J Nanopart Res (2013) 15:1455 DOI 10.1007/s11051-013-1455-2
PERSPECTIVES
Reference materials and representative test materials: the nanotechnology case G. Roebben • K. Rasmussen • V. Kestens • T. P. J. Linsinger • H. Rauscher • H. Emons H. Stamm
•
Received: 11 July 2012 / Accepted: 18 January 2013 European Union 2013
Abstract An increasing number of chemical, physical and biological tests are performed on manufactured nanomaterials for scientific and regulatory purposes. Existing test guidelines and measurement methods are not always directly applicable to or relevant for nanomaterials. Therefore, it is necessary to verify the use of the existing methods with nanomaterials, thereby identifying where modifications are needed, and where new methods need to be developed and validated. Efforts for verification, development and validation of methods as well as quality assurance of (routine) test results significantly benefit from the availability of suitable test and reference materials. This paper provides an overview of the existing types of reference materials and introduces a new class of test materials for which the term ‘representative test material’ is proposed. The three generic concepts of certified reference material, reference material (non-certified) and representative test material constitute a comprehensive system of
benchmarks that can be used by all measurement and testing communities, regardless of their specific discipline. This paper illustrates this system with examples from the field of nanomaterials, including reference materials and representative test materials developed at the European Commission’s Joint Research Centre, in particular at the Institute for Reference Materials and Measurements (IRMM), and at the Institute for Health and Consumer Protection (IHCP). Keywords Nanotechnology Nanomaterial Certified reference material Representative test material
Introduction The volume of nanomaterials1 on the market is growing as the nanotechnology industry develops,
G. Roebben (&) V. Kestens T. P. J. Linsinger H. Emons Institute for Reference Materials and Measurements, Joint Research Centre of the European Commission, Retieseweg 111, 2440 Geel, Belgium e-mail:
[email protected] K. Rasmussen H. Rauscher H. Stamm Institute for Health and Consumer Protection, Joint Research Centre of the European Commission, Via E. Fermi 2749, 21027 Ispra, VA, Italy
1
Several definitions exist for the term nanomaterial (e.g. ISO 2010a). For regulatory purposes, the European Commission published in 2011 a recommendation for a definition for nanomaterials (EC 2011): ‘‘‘Nanomaterial’’ means a 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 to 100 nm’.
123
Page 2 of 13
generating an increasing number of consumer products containing nanomaterials. Nanomaterials may have properties that are different from those of the corresponding macroscale materials (if the latter exist at all). In order to better understand the properties of nanomaterials an increasing number of chemical, physical and biological tests are performed, including e.g. toxicological and ecotoxicological tests. These efforts underpin current EU consumer protection and environmental legislation, such as REACH (EC 2006), which is applicable also to nanomaterials. Manufactured nanomaterials are often particulate materials, produced for example as a powder or as a dispersion of particles in a liquid matrix. Traditional methods for the testing of chemical substances are not necessarily adequate for assessing the properties and effects of these particulate nanomaterials (OECD 2009). Whether a certain method is applicable to a specific nanomaterial must be verified and, where necessary, the method has to be modified and (re-) validated (OECD 2010). Nanotechnologies rely on the combined expertise from different scientific disciplines, several of which have developed their own terminology. In addition, regulatory authorities have developed a number of specific terms relevant, for example, for safety assessment of nanomaterials. This sometimes leads to one term having more than one meaning, or to different terms covering similar concepts (e.g. the terms ‘measurement’ and ‘test method’, see ‘‘The terms ‘testing’ and ‘measurement’’’ and ‘‘Steps in a test or measurement process’’ sections). The main body of the paper will focus on the specific aspects of benchmark materials, and definitions of other, related terms are given in the Annex to this paper, which also explains a number of the basic concepts for materials testing and measurement used in this paper. Benchmark materials are materials or substances (see also ‘‘Substances, materials and samples’’ section) used for the assessment of the applicability of existing methods to new kinds of materials, modification of existing methods, development of new methods, validation of methods and quality control of routinely used methods. Table 1 gives an overview of the requirements for these materials with respect to the homogeneity and stability of their properties and the corresponding assigned values for different aspects of method validation and quality assurance. More information on the requirements summarised in Table 1 is provided in the Annex.
123
J Nanopart Res (2013) 15:1455
This paper aims to provide more clarity on the significance and the complementarity of the different kinds of benchmark materials. It describes the main features of previously defined classes of such materials, including (certified) reference materials, and the differences between their intended uses. In addition, we introduce the term ‘representative test material’, and provide a frame for its use. Examples are drawn from the activities of the European Commission’s Joint Research Centre institutes IRMM (Institute for Reference Materials and Measurements, Geel, Belgium) and IHCP (Institute for Health and Consumer Protection, Ispra, Italy), which provide a range of materials for the development, validation and quality assurance of test methods for nanomaterials.
