Difficulties in establishing regulations for engineered ...

Review Article Received: 20 March 2015,

Revised: 21 April 2015,

Accepted: 22 April 2015

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jat.3180

Difficulties in establishing regulations for engineered nanomaterials and considerations for policy makers: avoiding an unbalance between benefits and risks Luis Guillermo Garduño-Balderasa, Ismael Manuel Urrutia-Ortegaa,b, Estefany Ingrid Medina-Reyesa,b and Yolanda Irasema Chirinoa* ABSTRACT: Current evidence of engineered nanomaterials’ (ENM) toxicity has led to a latent concern about hazards for both humans and the environment. For this reason, some efforts have been made to suggest frameworks or other guidance to regulate ENM handling; however, the real exposure risk to humans has not been well established. The aims of this work were to analyze the difficulties in establishing regulations for ENM and to discuss some considerations that may be helpful for policy makers involved in the regulation of ENM. Difficulties in establishing regulations are based on the novel properties of ENM associated with cytotoxic effects, the insufficiency of standardized methods to test those effects and the lack of epidemiological evidence of ENM toxicity, especially in occupational settings. Nevertheless, we offer some suggestions for establishing regulations, which include frameworks oriented towards protecting personnel exposed to ENM without decreasing production. In addition, we propose an ENM data sheet to offer available information of ENM. Finally, ethical aspects should also be considered in developing ENM regulations because every person who is working around or consuming ENM has the right to be informed about the potential risk. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: engineered nanomaterials; regulations; standardized methods; technical ENM data sheet; ethical considerations

Introduction In the last decade, evidence of the toxicity of nanomaterials (NM) and engineered nanomaterials (ENM) has led to a latent concern about hazards for humans and the environment, and several surveys regarding usage (Schmid and Riediker 2008), precautions (Helland et al., 2008), safety practices (Conti et al., 2008) and risk perception (Engeman et al., 2012) have been conducted in companies related to ENM. Those studies note the uncertainty in ENM risk knowledge, suggest a preference for adapting safe handling practices and recognize that there is insufficient information to establish specific regulations. The main concern of ENM lies in the toxicological evidence, which is not easily translated into recommendations for several reasons. Another important fact is that the astonishing level of ENM production in the world expands the debate about their effects not only in human occupational settings but also on the environment. Moreover, the pace of ENM development overcomes the possibility of designing proper regulations because until now, there has been a deficit of realistic evidence regarding the effects of ENM on human health and the environment. Nevertheless, guidance documents provided by knowledgeable agencies, including the National Institute for Occupational Safety and Health (NIOSH), Health, Safety and the Environment (HSE) and the National Institute for Occupational Safety and Health, Japan ( JNIOSH), are available to implement or adapt practices for the safe development and handling of ENM. ENM regulation has multiple components, which complicates its success and requires maintaining a balance between the benefits of ENM and the safety of humans

J. Appl. Toxicol. 2015

and the environment. However, even although the landscape looks convoluted, we can take advantage of what we have learned in the past from other compounds/substances to try to avoid disrupting the balance between the benefits and risk of ENM. Here, we analyze some difficulties in establishing regulations for ENM and suggest some considerations that could be helpful for policy makers. In addition, we use several examples from the past as an analytical tool to keep our considerations in perspective, including how agencies have launched documents and how we have previously handled the toxicological evidence of substances when developing regulations, among others.

NM and ENM Definition Natural NM have existed much longer than humans, but intentionally manufactured NM, called ENM, have only recently been synthetized. The wide spectrum of applications of ENM has attracted attention across the world in fields of medicine, *Correspondence to: Y. I. Chirino, Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Av. de Los Barrios 1, Los Reyes Iztacala, Tlalnepantla 54090, Estado de México, México. E-mail: [email protected] a Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, CP 54090, Estado de México, México b Programa de Posgrado en Ciencias Biomédicas, Universidad Nacional Autónoma de México

Copyright © 2015 John Wiley & Sons, Ltd.

