalpha and PPARbeta are generally activated at lower concentrations of phthalate monoesters than human PPARalpha and PPARbeta, and that both mouse and human PPARgamma exhibit similar sensitivity to phthalate monoesters. Lastly, there is a good relationship between the relative ability of phthalate monoesters to trans-activate PPARalpha and PPARgamma, and the relative induction of PPARalpha target gene mRNA and PPARgamma-mediated adipocyte differentiation, respectively.

Phthalate treatment does not influence levels of IgE or Th2 cytokines in B6C3F1 mice.

Bronchial asthma is mediated, in part, by the immunoregulatory cytokines interleukins 4 and 13 (IL-4 and IL-13). These cytokines stimulate IgE synthesis that in turn is associated with airway hyper-responsiveness. Compounds that stimulate IgE synthesis and elicit bronchial reactivity are generally considered to be respiratory sensitizers. Recently, it has been hypothesized that exposure to phthalates may contribute to childhood asthma. To address this question, di-(2-ethylhexyl) phthalate (DEHP) was tested using a protocol adapted from work by Dearman that involves topical application (and challenge) of test substances to mice followed by measurements of total serum IgE. In addition, auricular lymph nodes were harvested for measurement of IL-4 and IL-13 proteins and their corresponding messenger RNAs. Because skin absorption of high molecular weight phthalates is limited, liver weight increase, a measure of peroxisomal proliferation, was monitored to assure that internal dosing had been achieved. ELISA and RNAse protection assays demonstrated that DEHP treatment did not significantly affect IgE, IL-4, or IL-13 levels. Similarly, IL-4 and IL-13 mRNA levels were not elevated. In contrast, all of these were significantly elevated by trimellitic anhydride (TMA), a respiratory sensitizer used as the positive control in this assay. Liver weights were significantly elevated by DEHP, providing evidence of sufficient percutaneous absorption to induce physiological responses. To extend these observations, three other commercial phthalate ester plasticizers, di-isononyl phthalate (DINP), di-isohexyl phthalate (DIHP), and butyl benzyl phthalate (BBP), were assessed using the same protocol. As above, ELISA and RNAse protection assays showed that IgE, IL-4, and IL-13 proteins, and IL-4 and IL-13 mRNAs in the phthalate-treated animals were all at levels similar to that of control values. The positive control, TMA, produced large, statistically significant increases in all parameters, demonstrating responsiveness of the assay. Another control, dinitrochlorobenzene (DNCB), a contact sensitizer, also responded as expected, producing smaller but statistically significant increases in IgE and in mRNA for IL-4 and IL-13 but not in the levels of these cytokines. In summary, treatment with DEHP, DINP, DIHP, and BBP did not result in significant elevations in total serum IgE, IL-4, or IL-13. As such it is unlikely that these substances would produce antibody-mediated respiratory allergy.

NTP center for the evaluation of risks to human reproduction reports on phthalates: addressing the data gaps.

Between 1998 and 2000 an Expert Panel convened by the National Toxicology Program's Center for the Evaluation of Risks to Human Reproduction (NTP-CERHR) reviewed information related to the developmental and reproductive toxicity of seven phthalate esters; DBP, BBP, DnHP, DEHP, DnOP, DINP, and DIDP. Information on exposures was also considered. The objectives were to determine whether any of these phthalates posed potential human reproductive risks, and if so, to define the circumstances. The Expert Panel also identified some areas of uncertainty. These assessments, ultimately published in 2002, concluded that reproductive risks were minimal to negligible in most cases although some specific uses were considered potentially more problematic. Since the evaluations were completed, numerous studies dealing with both hazard characterization and underlying mechanism have been carried out. Additionally, exposures of the general population have been much better characterized through the use of urinary measurements developed by the Centers for Disease Control (CDC). This additional information makes several important points. First, calculations based on the urinary metabolite measurements indicate that exposures within the general population are at levels similar to or lower than the estimates used by the NTP-CERHR. The demonstration that exposures were not underestimated by the CERHR has removed a substantial portion of the uncertainty. Second, new hazard characterization studies on several phthalates have established NOAELs similar to or higher than those used by the Expert Panel. Thus, these data demonstrate that, to the extent that the rodent data are useful for human health risk assessment, the no effect levels and dose-response relationships are now more precisely defined. In some cases, the no effect levels may be substantially higher than those estimated by the Expert Panel. Third, studies of underlying mechanism and/or hazard characterization studies in other species suggest that primates may be less sensitive than rodents to the reproductive effects of certain phthalates. Finally, the two specific situations that the CERHR identified as potentially problematic, the exposure of young children to DINP through the use of toys or to DEHP from medical devices, have been assessed by the responsible regulatory authorities. The Consumer Product Safety Commission concluded that exposure to DINP from toys was well below effect levels in animals, and, therefore, there was no risk. The Food and Drug Administration estimates of exposures from medical devices indicated that for some limited, intensive medical procedures, DEHP exposures could be similar to or greater than the NOAELs selected by the NTP-CERHR. However, the FDA also acknowledged that more recent information indicates that the NOAELs identified in rodent studies may be substantially higher than values previously proposed by the NTP-CERHR. In summary, much of the uncertainty identified by the CERHR has now been addressed, and the overall conclusions that levels of concern are minimal to negligible in most situations are much better established. The overall objective of this report is to summarize this new research and comment on its relevance to the NTP-CERHR assessments.