Reference materials Generic reference material terminology Materials used in method validation and quality control (see Table 1) have to be sufficiently homogeneous and stable, so that results obtained by testing different sub-samples, in different places, at different times and with different methods, can be meaningfully compared. For specific purposes, these materials also need to have reliable, assigned property value(s). Several scientific communities have developed their own terminology for the various kinds of materials fulfilling these requirements, which hampers mutual understanding. To remedy this situation, a dedicated ISO (International Organization for Standardization) Committee dealing with Reference Materials (ISO/REMCO) was created. To ensure that REMCO addresses all generic measurement and testing needs, across the main scientific disciplines physics, chemistry and biology, REMCO established liaisons with among others the World Health Organization (WHO), the International Organization of Legal Metrology, the Pharmacopoeia Discussion Group, the World Association of Societies of Pathology and Laboratory Medicine, the International Federation of Clinical Chemistry and Laboratory Medicine, and the International Union of Pure and Applied Chemistry (IUPAC), but also with specific ISO Technical Committees, such as ISO/TC 229 Nanotechnologies. ISO/REMCO introduced the following generic reference material definition (ISO 2008a):
Known between-sample heterogeneity, negligible heterogeneity for all subsamples of a specific sample
Negligible heterogeneity for all (sub-) samples used in one assessment
Negligible heterogeneity for all (sub-) samples used in one assessment
Known between-sample heterogeneity, negligible heterogeneity for all subsamples of a specific sample
Negligible heterogeneity for all (sub-) samples used in one assessment
Known between-sample heterogeneity, negligible heterogeneity of all (sub)samples of a specific sample
Calibration (see annex, ‘‘Calibration’’ section)
Assessment of robustness (see annex, ‘‘Method validation’’ section)
Assessment of precision (see annex, ‘‘Method validation’’ section)
Assessment of trueness (see annex, ‘‘Method validation’’ section)
Quality control (see annex, ‘‘Quality control of methods’’ section)
Interlaboratory comparison (ILC) (see annex, ‘‘Interlaboratory comparisons’’ section)
Homogeneity
For the time of the study (weeks)
For the time of use (months-years)
From value assignment until testing (hours to months or even years)
For the time of the study (typically weeks)
For the time of the study (typically days)
From value assignment until measurement (hours to months or even years)
Stability
Table 1 Technical requirements of materials for method validation and quality assurance
Preferably assigned independently from the ILC, but usually obtained as an average of the results from (selected) ILCparticipants
Value derived from average of several tests sufficient
Value with metrological traceability required
Not required—relative assessment only
Not required—relative assessment only
Value with metrological traceability required
Assigned values
Proficiency test (PT) material
(Laboratory) control material, inhouse reference material, quality control material, positive/negative control
Certified reference material, reference standard
Quality control Material
Calibrant, standard, reference substance, reference chemical, reference item
Frequently used names
J Nanopart Res (2013) 15:1455 Page 3 of 13
123
Page 4 of 13
A reference material is a ‘material, sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process’. Homogeneity and stability must be determined for each of the measurands (the quantities intended to be measured), corresponding with the properties of interest, and a reference material (RM) is a RM only for these specified properties. RMs produced and used applying the conditions and terms described in ISO Guide 34 meet these requirements (ISO 2009). As indicated in Table 1, materials with a known property value are indeed required for several calibration, validation and quality assurance applications. ISO/REMCO is promoting the term ‘certified reference material’ for these materials and defines it as follows (ISO 2008a): A certified reference material is a reference material characterized by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability. The basic difference between a reference material (RM) and a certified reference material (CRM) is the status of the property values assigned to the material. Metrologically valid procedures for the production and certification of reference materials can be found for instance in ISO Guides 34 and 35 (ISO 2009, 2006). CRMs are cornerstones of modern quality assurance because they allow calibration of instruments and quality control of methods and laboratories based on metrological traceability (JCGM 2008), and therefore ensure comparability of test results (Emons et al. 2004). Synonyms for reference materials The terms ‘control material’ as defined by IUPAC (IUPAC 1995, 2012), ‘in-house reference material’ as defined by the United Nations Office on Drugs and Crime (UNODC 2009), as well as the term ‘quality control material (QCM)’ (Emons 2006), are application-specific terms for subgroups of reference materials (Emons et al. 2004).