L. G. Garduño-Balderas et al. agriculture and electronics, among others (Guan et al., 2014; Kim and Hyeon 2014; Liu and Lal 2015). The necessity of providing a definition of NM has resulted in the formation of various committees and agencies, and since 2004, NM and ENM definitions have been suggested with sequential changes, which include or exclude some features in order to develop a precise definition (Table 1). For example, Health Canada classifies an NM, even if the material is not in the nanoscale range, as an ENM if it has ’properties/phenomena‘ similar to ENM. Some European agencies consider the aggregation/agglomeration state in the NM definition while others only consider a substance to be an NM if this is in an unbounded state. In addition, insolubility or biopersistance is also considered in NM definitions. The European Chemicals Agency specifies that some materials with one dimension in the sub-nanometric range, such as fullerenes, graphene flakes and single-wall carbon nanotubes, shall be considered NM. In 2011, the European Union established that any material containing 50% of nanoparticles should be considered ENM; however, the European Parliament ’amended the definition‘ in 2014 to reduce the percentage from 50% to 10% (http://www.nanowerk.com/ spotlight/spotid=38308.php). A unified ENM definition is increasingly needed and could be helpful for the development of frameworks, legislative guides, guidance documents or recommendations. Current Situation of Worldwide ENM Production According to the nanowerk database, 216 companies responsible for manufacturing and/or providing ENM (Fig. 1) are currently registered (revised by the Environmental Protection Agency, 2009). Asia has 79 of these companies, of which China operates 34.1%. North America has 77 companies, of which 89.6% are in the USA. Europe has 52 companies, and Germany holds 26.9%. Africa has 2 companies in South Africa, while Oceania has 6 companies, with 83.3% of them in Australia (http://www.nanowerk. com). Manufacturing and supply companies provide information ranging from the sole ENM diameter to toxicological data (Table 2). Shape and size are the most common pieces of information provided; however, the method used for these determinations is frequently omitted. The second most common piece of information provided is an image of the ENM taken by scanning electron microscopy (SEM) or transmission electron microscopy (TEM), as proof of the ENM shape and size. The third most common piece of information provided is purity; however, the method used is also frequently absent. A good example of ENM information provided by a company is taken from Mach 1, Inc. in the USA, which supplies nanospheres of metals, metal oxides and ceramics. The technical data sheet from this company contains particle size distribution, composition, physical and chemical properties, toxicity and TEM (Table 2). The most produced metal oxides include titanium dioxide (TiO2), zinc oxide (ZnO), iron oxides (FeOx), aluminium oxides (AlOx), silicon oxide (SiO2) and cerium oxide (CeO2). Carbon-based nanotube production includes single-walled carbon nanotubes (SWCNT) and multi-walled carbon nanotubes (MWCNT). Nanoparticles of silver (Ag) and gold (Au), quantum dots and fullerenes are also of great importance to the ENM industry. The USA and the European Union are recognized as worldwide ENM producers, with Switzerland being the distinguished country of ENM production in Europe (Fig. 2). In the last 5 years, Asia has become an important ENM producer while Oceania and Central and South America are ascending. The methodology for determining

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worldwide ENM production has been based on surveys and collected data from academic publications, professional reports, company Web sites, production process patents and personal communication with company leaders (Templeton et al., 2006; Thompson 2007; Schmid and Riediker 2008; Hendren et al., 2011; Piccinno et al., 2012). However, it is difficult to identify precisely the total ENM production in the world because this industry is constantly growing, and databases have different criteria for analysis. It is necessary to make an effort to determine worldwide ENM production, which will make it easier to stipulate a better relationship between exposure and risk. Challenges of nanotoxicology includes the predictive capability of its methods to identify the most hazards ENM based on the physicochemical characteristics, because these characteristics, including shape and size have a specific impact on biological effects. Then, identifying workplaces, in which most hazardous shapes of ENM are produced, could be relevant to analyze the risk of occupational exposure. In addition, the economic impact of different shapes of ENM is significant for innovation and technology programs over the world and enables the development of science and nanotechnology. Nanospheres, the most produced shape in all regions, are widely used in medical products (Singh et al., 2010; Dhanaraj et al., 2014). The USA reports 44 medical products on the market, and the global market for nanomedicine will be approximately $630 million in 2016. The USA also invested approximately $68.8 million in 2011 (3.7% of the overall National Nanotechnology Initiatives budget) for conversion energy efficiency, solar thermal, thermoelectric and energy storage, among others and the budget for this initiative increased to 5.9% in 2012. Earnings by solar cell production, which involves silicon nanospheres for the global solar market (Winkless 2014), have been estimated at $15 billion by 2015 in the USA (Fraas and Partain 2010). For these reasons, classification of ENM by shape may constantly be updated. Difficulties of Establishing Regulations for ENM Regulations for ENM companies cannot be circumscribed to a specific country or region in the world. Indeed, according to the nanowerk database, more than 80 companies are operating in America, and all of them are involved in the production of different ENM shapes, including nanotubes, nanospheres, fullerenes, graphenes, quantum dots, nanofibers and nanowires (Fig. 3). All these ENM are produced in Europe, Asia and Oceania, and some ENM companies have intercontinental activities, such as CNano Technology, which produces ENM in the USA, China and Japan, and Nanocs, which manufactures and supplies ENM in the US, Japan, the UK, France, Italy, India, Malaysia, Australia and South Africa. This situation suggests that one company could potentially adhere to a strict set of regulations in one country, but follow less stringent procedures in a more flexible country. There are several difficulties in establishing ENM regulations, including (i) the constant synthesis of innovative ENM with different properties, (ii) a shortage of standardized methods to establish toxicity, and (iii) the absence of information about the degree of the risk of exposure in occupational settings, or the risk of use and consumption of products for humans. Companies are aware of the risk, but they argue that the available information is insufficient to establish a guidance regarding ENM (Engeman et al., 2012). Here, we aim to analyze those complications and provide some examples of how society has solved similar problems in the past.