Reproductive toxicity evaluation of dietary butyl benzyl phthalate (BBP) in rats.

Butyl benzyl phthalate (BBP) was administered in the diet at 0, 750, 3750, and 11,250 ppm ad libitum to 30 rats per sex per dose for two offspring generations, one litter/breeding pair/generation, through weaning of F2 litters. Adult F0 systemic toxicity and adult F1 systemic and reproductive toxicity were present at 11,250 ppm (750 mg/kg per day). At 11,250 ppm, there were reduced F1 and F2 male anogenital distance (AGD) and body weights/litter during lactation, delayed acquisition of puberty in F1 males and females, retention of nipples and areolae in F1 and F2 males, and male reproductive system malformations. At 3750 ppm (250 mg/kg per day), only reduced F1 and F2 offspring male AGD was present. There were no effects on parents or offspring at 750 ppm (50 mg/kg per day). The F1 parental systemic and reproductive toxicity no observable adverse effect level (NOAEL) was 3750 ppm. The offspring toxicity NOAEL was 3750 ppm. The offspring toxicity no observable effect level (NOEL) was 750 ppm, based on the presence of reduced AGD in F1 and F2 males at birth at 3750 ppm, but no effects on reproductive development, structures, or functions.

In vitro bioavailability of heavy metals in pressure-treated wood dust.

Pressure treatment with chromium, copper, and arsenic (CCA) is the most prevalent method for protecting wood used in outdoor construction projects. Although these metals are tightly bound to the wood fibers and are not released under most conditions of use, we examined the bioavailability of metals in CCA pressure-treated wood dust in vitro. Cytotoxicity and metallothionein (MT) mRNA expression were examined in V79 Chinese hamster lung fibroblast cells incubated with respirable-size wood dust generated by sanding CCA-treated and untreated (control) Southern yellow pine. In colony survival studies, increased cytotoxicity (p < 0.05) occurred in V79 cells treated with CCA wood dust (351 +/- 77 microg/ml, mean +/- SE) compared with control wood dust (883 +/- 91 microg/ml). Increased cytotoxicity with CCA wood dust also occurred in an arsenic resistant subline of V79 cells, thus suggesting that arsenic was not responsible for the increased cytotoxicity. Metallothionein mRNA was significantly increased after 48 h of treatment with CCA wood dust compared with control wood dust. Incubation of CCA wood dust in cell culture media resulted in the transfer of copper, but not chromium or arsenic, into the media. Moreover, the treatment of cells with this filtered extract resulted in significantly increased metallothionein mRNA, suggesting that bioavailable copper is responsible for inducing metallothionein mRNA in V79 cells. Thus, these bioassays suggest that metals become bioavailable during in vitro culture of phagocytic V79 cells with CCA wood dust.

Exposure to wood dust and heavy metals in workers using CCA pressure-treated wood.

Chemical pesticide treatment enables relatively nonresistant woods to be used in outdoor construction projects. The most prevalent procedure used to protect these woods is pressure treatment with chromium, copper, and arsenic (CCA). This pilot study examined the airborne concentration and particle size distribution of wood particles, chromium, copper, and arsenic at both outdoor (measured over the whole work day) and indoor (measured during the performance of specific tasks) work sites. At the outdoor residential deck construction sites, the arithmetic mean total dust concentration, measured using personal filter cassette samplers, was 0.57 mg/m3. The mass median aerodynamic diameter (da) of the outdoor wood dust was greater than 20 microm. Indoor wood dust concentrations were significantly greater than those measured outdoor and were job category-dependent. The highest mean breathing zone dust concentration, 49.0 mg/m3, was measured at the indoor sanding operation. Personal impactor sampling demonstrated that the mean total airborne concentration of arsenic, but not chromium or copper, was consistently above recommended occupational exposure levels at the indoor work site, and occasionally at the outdoor work sites. At the indoor sanding operation, the mean total chromium, copper, and arsenic concentrations were 345, 170, and 342 microg/m3, respectively. Thus, significant exposure to airborne heavy metals can occur as a result of indoor and outdoor exposure to CCA pressure-treated wood dust. Therefore, current standards for wood dust may not adequately protect workers from the heavy metals commonly used in CCA pressure-treated wood.