123
J Nanopart Res (2013) 15:1455
The OECD test guidelines use the terms reference substance and reference chemical which sound very similar to the term reference material. OECD reference substances and reference chemicals are used in tests where observed effects of an unknown substance are compared to known effects of a known substance in biological test systems (see OECD Guidance Document 34, OECD 2005). Since reference substances are chemicals for which responses in the selected biological test system are already known, they can be used in validation processes. This type of application of a material is similar to the use made of certified reference materials. According to the Mutual Acceptance of Data (MAD) agreement established for OECD test guidelines, new tests should be performed applying Good Laboratory Practice (GLP). GLP uses the terms reference item (also called control item) and test item. The latter means an object (substance) that is the subject of a study, whereas the former means any object (substance) used to provide a basis for comparison with the test item (EC 2004). When samples of a reference substance or chemical are used as reference test items, principles of OECD Guidance Document 34 and GLP apply in a manner similar to the ISO Guides for reference materials mentioned above. The most useful reference substances meet all technical requirements of a CRM (see Table 1 and see annex). Usually (eco) toxicological tests do not use formally certified reference substances, which would have to be accompanied by a certificate carrying all relevant information. The outcome of (eco) toxicological tests depends strongly on many factors such as the selected species, intra-species variations (individuals in any population have different susceptibilities), and the test method applied. It could be difficult to summarise these in a certification process in a cost-effective way. On the other hand, it is often the case that (eco) toxicological effects of a certain dose of material depend primarily on the chemical composition of the material. In this case, the reference character of a reference substance is sufficiently defined by its chemical composition, including purity and impurities, or by its activity in a well-defined test system and a fine chemical with a known composition (including impurities) may be used as reference material. In the specific case of nanomaterials, knowledge of only the elemental or molecular composition may be
J Nanopart Res (2013) 15:1455
insufficient for use as a reference substance in safety assessment, as nanomaterials may have (eco) toxicological and other properties that depend also on their size, shape, surface area, etc. The variations in these properties could have a larger influence on the test results than variations in the elemental or molecular composition within or between batches or between producers of nominally the same material (see also ‘‘Need for a new term’’ section). Reference materials to assure metrological traceability in nanomaterial testing Assuring the quality of testing of nanomaterials requires the same type of reference materials as the ones discussed in ‘‘Generic reference material terminology’’ section. One important feature is the metrological traceability of test results obtained on nanomaterials (see also Annex, ‘‘Metrological traceability’’ section). For nanomaterials, lists of endpoints (properties), relevant for their safety assessment, are being established, for example by the OECD Working Party on Manufactured Nanomaterials (WPMN) (OECD 2008) and in ISO/TC 229 (Nanotechnologies) Working Group 3 (Health, Safety and Environmental Aspects of Nanotechnologies) (ISO 2011). Many of the listed endpoints, both physico-chemical and (eco) toxicological ones, are method-defined: the value of the property measured is determined by how (by which method) it is measured (Roebben et al. 2010). One example is the size of a particle. For regularly shaped particles, one value (e.g. the diameter of a sphere) or a few values (e.g. the length, width and height of a cuboid) can define the particle size. However, for particles with irregular shapes the so-called apparent size values are obtained, depending on how the particle size is measured and the measurement is evaluated. In practice, size measurements are often indirect measurements (e.g. of a sedimentation or diffusion rate), and therefore, even for near-spherical particles, the result of the measurement depends on the applied method. An example is ERM-FD100 (Braun et al. 2011), a CRM for which four different particle size values are certified, each corresponding to a different measurement technique (electron microscopy (EM), centrifugal liquid sedimentation (CLS), small-angle X-ray scattering (SAXS) and dynamic light scattering
Page 5 of 13
(DLS)). For each of these methods, the interlaboratory study to characterise ERM-FD100 has demonstrated that the particle size can be reproducibly determined in different laboratories, if the laboratories follow the agreed measurement procedure, including a correct calibration of the instrument. The method-defined nature of the certified property values has important implications for the use of test results, as it is in principle impossible to convert results from one method to another, e.g. convert size as measured by DLS into size as measured by SAXS. However, the added value of this CRM, ERM-FD100, is that it provides the tool that laboratories need to ensure that the results of their DLS, CLS, EM and SAXS measurements of (near-) spherical nanoparticles are traceable to the SI unit metre, and thereby comparable with other results obtained with the same methods. More recently, IRMM released ERM-FD304 (Franks et al. 2012), a colloidal silica with monomodal size distribution, alike ERM-FD100, but with a higher polydispersity than ERM-FD100. Due to the higher polydispersity the differences between the certified values for different particle size methods are more pronounced for ERM-FD304 than for ERM-FD100.
Representative test materials Need for a new term The two terms ‘reference material’ and ‘certified reference material’ cover all uses mentioned in Table 1. This means that the materials needed for calibration, method validation and quality control must fulfil the requirements for RMs or even for CRMs. The high number of method-defined material properties (e.g. in the field of nanotechnology) implies that there is an equally large need for reference materials. However, reference materials are scarce, even in well-established fields like particle size analysis (Linsinger et al. 2011), whereas for many other measurands no reference materials exist at all. Indeed, the decision to develop a reference material is not taken lightly, largely due to the considerable resources needed to produce a reference material. Such decision depends on the size of the possible user community and on the consensus in this user community about the type of material needed and on the parameters to be assigned or certified. Therefore, it is
123
Page 6 of 13
J Nanopart Res (2013) 15:1455
current praxis in all technologies (including nanotechnology) that analysts use materials other than reference materials as a provisional benchmark, e.g. awaiting additional testing to ensure that the candidate reference materials and candidate standard methods meet the necessary requirements. We propose to give these materials their own name and status and call them representative test materials, with the following definition: A representative test material (RTM) is a material from a single batch, which is sufficiently homogeneous and stable with respect to one or more specified properties, and which implicitly is assumed to be fit for its intended use in the development of test methods which target properties other than the properties for which homogeneity and stability have been demonstrated. A RTM is not a reference material for the tests for which it is intended to be used, because homogeneity and stability are not demonstrated for the corresponding measurand. An RM can therefore be an RTM if it is used outside the range of properties for which it is an RM. In any case, an RTM is more valuable than an ordinary test material, since it has been checked for homogeneity and stability in terms of one or more specified properties. RTMs are extremely useful tools in intra- or interlaboratory development of methods for which reference materials are not (yet) available. Table 2 compares the essential characteristics of representative test materials with those of certified and non-certified reference materials. As seen from table 2, it is assumed for RMs as well as RTMs that the material is representative for the class of materials that are or will be subject to testing with a selected method. For the RMs, homogeneity and stability are assessed and demonstrated for the measurands corresponding