Difficulties in establishing regulations for engineered nanomaterials Table 1. Nanomaterials (NM) and engineered nanomaterials (ENM) definition Organization Royal Society & the Royal Academy of Engineering of the United Kingdom (United Kingdom)

Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR; European Union) Cosmetic Regulation (European Union)

National Industrial Chemicals Notification and Assessment Scheme (NICNAS; Australia)

International organization for standardization (ISO) Health Canada (Canada)

European Union

Chemicals Watch (European Union)

The French decree on Nano (France)

Organization Environmental Protection Agency (EPA; United States of America)

NM definition

Year

Those which have structured components with at least one dimension less than 100 nm. Materials that have one dimension in the nanoscale and are extended in the other two dimensions, such as a thin films or surface coatings. Some of the features on computer chips come in this category. Materials that are nanoscale in two dimensions (and extended in one dimension) include nanowires and nanotubes. Materials that are nanoscale in three dimensions are particles, for example precipitates, colloids and quantum dots (tiny particles of semiconductor materials). Nanocrystalline materials, made up of nanometer-sized grains, also fall into this category. Any form of a material that is composed of discrete functional parts, many of which have one or more dimensions of the order of 100 nm or less. Insoluble or biopersistent and intentionally manufactured material with one or more external dimensions, or an internal structure, on the scale from 1 to 100 nm. Industrial materials intentionally produced, manufactured or engineered to have unique properties or specific composition at the nanoscale that is a size range typically between 1 nm and 100 nm, and is either a nano-object (i.e. that is confined in one, two or three dimensions at the nanoscale) or is nanostructured (i.e. having an internal or surface structure at the nanoscale). Material with any external dimension in the nanoscale or having internal or surface structure in the nanoscale. Manufactured substance or product, and any component material, ingredient, device, or structure to be nanomaterial if it is at or within the nanoscale in at least one external dimension, or has internal or surface structure at the nanoscale, or if it is smaller or larger than the nanoscale in all dimensions and exhibits one or more nanoscale properties/phenomena. Natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness, the number size distribution threshold of 50 % may be replaced by a threshold between 1 and 50%. Nanomaterial’ means a natural or manufactured active substance or non-active substance containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1-100 nm. Fullerenes, graphene flakes and single-wall carbon nanotubes with one or more external dimensions below 1 nm shall be considered as nanomaterials. Intentionally manufactured to a nanometric scale and containing particles in an unbound state or as an aggregate or as an agglomerate and where, for a minimum proportion threshold of the particles in the number size distribution, one or more external dimensions is in the size range 1-100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the minimum proportion of number size distribution threshold can be reduced.

2004

ENM definition Any particle, substance, or material that has been engineered to have one or more dimensions in the nanoscale. The term “engineered” is intended to mean that the material is 1) purposefully produced and 2) purposefully designed to be a nanoscale material.

2007

2009

2009

2010 2011

2011

2011

2012

Year 2007

(Continues)




L. G. Garduño-Balderas et al. Table 1. (Continued) Organization Cosmetics Regulation (European Union)

International organization for standardization ISO) American Chemistry Council’s (ACC’s; United States of America)

ENM definition

Year

Any intentionally produced material that has one or more dimensions of the order of 100 nm or is composed of discrete functional parts, either internally or at the surface, many of which have one or more dimensions of the order of 100 nm or less, including structures, agglomerates or aggregates, which may have a size above the order of 100 nm but retain properties that are characteristic to the nanoscale. Nanomaterial designed for specific purpose or function.

2009

Any intentionally produced material that has a size in 1, 2 or 3 dimensions of typically between 1-100 nanometers. It is noted that neither 1 nm nor 100 nm is a ‘bright line’ and data available for materials outside of this range may be valuable.

2012 2013

NM and ENM definition provided by different organizations since 2004.

Figure 1. Worldwide distribution of companies supplying and manufacturing ENM. The distribution of companies is the following: USA, 69 companies; China, 27 companies; South Korea, 15 companies; India and Germany, 14 companies; Japan, 10 companies; Canada, UK and Spain, 6 companies; Australia, 5 companies; Turkey, Italy, Portugal, Iran and France, 3 companies; Switzerland, Czech Republic, Estonia, Thailand, Singapore, Russia, Finland and South Africa, 2 companies; Mexico, Greece, Belgium, Norway, Ukraine, The Netherlands, North Korea, Malasya, Vietnam, Cyprus, Israel, New Zealand and Argentina, 1 company. Data unavailable for grayed-countries.

Novel Compounds with Novel Properties One of the primary complications of ENM regulation is the rapid development of new ENM every year and sustained production in subsequent years is expected, in part because ENM synthesis uses relatively simple methods, such as the hydrothermal method, sol-gel, seeded growth, alumina templating, surfactantdirected synthesis and chemical vapor deposition, among others (Pichat 2014). In this regard, by modifying a single parameter, such as temperature, reaction time, voltage, NaOH concentration or pH, an ENM with different properties, including shape,


crystallinity, tube spacing and allotropic form, can be obtained (Pichat 2014). In the past, the classification of agents has taken several decades, and when risk evidence is solid, committees develop policy frameworks. As an example, establishing ionizing radiation risk was led by the United Nations Scientific Committee on the Effects of Atomic Radiation. This committee, whose members consist of scientists from 27 countries designated by the UN General Assembly, has collected and analyzed documents regarding ionizing radiation since the 1960s (http://www.unscear.org/). The problem is that ENM are new in the world, and we are now





Europe

America

Asia

Region

http://www.meliorum.com/ http://www.microtechnano.com/ http://www.nano-oxides.com/index.html http://nanocomposix.com/ http://www.nanopowder.ca/ http://nanotekinstruments.com/ http://www.plasmachem.de/ http://www.goodfellow.com/sp/ http://www.metalnanopowders.com/ http://www.mbn.it/eng/index.php http://www.nanoiron.cz/en/home-page http://www.turbobeads.com/ http://www.enteknomaterials.com/?ID=1 http://www.n-tec.no/ http://anftechnology.com/fibers-and-powders/

Meliorum Technologies Microtechnano Nano-Oxides

NanoComposix

Nano Powder Canada Nanotek Instruments PlasmaChem

Goodfellow Metal Nanopowders MBN Nanomaterialia S.p.A.

NANO IRON,s.r.o. TurboBeads Entekno n-Tec Nafen

Nanospheres Fullerenes Nanotube Fullerenes Nanofiber

http://www.fusokk.co.jp/eng/index.html http://www.f-carbon.com/index.html http://ocsial.com/en/ http://www.neotechproduct.ru/eng_main_page http://www.anstco.com/english/p5.html

Nanospheres Graphene Nanospheres Nanowires Quantum dots Fullerenes NanoClusters Nanospheres Fe nanopowder FeNi nanopowder Nanospheres Nanospheres Nanopowder MWCNT SWCNT Nanofiber

Nanospheres

Nanospheres MWCNT Nanopowder

Nanospheres Nanospheres Nanospheres

Nanopowder

http://www.hznano.com/en/Index.asp

http://www.nanospheres.com http://www.advancedmaterials.us/nanomat.htm http://www.machichemicals.com/

Nanospheres

Nanospheres Nanospheres Nanopowder Nanospheres Nanotubes Nanospheres Nanofiber

ENM

http://www.abcnanotech.com http://www.anapro.com/kor/ http://www.avansa.co.in/index.html http://www.nanoshel.com/ http://www.nanoclay.net/sdp/96374/4/ main-998443/0/Home.html http://www.nanoparticles-microspheres.com/

Web site

EPRUI Nanoparticles & Microspheres Shanghai Huzheng Nano Technology Fuso Chemical Frontier Carbon Corporation OCSiAl NeoTechProduct Asian Nanostructures Technology Company (ANSTCO) Cospheric Nano Inframat Advanced Materials Mach 1, Inc.

ABCNANOTECH Advance Nano Products ANP) AVANSA Nanoshel FCCinc Additives & Instruments

Company

Table 2. Engineered nanomaterials (ENM) characterization provided by manufacturers and/or suppliers worldwide

SEM TEM and particle size SEM and particle size SEM SEM and diameter (Continues)

SEM, mean diameter and sphericity Particle size, and composition TEM, size distribution, composition, physical and chemical properties and toxicity Diameter SEM, diameter, purity, and electrical conductivity TEM, particle size, specific surface area, purity and applications TEM, size distribution, optical properties and characterization Instrumentation SEM and X-ray diffraction SEM and specific energy density TEM, particle size, Raman spectrum and composition Particle size TEM SEM, composition and particle size distribution

SEM and particle size Data unavailable Data unavailable Applications, purity Data unavailable

Particle size

Purity and particle size

SEM, molecular shape and applications TEM, X-ray diffraction and particle size Size and purity Purity and particle size Particle size

Information provided

Difficulties in establishing regulations for engineered nanomaterials


Africa

http://www.micronisers.com/ www.nanoclusterdevices.com/ http://www.comarchemicals.com/index.php/en/ Micronisers Nano Cluster Devices Comar Chemicals


ENM characterization provided by manufacturers and/or suppliers worldwide. Website of each company is indicated. SEM, scanning electron microscopy; TEM, transmission electron microscopy; MWCNT, multi-walled carbon nanotubes; SWCNT, single-wall carbon nanotubes; DWCNT, double-walled carbon nanotubes.

Particle size Data unavailable Particle size

TEM, particle size and size distribution Data unavailable

Nanospheres Fullerenes DWCNT Nanowires Nanopowder Nanopowder Nanowires Nanopowder http://www.antaria.com/irm/content/default.aspx http://www.kemix.com.au ANTARIA Kemix Oceania

Region

Table 2. (Continued)