with the method that will be evaluated with the reference material. In the case of RTMs, homogeneity and stability for the measurands of interest are implicitly assumed based on data from other methods. The authors have proposed the term RTM for use in a document being prepared by ISO/TC 229 (Nanotechnologies), where it is being discussed in the Joint Working Group 2 Measurement and Characterisation (ISO/AWI TS 16195), but the concept is not unique to nanomaterials. An example of a material that corresponds with the RTM concept is the IRMM-481 Peanut Test Material Kit (IRMM 2005), which consists of carefully prepared peanut powders, with a homogeneous particle size and amount per vial. This material was developed for use in the research to identify and quantify allergenic proteins in peanut matrices (Scaravelli et al. 2009). The product information sheet (IRMM 2005) clearly states that the material is not a reference material, but it does not give an alternative denomination. Representative test materials for nanomaterials testing Obstacles to the production of reference nanomaterials There is a lack of comparability of reported results on nanomaterials which have the same elemental composition (silver nanoparticles, carbon nanotubes, colloidal silica, etc.). Clearly, a nominal or purely elemental composition, without structural and other characteristics, is not sufficient to fully describe materials, let alone nanomaterials (Warheit 2010; Johnston et al. 2009, 2010a, b). For example, the surface structure of nanoparticles may be affected by a different history of environments in which the
Table 2 Essential characteristics of the new concept ‘representative test material’ compared to the existing concepts of reference material and certified reference material Representative test material
Reference material Not certified
Certified
Parent material
Representative for class of materials to be investigated with the target method(s)
Homogeneity/ stability
Assumed for the measurands of interest, demonstrated for other measurands
Demonstrated for the measurands of interest
Demonstrated for the measurands of interest
Assigned property value
None
None, or indicative only.
Certified for the measurand of interest
123
J Nanopart Res (2013) 15:1455
material has been; or the particle size distribution of a particulate nanomaterial may be different from one batch to another. It has e.g. been recognised for carbon nanotubes (CNT) that certain aspect ratios could give the CNT asbestos-like properties, which are absent for other aspect ratios (Poland et al. 2008). Related to this last example, there are a number of open questions related to hazard assessment (Aschberger et al. 2011) regarding for example (1) which parameters should be tested for nanomaterials, (2) how to express the doses for the effects data (e.g. in terms of total mass or mass fraction, particle number, specific surface area), (3) which methods and test guidelines to use, (4) and which test conditions to use. Since there is no agreement yet on which measurands would be the most relevant ones, development of standardised methods and corresponding reference materials is hampered. As a result, only few reference nanomaterials have been produced (Roebben et al. 2011) other than some reference materials for particle size (NIST 2011; Linsinger et al. 2011). In the absence of a clear understanding and agreement on which would be the relevant characteristics of a suitable reference nanomaterial, and on which methods to use for its characterisation, representative test materials will have to suffice.
Representative test materials for OECD WPMN In order to assess the relevance and reliability of applying existing OECD test guidelines, which were developed for the testing of the safety of chemicals, to nanomaterials, the OECD WPMN initiated a vast collaborative project, called ‘OECD WPMN Safety Testing of a Set of Representative Manufactured Nanomaterials’. In this project, a set of data will be generated for 13 commercially important nanomaterials: carbon nanotubes (single wall and multiwall), fullerenes, dendrimers, nanoclays and nanoparticles of aluminium oxide, silver, titanium dioxide, cerium dioxide, zinc oxide, silicon dioxide, iron and gold. The collective development of data in the first phase will support evaluation of the methods’ applicability to nanomaterials for physico-chemical characterisation and (eco) toxicological testing. The tests performed will be the basis for discussions on if and how existing OECD methods should be adapted to nanomaterials and the need to develop new methods. The Guidance
Page 7 of 13
Manual for Sponsors (OECD 2010) describes in detail how to address each endpoint agreed for the project. The concept of ‘representative test materials’ was implicitly adopted by this testing programme by recognising that using a common source of nanomaterials would exclude one of the main variables in (eco) toxicity testing of nanomaterials performed by different laboratories. Furthermore, by making these nanomaterials available for testing also in additional projects, a knowledge base on those materials is being built, paving the way for using them later to better correlate different properties and effects of the tested nanomaterials. In order to assist the testing under the OECD WPMN, the IHCP set up the JRC repository of representative nanomaterials. The nanomaterials in the NM-series are made from portions of industrial materials, produced to commercial specifications, and each series comes from the same production batch. The further processing of the industrially sourced base materials into the NM-series of materials ensures that the material has been homogenised, before being subsampled into vials, under GLP conditions. For example, the between-vial heterogeneity was explicitly assessed in terms of the primary particle size for the zinc oxide materials NM-110 and NM-111, which are dry powders (Singh et al. 2011). Based on the evaluation of SEM measurements, the materials can be considered as homogeneous (between-bottle standard deviation less than 4 %) with regard to the Feret’s diameter of the primary particles. Also, the stability of the sub-sampled materials is monitored. Another example is a colloidal dispersion of silver nanoparticles (NM-300). The amount of silver (measured with UV–vis), the number of particles and the size distribution for samples before storage and after 12 months of storage are statistically not different, demonstrating the stability of this aspect of the NM-300 material under the specified storage condition (Klein et al. 2011). Thus, with homogeneity and stability being assessed and monitored for at least one relevant material property, all vials from the NM material are similar in terms of the monitored parameter. Having shown that the material is homogeneous and stable for a certain parameter, the RTM concept assumes that this holds true also for other properties. As a consequence, if betweenvial differences in test results are obtained with other methods or for other properties, they should not be attributed lightly to the heterogeneity of the test material. This is the essence of the RTM concept.