Company

Web site

ENM

Information provided

L. G. Garduño-Balderas et al. creating committees to analyze the scientific evidence of risk for humans and the environment, but the development of this evidence remains an ongoing process. In addition, each ENM has broad design possibilities in terms of composition, shape, size and coating, which broaden their properties and applications but also complicate the understanding of their effects on humans and the environment. For example, TiO2 nanoparticles – which are extracted from Ilmenite rock, mainly in Russia, Norway, Canada, Brazil and Ukraine (Chang 2002), and are one of the most produced nanoparticles worldwide (Piccinno et al., 2012) – are obtained in three crystalline phases: anatase, rutile and brookite. TiO2 nanoparticles were synthesized mainly shaped as nanospheres from anatase; however, since the last decade, shapes such as nanotubes, nanosheets, nanofibers, nanowires and nanoflowers have been produced not only from anatase but also from rutile or brookite. These new synthesized shapes are under research to develop an understanding of their physicochemical properties and applications, but some of them are already in the market. The shape has a strong influence on (1) surface area, (2) agglomerate/aggregate state, (3) zeta potential and (4) chemical properties, and consequently, all these properties have an impact on biological effects. In addition, coating ENM has been used as a strategy to confer new characteristics (Fig. 4). TiO2 nanoparticles coated with dissolved matter organic enhances phenanthrene sorption, which is useful in soil remediation (Wang et al., 2014). Similarly, TiO2/SiO2 nanofibers coated with polyaniline enhances the degradation of methyl orange under the visible light by photocatalytic activity, suggesting promising applications for water remediation (Liu et al., 2014). Differences between the crystalline phase, shape and coating influence the physicochemical properties and the toxicity. With this in mind, some reports have concluded that anatase induces more toxicity than rutilo or brookite (Iavicoli et al., 2011; Skocaj et al., 2011; Warheit 2013). Furthermore, shape partially defines cell internalization. For instance, TiO2 nanospheres are internalized by endocytosis clathrin-dependent, caveolae formation (Thurn et al., 2011), and Toll-like receptor 4 (Fröhlich 2012; Mano et al., 2013), while nanobelts are internalized mainly by micropinocytosis (Hamilton et al., 2009); however, new ENM, such as gold nanoflowers, have an unknown internalization mechanism (Wang et al., 2010). Toxicity is also regulated by shape – a good example is nanobelts, which induce more toxicity than nanospheres both in vitro and in vivo (Hamilton et al., 2009; Silva et al., 2013; Medina-Reyes et al., 2015). Coating also has repercussions in biological effects. For example, TiO2 nanobelts coated with carboxyl groups induce less inflammation than their uncoated counterparts (Hamilton et al., 2014). All of these properties must be considered together to determine ENM effects, and a single variation needs a full characterization and toxicological analysis, which in part explains why we cannot generalize the effect of one type. Insufficiency of Standardized Methods to Assess Toxicity ENM methods for establishing characterization and toxicity have limitations. ENM characterization includes particle size, size distribution, particle morphology, particle composition, surface area and surface chemistry (Murdock et al., 2008). Until now, there has been no standardized technique to determine some of those ENM characteristics. Particle size and shape are determined by SEM, TEM and atomic force microscopy (AFM) (Kim et al., 2014); chemical composition and purity are determined by X-ray



Difficulties in establishing regulations for engineered nanomaterials

Figure 2. Main engineered nanomaterials (ENM) produced in worldwide. ENM production (tons/year) and the accessible information is only from Switzerland, USA, Europe and world. The main ENM produced are TiO2 (titanium dioxide) ZnO (zinc oxide), SiO2 (silica oxide) , FeOx (iron oxide), AlOx (aluminium oxide) and CeOx (cesium oxide), SWCN (single-wall carbon nanotubes), MWCN (multi-wall carbon nanotube), silver nanoparticle (Ag), gold nanoparticle (Au), Quantum dots (QDs), fullerenes and dendrimers. Data presented were collected from Piccino et al., 2012; Schmid and Riediker 2008; Hendren et al. 2011; Kennedy et al., 2008; Thompson 2007).

Figure 3. Distribution of companies manufacturing and/or supplying engineered nanomaterials (ENM) classified by shape. All shapes including nanotubes, nanospheres, fullerenes, graphenes, quantum dots, nanofibers and nanowires are produced and/or supplied in America and Europe; Asia does not report quantum dots production and Oceania produced nanotubes, nanosphres, fullerenes and nanowires. Some companies, such as Nanocs produces and/or supplies nanotubes and nanospheres in America, Europa, Asia, Oceania and South Africa. Nanospheres are still the most produced ENM followed by nanotubes.


diffraction, AFM, X-ray photoelectron spectroscopy and UV-Vis spectroscopy (Shrestha et al., 2014). ENM suspensions for experimental analysis are sonicated, forming aggregates/agglomerates with different characteristics depending on the type of dispersion solution, frequency, time and temperature of sonication (De Temmerman et al., 2012; Williges et al., 2013), which has an impact on toxicity (http://www.oecd.org/officialdocuments/public displaydocumentpdf/?cote=env/jm/mono%282012%2940&doc language=en). Concern about the deficient correlation between in vitro and in vivo studies of ENM genotoxicity lead to the Nanogenotox Joint Action collaborative project coordinated by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) in the European Union to assess genotoxicity jointly to try to identify the most common problems in the laboratory. ENM genotoxicity was designed by intratracheal instillation, oral and intravenously administration and despite the efforts and commitments of this 3-year project, conclusions were ambiguous and largely dependent on in vivo and in vitro model employed (http://www.oecd. org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/ mono%282014%2934&doclanguage=en). Another effort to establish methods for minimizing variability and improving toxicity testing was made by a consortium program funded by Grantees of the National Institute of Health Sciences (NIEHS), with a $13 million budget. The aim of this consortium program, formed by 13 laboratories, was to develop reliable and reproducible research methods to develop a better interpretation of ENM effects. In the first phase, the results from in vivo experiments were analyzed to improve and minimize the variability in further studies. In the second phase, the results had minimum



L. G. Garduño-Balderas et al.

Figure 4. Divergence of titanium dioxide to different physical nanoparticles features. Ilmenite rock is the main titanium dioxide (TiO2) source. TiO2 nanoparticles are obtained as a crystalline phase of anatase, rutile and brookite. Different shapes can be synthetized using any of those crystalline phases, including nanofibers, nanoflowers, nanosheets, nanospheres, nanotubes and nanowires. Each form can be coated with different compounds such as polyaniline, carboxyl groups or organic matter even with other ENM, including silica or zinc oxide. The final shape of ENM determines the number of dimensions and each dimension may measure between 1–100 nm.

variability among different laboratories and were published (Bonner et al., 2013; Xia et al., 2013). Some of the analysis showed that 1) centrifugation is needed after MTT incubation to be compared with the LDH assay; 2) usage of multiple cell types avoids false-negative results; 3) undoubtedly, in vivo studies are helpful in predicting in vivo outcomes; and 4) well-characterized ENM, including positive and negative control, in addition to a wellestablished dispersion, ensures stable suspensions in cell culture media, which is crucial for the reproducibility of results (Bonner et al., 2013; Xia et al., 2013). This consortium suggests that by taking into account some of these considerations, reproducibility can be improved and results of ENM exposure can reach greater reliability. In addition, nanoscience is not only facing the challenge of having standardized methods for testing toxic effects, but also for classifying ENM such as graphene, which according to GRAPHENE Flagship is the strongest material in the world, even more than diamond (http://graphene-flagship.eu/). Indeed, graphene-based materials require a description of additional physicochemical characteristics for classification, which include the number of layers, lateral size and the atomic C/O ratio (Wick et al., 2014). However, until now, we have had insufficient standard methods to characterize ENM and measure ENM toxicity. Furthermore, next-generation sequencing techniques, ’omics‘, and high-throughput screening analysis are currently being used


to evaluate ENM toxicity (Klaper et al., 2014) and genotoxicity (Vecchio et al., 2014; Watson et al., 2014), while structure–activity relationships (SARs) are establishing associations between ENM physicochemical properties and biological effects (Nel 2013). However, even if these tools are on the cutting edge of nanotoxicology, they are not ready to be used for predicting adverse effects on human health. Pre-clinical Assays Still Under Development ENM have opened a distinctive field of medicine, with potential usage as nanodrugs/nanopharmaceuticals/nanocarriers and promising outcomes; however, pre-clinical assays are still under development for nanomedicine. This field requires attention because pre-clinical assays face additional challenges compared with other types of drugs because the absorption, distribution, metabolism and excretion (ADME) could differ from an average drug. For instance, titanium dioxide, zinc oxide and silica oxide have different hemodynamic effects (Haberl et al., 2015) even although those ENM belong to same metal oxide classification. This phenomenon underscores the importance of additional assays, such as characterization in pre-clinical assays. In this regard, the Nanotechnology Characterization Laboratory, which belongs to the National Cancer Institute, has led and developed



Difficulties in establishing regulations for engineered nanomaterials standardized pre-clinical assays for ENM (http://ncl.cancer.gov/). However, in vitro toxicity is being evaluated in hepatic and kidney cells using standardized methods as other drugs, but toxicity in other cell types might be included specifically because some ENM are proposed as nano-pharmaceuticals/nano-carriers that may be used intravenously and pre-clinical assays could be more focused on blood toxicity. In contrast, cytotoxicity in pre-clinical studies includes oxidative stress, apoptosis, autophagy and necrosis, but pyrogenicity and genotoxicity could also be included. Pre-clinical in vivo studies are frequently performed in healthy experimental animals, but diseased animals could also be included to mimic some characteristics of the targeted disease, specifically cancer, as many of the ENM have promising effects against neoplasias. Moreover, the real success of preclinical studies would be to provide relevant data applicable to human health. Lack of Epidemiological Evidence One of the main complexities in launching ENM guidance documents is related to the lack of epidemiologic evidence to establish how great the risk is for humans after ENM exposure. In this regard, the International Agency for Research in Cancer (IARC) has succeeded in classifying diverse agents as risk entities for human health. This agency, which has been working since 1971, has evaluated 900 agents, of which 400 are carcinogenic, probably carcinogenic, or possibly carcinogenic to humans. Classification is based on the extensive interdisciplinary analysis performed by experts (http://www.iarc.fr/). Epidemiological evidence plays a central role in getting an agent in the top classification, which is group 1 – a carcinogen to humans. We are still far from having epidemiological evidence, in part because the innovation of ENM synthesis, and also because epidemiological studies must be performed not only in terms of each ENM, but in terms of routes of exposure. For instance, titanium dioxide nanoparticles can be inhaled in occupational settings during production, can be ingested during food consumption, or can be spread through the skin using sun blockers, and dose and time of exposure determine the effects in humans (Skocaj et al., 2011; Shi et al., 2013). However, the scientific community is aware that occupational settings have special vulnerabilities because the number of workers is increasing. In this regard, the National Science Foundation (USA) estimates that the number of personnel in the ENM industry will increase to 2 million worldwide by 2015 (Roco 2011), which means that epidemiological studies could be focused first on environmental settings. Simultaneously, epidemiological studies could be focused on oral exposure, as ENM are being used in agriculture, added to food and used in food processing and packing (Duncan 2011). The amount of ENM ingested in a normal diet is also increasing; for example, food-grade titanium dioxide, named E171, was used in the past mainly as micro-sized particles but currently is a mixture of micro and nanoparticles (Peters et al., 2014). Adult consumption of titanium dioxide is approximately 1 mg body weight–1 day–1, while children may consume up to 2 mg body weight–1 day–1 (Weir et al., 2012). Evidence that healthy children and children with inflammatory bowel disease have deposits of titanium dioxide derived from oral consumption (Hummel et al., 2014) is attracting the attention of science, as are silica nanoparticles, named E551, which are used as an anticaking agent in food, because it is suspected to have some effect in the gastrointestinal tract (Bellmann et al., 2015). The scientific community is aware of occupational and oral consumption of ENM, and