123
Page 8 of 13
The JRC nanomaterials repository hosts many of the nanomaterials which are tested within the OECD WPMN and provides them to the participants wishing to investigate one or more aspects of the materials (IHCP 2011). Currently, the repository hosts multiwalled carbon nanotubes (4 types), nanoclay (1 type) and nanoparticles of silver (1 type, two batches), titanium dioxide (6 types), cerium dioxide (2 types), zinc oxide (2 types) and silicon dioxide (5 types). The nanomaterials can be provided to researchers looking for materials, which are well characterised and for which tests are being performed for endpoints potentially relevant also in a regulatory context. The knowledge base on these nanoscale representative test materials is increasing through the tests performed. It is conceivable that, for a number of the assessed properties, materials from the NM-series may become reference materials. The reference material status may include selected (eco) toxicological endpoints, an area where RMs have not been developed in any significant number.
Summary The current paper clarifies the meaning of three categories of test materials: reference material (RM), certified reference material (CRM), and representative test material (RTM). The new term RTM is put forward in this paper to close a gap in existing terminology. RTM is proposed to cover those materials that are used as a benchmark, for example in inter- or intralaboratory development of new or modified methods, for which the homogeneity and stability studies required for a reference material cannot be or have not yet been performed. These three types of materials (RTM, RM, CRM) cover the whole range of test materials typically needed in the development, validation and routine use of measurement and testing methods. The JRC is actively involved in work related to all three types of materials in the field of nanotechnology, as the IHCP is providing representative test materials to the OECD WPMN and the IRMM is producing both certified and non-certified reference materials for nanotechnology. Thus, the two institutes contribute to providing the measurement and testing community with the means to ensure the reliability of test results on nanomaterials. Acknowledgments The authors wish to thank Juan Riego Sintes (JRC-IHCP) for valuable comments.
123
J Nanopart Res (2013) 15:1455
Annex Basic concepts for measurement and testing and related quality assurance A number of basic measurement and testing concepts are explained in this annex. This will help the reader of the paper to better understand some of the terms used in the main body of the text. The authors also wanted to illustrate the similarities between the concepts behind the different terms used in different fields of measurement and (regulatory) testing. The terms ‘testing’ and ‘measurement’ Two fundamental concepts in this article are ‘test’ and ‘measurement’, which may have different connotations in different scientific disciplines. For regulatory testing, the OECD guidance document 34 (OECD 2005) defines a test method as (p. 17): ‘… an experimental system that can be used to obtain a range of information from chemical properties through the adverse effects of a substance. The term ‘test method’ may be used interchangeably with ‘assay’ for ecotoxicity as well as for human health studies. …’. Testing means applying a test method. Measurement is defined (JCGM 2008) as the process of experimentally obtaining one or more quantity values (number and reference together expressing magnitude) of a quantity, the latter being a property of a phenomenon, body, or substance, where the property has a magnitude that can be expressed as a number and a reference. The science of measurement and its application is called metrology. When speaking in the most general sense, there are tests that are not usually seen as measurements, and measurements that are not usually called tests. However, the basic elements of the above definitions are highly similar. Substances, materials and samples The term substance generally describes a chemical element or compound. This paper uses the REACH (EC 2006) definition of substance: In the EU regulatory context (EC 1967), now part of REACH, the term ‘substance’ has a more specific meaning: substance means ‘a chemical element and its compounds in the natural state or
J Nanopart Res (2013) 15:1455
obtained by any manufacturing process, including any additive necessary to preserve its stability and any impurity deriving from the process used, but excluding any solvent which may be separated without affecting the stability of the substance or changing its composition’. Hence, REACH substances may consist of one or more chemical compounds, and guidance is found on http://echa.europa.eu. There is no unique definition of the term material. Its exact meaning varies between different scientific fields or industrial sectors (ISO 2012), but most of the existing ISO definitions [for example in ISO 1182:2010 (ISO 2010b)] describe a material as a single substance or a uniformly dispersed mixture of substances. This definition implies that the term material has a broader coverage than the term substance as defined in REACH. Examples of materials that are not typically called substance are concrete, timber, stone, milk powder, etc. Nevertheless, as REACH addresses all forms of substances placed on the market, materials, including nanomaterials, are covered by REACH. Both materials and substances may be quite complex in composition, including for example impurities and necessary stabilisers and may consist of multiple phases or components with a certain variability in composition. In any case, material and substance are collective terms for kinds of matter with properties that are considered uniform above a certain scale. A relevant question to ask is when substances or materials produced as different batches, or in different places, or by different processes can be considered as identical, equivalent or similar. The answer depends amongst others on the acceptable heterogeneity, for example in the variation of property values between different batches, as well as on the context, which for example may relate to specific legal requirements. To assess the heterogeneity within or between batches, tests need to be performed on samples of the material(s): a sample is a specific, unique portion of material or substance selected from a larger amount of parent material. A representative sample is a sample that reflects the average property of the parent material. The terms test sample, test material, and test substance are straightforward combined terms, to be interpreted in accordance with the above understanding of the terms sample, material, or substance. Hence, a test material or test substance is a material or
Page 9 of 13
substance to be tested. A test sample is a portion of a test material or test substance. The terms test item [see e.g. Good Laboratory Practice (GLP) (OECD 1998)], test piece [see e.g. ISO 148–2 (ISO 2008b)] and test specimen are used synonymously, and have the same meaning as test sample. Steps in a test or measurement process On the most generic level, any test or measurement consists of the following steps: a)
A representative sample is taken from the material to be tested and often undergoes pre-treatment (dispersion, dissolution, digestion, etc.). b) The test sample is fed into a test system where it creates a response. A wide range of test systems exists, for example physical instruments (electron microscopes, etc.) or biological systems (microbiological assays for antibiotics, rats for toxicity testing, etc.). The nature of the response can be chemical (e.g. production of a precipitate), physical (e.g. absorption of light of a certain wavelength) or biological (e.g. change of growth of bacteria in the presence of the sample). c) The response is expressed in quantitative terms (e.g. mass of precipitate is 1.245 g) or in qualitative terms (e.g. there is a blue precipitate). d) The response is compared with the response of a (sample of a) second material, with known properties and response, either in a quantitative or in a qualitative way. As the discussion below shows, numerous terms like ‘calibrant’, ‘calibrator’, ‘standard’, ‘reference substance’, ‘reference chemical’ or ‘certified reference material’ and ‘control’ exist for this second sample (Emons et al. 2004), and possibly each term has a slightly different meaning in its context of origin. This comparison is an important part of the metrological traceability chain of the test result (see ‘‘Metrological traceability’’ section). In addition to this generic procedure, there are of course features which are particular to the test system (e.g. higher variability in observed responses when using biological test systems as compared to physical test systems). Also in these cases, the above generic procedure would still provide a descriptive framework.
123
Page 10 of 13
J Nanopart Res (2013) 15:1455
Metrological traceability
Calibration
A key concept in any testing is ‘metrological traceability’, defined by the Joint Committee for Guides in Metrology (JCGM 2008) as ‘property of a measurement result whereby the result can be related to a reference through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty’. The level of traceability establishes the degree of comparability of the measurement for example over time and geographically: whether the result of a measurement can be compared to the previous one, to a measurement result from a year ago, or to the result of a measurement performed anywhere else in the world. Metrological traceability includes the identity of the measurand (the property to be measured) and its quantity value. The identity of the measurand can be structurally defined (such as mass, or bond lengths in a specific molecule) or procedurally defined. Examples of test procedures defining the measurand are the mass fraction of dietary fibre in fruits, enzyme activity or skin sensitization according to OECD Test Guideline 429, where the measurand is the proliferation of lymphocytes in the lymph node in test groups of animals exposed to the test substance. This is compared to the proliferation of lymphocytes in the lymph node in test groups of animals which are vehicle treated controls, and gives a Stimulation Index. Traceability of the quantity value is defined by the calibration system. In its simplest form, an international agreement has been reached to use a certain material as standard, and a well-known example is the international kilogram stored in Paris, but also many primary WHO preparations (WHO 2011) fall under this category.
Calibration is the process where the response of the calibrant or reference substance is related to its property value. The curve of the known property values of a set of calibrants versus responses of the test system allows assigning a quantity value to other substances measured thereafter. A first key requirement is that the calibrant or reference substance is homogeneous and stable to ensure that any portion of it gives the same result. A second key requirement is that the value of the measurand of interest is well established. The value of the measurand can for example be a concentration or an antibiotic potency. The measurement unit does not need to be a physical unit, and for example, the result of a test may be that a substance A is ‘x times more potent than reference substance B’. Often, calibrants or reference substances are idealised samples, for example consisting of a single chemical compound. For (eco-) toxicological testing (using for example the OECD test guidelines), the tests may not have designs that lend themselves to classical calibration, and the term ‘calibrant/calibration’ is not used.
Quality assurance of test and measurement methods In order to ensure that the testing is as reliable as required by the user of the test results, methods are validated, calibrated and subjected to continuous quality control, as relevant. The main aspects of such quality assurance efforts, as well as the requirements of the corresponding benchmark materials, are described below.