human studies will likely reveal the effects of ENM in the following years. However, without human findings, it will remain difficult to establish precise regulations. Considerations that may be Helpful for Policy Makers Policy frameworks cannot be oriented to decrease the ENM production. One of the primary concerns about regulation is related to the economic impact regulation will have on worldwide ENM production. We cannot dismiss the fact that benefits are clearly proved for both humans and the environment. One of the most important examples is the development of new therapeutic treatments based on ENM. In terms of the environment, ENM such as graphene nanosheets have improved the removal of contaminants from water (Yu et al., 2015) and also have provided tools for clean energy development (Iavicoli et al., 2014). Based on this, policy frameworks cannot be oriented to decrease ENM production or hinder the development of new drugs, therapeutic strategies, the diagnosis of diseases, or food production for humans. The frameworks should not interfere with nanotechnology strategies leaning to energy consumption reduction or combating air, water or soil pollution. Frameworks Must be Oriented to Protect Workers in Occupational Settings and Environments In contrast, we have learned from the past that some chemicals or drugs appear to be safe, but after scientific studies and analysis, it is revealed that they are not. Asbestos began to be largely produced between the 1920s and 1930s and stopped around the 1980s because scientific evidence of mesothelioma induced by its exposure was discovered, resulting in asbestos being banned in 50 countries. Currently, asbestos is still used in India, Brazil, China, Indonesia, Russia, Uzbekistan and Vietnam, among others and is not banned in India and United States (Frank and Joshi 2014). This situation clarifies that when scientific evidence becomes conclusive, regulatory aspects must prioritize human health over economic impacts, but countries that have implemented regulations cannot force other countries to take part in those frameworks. However, confirmation of human ENM exposure as a real risk for developing diseases is still distant compared with the vast amount of evidence of asbestos as an agent causing mesothelioma. In this regard, concern about ENM inhalation in occupational settings has increased, as it has been classified by the IARC in group 2B as a possible carcinogen for humans (Baan 2007). A plausible regulation could be related to providing respiratory protection to workers; the National Institute for Occupational Safety and Health (NIOSH), which is part of Centers for Disease Control and Prevention (CDC) within the USA, is an experienced institution that offers information about a Respiratory Protection Program (https://www.osha.gov/Publications/3384small-entity-forrespiratory-protection-standard-rev.pdf). This program also includes health work monitoring, which may help to further epidemiology studies. In terms of inhalatory toxicology, we also can learn from airborne particulate matter (PM) exposure, which it is a risk for the development of lung and cardiovascular diseases as well as lung cancer (Brook et al., 2010; Cui et al., 2015). In this regard, the World Health Organization (WHO) has provided guidelines for PM air concentration for 24-h and 1-year periods (http://whqlibdoc.who.int/ hq/2006/WHO_SDE_PHE_OEH_06.02_eng.pdf). This recommendation has been supportive of the development of Environmental



L. G. Garduño-Balderas et al. Policies in some countries, including polluted cities such as Mexico City (http://www.dof.gob.mx/nota_detalle.php?codigo=5357042& fecha=20/08/2014). Limits of PM concentration established by the WHO are based on scientific and epidemiological evidence of the harmful effects in humans, especially in polluted cities; however, in terms of ENM, regulations could encourage the ENM industry to have a registry of ENM air concentration, room dimensions, number of workers, hours of exposure and ENM type produced in those environmental settings, and together, actions can contribute to developing outlines to protect workers and propel aspirations of having appropriate recommendations. Recommendations Should Encourage Availability of Technical ENM Data Sheet and ENM Label for Products ENM manufacturing and supply companies could provide in a Technical Data Sheet as much information as is available, even if there is not a standard design for the technical data sheet of

ENM, including primary size and shape and solubility. This information does not represent a costly investment for an ENM company and could offer an advantage to a purchaser. In addition, we can use as an example information provided by chemical products. The Technical Data Sheet could suggest the following: (i) the usage of standard handling and personal protective equipment for eyes, skin and inhalation recommended by NIOSH/MSHA or the European Standard EN 149. Some links to well-recognized agencies could be included, and information about respirators suggested by specialized agencies could also be provided. (ii) First aid measures in case of contact with eyes, skin, ingestion and inhalation by accident. First aid measures can also be general recommendations, which could include washing the affected zone of the body extensively, being moved to a well-ventilated area if inhalation occurs, calling emergency services if oral exposure occurs, and in any case, being checked by a doctor. (iii) Accidental release measures, which could indicate avoiding sweeping and washing to avoid dispersing ENM and calling Biosafety Authorities.

Figure 5. Suggested Material Safety Data Sheet for Engineered Nanomaterials (ENM). Recommended ENM information that could be provided by supplier/ manufacturer companies. We suggest that companies could collect information from scientific databases to design this ENM data sheet.




Difficulties in establishing regulations for engineered nanomaterials (iv) Toxicological information, which could indicate a lethal dose 50 and lethal concentration 50 in animals. (v) Disposal information, which could indicate, at least, avoiding throwing out in water and soil. We suggest that some of those general recommendations can be offered according to each ENM in a general Technical Data Sheet (Fig. 5). In contrast, some other ENM products for human consumption should be labeled with periodic re-evaluations. For example, food-grade titanium dioxide additive, named E171, was approved by the FDA in 1966, but currently, oral exposure has caused controversy in the last 5 years. This additive has been produced in the nano-sized range, and scientific evidence shows that oral consumption could cause some toxicity (Borm et al., 2006; Giovanni et al., 2014; Teubl et al., 2014). In the same category, there are other additives that are currently used on the nanometric scale, including E170 (calcium carbonate), E551 (silicon dioxide) and E552 (calcium silicate) (Table 3). Ethical Considerations for ENM In terms of ethics, any person who is working with or consuming a product containing ENM has the right to know, and some angle of regulation should be directed to spread that information – for instance, an employee working with ENM or a consumer acquiring an ENM in food products or devices has the right to know that they are being exposed to an ENM, however, products or devices containing ENM are not necessarily labeled and for now, labeling is an option for the producer or the seller. Ethical concerns about the right to know that any person is being exposed or is consuming a specific product have been observed in the past. A good example is the consumption of genetically modified organisms (GMO). In Europe, there are 50 genetically modified plants approved by the European Food Safety Authority (EFSA) for commercial use (http://ec.europa.eu/food/dyna/gm_register/index_en. cfm), and this information should be explicitly labeled on products containing GMOs.

In contrast, as scientific evidence is constantly generated, periodic revision should be contemplated for compounds or additives to human foods. As an example, aspartame, which was approved by the FDA, has been considered for a re-evaluation based on the scientific evidence of its toxic effects (Chattopadhyay et al., 2014; Soffritti et al., 2014). Final Remarks The definition of ENM has been progressively refined, but a consolidated definition of the term still needs to be adopted globally. In the future, new ENM in a sub-nanometric size should be considered in the definition, as should other materials that may behave as ENM. A global database of ENM in terms of synthesis, production and handling, is urgently needed. In this regard, a registry containing information regarding the type of ENM, number of workers, work-day and work-place, among other details, could be helpful for a better estimation of human occupational exposure. Difficulties in establishing regulations are related to the pace of production of new ENM, the insufficiency of standardized in vivo and in vitro methods to measure ENM toxicity and the lack of epidemiological evidence. Importantly, ENM regulations should not be oriented to decrease ENM production, because ENM constitutes the base of nanotechnology, and their benefits to health, the environment, agriculture, informatics, physics and engineering are clearly evident. By contrast, ENM regulations should encourage the availability of information for workers, particularly in occupational settings, and for consumers using certain products. In addition, periodic evaluation of ENM safety should be considered. The concern of providing ENM information to workers and consumers arises from the right of every person who is working with or consuming ENM to know. Finally, we apologize for excluding environmental issues, which we consider critical for regulations, and also for not including nanocomposites in ENM in the analysis, for which information is still limited.

Table 3. Codex Alimentarius classification for food additives Catalogue number

Substance

Particle size

Usage

Year of adoption/ approval

E170 E171 E172 E174 E175 E551

Calcium carbonate Titanium dioxide Iron oxide and hydroxide Silver Gold Silicon dioxide

40-10,000 nm 40-220 nm Not confirmed Not confirmed Not confirmed 10-200 nm

1967a, 1981b, 2005c 1966a, 1979b, 1993d, 2007d, 2010d, 2013d 1986a, 1983b, 1987d, 2005d, 2009d 1979a Data unavailable 1981b, 2003c

E552

Calcium silicate

20-70 nm

E553a

Magnesium silicate

Not confirmed

E553b

Talc

Not confirmed

Colorant Colorant Colorant Colorant Colorant Emulsifiers, stabilizers, thickeners and gelling Emulsifiers, stabilizers, thickeners and gelling Emulsifiers, stabilizers, thickeners and gelling Emulsifiers, stabilizers, thickeners and gelling

Data unavailable 1985b, 2003c, 2004d, 2012d 1967a, 2001b, 2004d

a

Approval year from Food and Drug Administration (FDA, USA). Adoption year of Codex Alimentarius classification. c Revision for arsenic and heavy metal content. d Year of revision and reapprove of Codex Alimentarius. Codex Alimentarius classification for food additives. Some additives are used in nano-sized form and some others are not confirmed by European Food Safety Agency (EFSA). b




L. G. Garduño-Balderas et al.

Acknowledgments This work was supported by National Council of Science and Technology of Mexico (CONACyT 166727) and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT IN218015). IMUO (489350) and EIMR (576227) have fellowship from CONACyT in the PhD Program of Biomedical Sciences at UNAM.

Conflict of interest The authors did not report any conflict of interest.

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Review Article Difficulties in establishing regulations for engineered nanomaterials and considerations for policy makers: avoiding an unbalance between benefits and risks Luis Guillermo Garduño-Balderas, Ismael Manuel Urrutia-Ortega, Estefany Ingrid Medina-Reyes and Yolanda Irasema Chirino

The hazards of exposure to engineered nanomaterials (ENM) have led to some efforts to offer frameworks or guidelines to better maintain human health. Te aim o this work was to analyze the difficulties in establishing regulations for ENM, to discuss some considerations that may be helpful for policy makers and to provide suggestions for establishing normative regulations without decreasing ENM production.

Wiley Online Library Short Abstract