123
Method validation For (eco-) toxicological testing, the OECD Guidance Document 34 (OECD 2005) defines test method validation as ‘… a process based on scientifically sound principles by which the reliability and relevance of a particular test, approach, method or process are established for a specific purpose. Reliability is defined as the extent of reproducibility of results from a test within and among laboratories over time, when performed using the same standardised protocol. The relevance of a test method describes the relationship between the test and the effect in the target species and whether the test method is meaningful and useful for a defined purpose, with the limitations identified. In brief, it is the extent to which the test method correctly measures or predicts the (biological) effect of interest, as appropriate. Regulatory need, usefulness and limitations of the test method are aspects of its relevance. New and updated test methods need to be both reliable and relevant, i.e. validated’. Validation of regulatory test methods, for example within the OECD, takes place once to ensure that the methods are reliable and relevant for the endpoint and
J Nanopart Res (2013) 15:1455
that the method is recognised within the relevant jurisdiction. Method validation has also a more generic meaning: laboratories validate test methods, i.e. they verify whether the specified method is adequate for an intended use. According to ISO and others, method validation requires materials to test robustness, precision and trueness. One can note that robustness and precision are the equivalent of the term reliability used in OECD (OECD 2005), and that trueness is conceptually similar to the term relevance in OECD (OECD 2005). Testing of robustness comprises including slight variations in the test protocol and observing the effect of this on the outcome of the test. The materials used for this test should resemble the actual test samples as closely as possible. In addition, the material needs to be stable over the time of the study and must be sufficiently homogeneous, so that a variation of results, if detected, indeed reflects the effect of the variations in the test protocol and not sample heterogeneity. In a precision study, tests are performed on different subsamples of the same material to investigate the test result variation within one test series in the same laboratory (repeatability), between series in the same laboratory (intermediate precision) and between laboratories (reproducibility). Also here, the sample should closely resemble actual test samples in composition and response. To make the results meaningful, the material chosen must be stable over the time of the study and must be homogeneous to assure that observed variations reflect the variability of the method in or between laboratories, and not heterogeneity or changes of the material. The material tested does not need to be accompanied by beforehand well-established quantity values of the measurands of interest as the goal is a relative assessment. In a trueness experiment, the response of the test system to a sample is compared to an assigned or expected response to this sample, and this necessarily requires a well-established value of the measurand in question. The material used to check trueness must be stable until the time of use and sufficiently homogeneous to ensure that different portions give the same result. Typically, a material for a trueness experiment is more complex or resembling more the real-life materials to be tested later, than a material used for calibration. The reason for this is that the trueness
Page 11 of 13
experiment must not only cover the signal-producing step in the measurement process, but also all sample preparation steps. Quality control of methods For quality control of a routinely used method, a known sample is analysed periodically or as part of every test series and results are compared with target values. If the result is within specified limits, the method is under control. The material used must be homogeneous, so that heterogeneity does not contribute to the variation of results. The material must also be stable, often over months or even years, as otherwise degradation would make comparison of results obtained over time invalid. Target values and acceptance limits are usually obtained from repeated tests. Interlaboratory comparisons Interlaboratory comparisons are faced with the same issues as interlaboratory method validation: A homogeneous set of samples, which is stable over the duration of the period of comparison, is required to ensure that any difference between participants’ test results are not due to changes in the inherent material properties or due to differences between the distributed samples.
References Aschberger K, Micheletti C, Sokull-Klu¨ttgen B, Christensen FM (2011) Analysis of currently available data for characterising the risk of engineered nanomaterials to the environment and human health—lessons learned from four case studies. Environ Int 37:1143–1156 Braun A, Franks K, Kestens V, Roebben G, Lamberty A, Linsinger TPJ (2011) Certification of equivalent spherical diameters of silica nanoparticles in water, certified reference material ERM-FD100, EUR 24620 EN, ISBN 978-92-79-18676-9. Publications Office of the European Union, Luxembourg EC (European Commission) (1967) Council Directive 67/548/ EEC of 27 June 1967 on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. OJ L 196, 16.8.1967, 1–98 EC (European Commission) (2004) Directive 2004/10/EC of the European Parliament and of the Council of 11 February
123
Page 12 of 13 2004 on the harmonisation of laws, regulations and administrative provisions relating to the application of the principles of good laboratory practice and the verification of their applications for tests on chemical substances. OJ L 50, 20.2.2004, p. 44–59 EC (European Commission) (2006) Regulation (EC) No 1907/ 2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/ EC and 2000/21/EC. OJ L 396, 30.12.2006, 1–849 EC (European Commission) (2011) Commission Recommendation of 18 October 2011 on the definition of nanomaterial (2011/696/EU). OJ L 275, 20.10.2011, 38–40 Emons H (2006) The ‘RM family’—identification of all its members. Accred Qual Assur 10:690–691 Emons H, Linsinger TPJ, Gawlik BM (2004) Reference materials: terminology and use. Can’t one see the forest for the trees? Trends Anal Chem 23:442–449 Franks K, Braun A, Charoud-Got J, Couteau O, Kestens V, Lamberty A, Linsinger TPJ, Roebben G (2012) Certification of the equivalent spherical diameters of silica nanoparticles in aqueous solution, certified reference material ERM-FD304, EUR 25018 EN, ISBN 978-92-79-218675. Publications Office of the European Union, Luxembourg IHCP (Institute for Health and Consumer Protection) (2011) http://ihcp.jrc.ec.europa.eu/our_activities/nanotechnology/ nanomaterials-repository/list_materials_JRC_rep_oct_ 2011.pdf IRMM (Institute for Reference Materials and Measurements) (2005) IRMM-481 Product Information Sheet, December 2005, http://irmm.jrc.ec.europa.eu/Pages/rmcatalogue. aspx ISO (International Organization for Standardization) (2006) ISO Guide 35, reference materials—General and statistical principles for certification. ISO, Geneva ISO (International Organization for Standardization) (2008a) ISO Guide 30:1992/Amd.1:2008, Terms and definitions used in connection with reference materials. ISO, Geneva ISO (International Organization for Standardization) (2008b) ISO 148-2:2008 Metallic materials, Charpy pendulum impact test, Part 2: Verification of testing machines, ISO, Geneva ISO (International Organization for Standardization) (2009) ISO Guide 34. General requirements for the competence of reference material producers, ISO, Geneva ISO (International Organization for Standardization) (2010a) ISO/TS 80004–1:2010 Nanotechnologies—Vocabulary— Part 1: Core terms. ISO, Geneva ISO (International Organization for Standardization) (2010b) ISO 1182:2010 Reaction to fire tests for products. Noncombustibility test, ISO, Geneva ISO (International Organization for Standardization) (2011) ISO/DTR 13014, Nanotechnologies, Guidance on physicochemical characterization for manufactured nano-objects submitted for toxicological testing, (approved for publication) ISO, Geneva
123
J Nanopart Res (2013) 15:1455 ISO (International Organization for Standardization) (2012) ISO Concept Database, http://cdb.iso.org, accessed 11 January 2012 IUPAC (International Union for Pure and Applied Chemistry) (1995) Harmonized guidelines for internal quality control in analytical chemistry laboratories. Pure Appl Chem 67:649–666 IUPAC (International Union for Pure and Applied Chemistry) (2012) IUPAC compendium of chemical terminology—the gold book, http://goldbook.iupac.org/ Accessed 11 January 2012 JCGM (Joint Committee for Guides in Metrology) (2008). International vocabulary of metrology—Basic and general concepts and associated terms (VIM), 3rd Edition, JCGM 200:2008, Sevres, France, 2008. http://www.bipm.org/ utils/common/documents/jcgm/JCGM_200_2008.pdf Johnston HJ, Hutchison GR, Christensen FM, Peters S, Hankin S, Stone V (2009) Identification of the mechanisms that drive the toxicity of TiO2 particulates; the contribution of physicochemical characteristics. Part Fibre Toxicol 6:33. doi:10.1186/1743-8977-6-33 Johnston HJ, Hutchison GR, Christensen FM, Peters S, Hankin S, Aschberger K, Stone V (2010a) A critical review of the biological mechanisms underlying the in vivo and in vitro toxicity of carbon nanotubes: the contribution of physicochemical characteristics. Nanotoxicology 4:207–246 Johnston HJ, Hutchison GR, Christensen FM, Peters S, Hankin S, Stone V (2010b) A review of the in vivo and in vitro toxicity of silver and gold particulates: particle attributes and biological mechanisms responsible for the observed toxicity. Crit Rev Toxicol 40:328–346 Klein CL, Comero S, Stahlmecke B, Romazanov J, Kuhlbusch TAJ, Van Doren E, De Temmerman PJ, Mast J, Wick P, Krug H, Locoro G, Hund-Rinke K, Ko¨rdel W, Friedrichs S, Maier G, Werner J, Linsinger T and Gawlik BM. (2011). NM-Series of Representative Manufactured Nanomaterials, NM-300 Silver, Characterisation, Stability, Homogeneity. EUR 24693 EN. ISBN 978-92-79-19068-1 Linsinger TPJ, Roebben G, Solans C, Ramsch R (2011) Reference materials for measuring the size of nanoparticles. Trends Anal Chem 30:18–27 NIST (National Institute for Standardisation) (2011) NIST releases First Certified Reference Material for Single-Wall Carbon Nanotubes, http://www.nist.gov/mml/polymers/ cnt-122011.cfm OECD (Organisation for Economic Co-operation and Development) (1998) OECD Principles on Good Laboratory Practice, in OECD Series on principles of Good Laboratory Practice and Compliance Monitoring, Number 1 ENV/MC/ CHEM(98)17 OECD (Organisation for Economic Co-operation and Development) (2005) Guidance Document on the Validation and International Acceptance of new or Updated Test Methods for Hazard Assessment, ENV/JM/MONO(2005)14, in OECD Series on Testing and Assessment, Number 34 OECD (Organisation for Economic Co-operation and Development) (2008) OECD-WPMN, Series on the Safety of Manufactured Nanomaterials, Number 6, List of manufactured nanomaterials and list of endpoints for phase one of the OECD testing programme, ENV/JM/MONO [2008]13 (2008)
J Nanopart Res (2013) 15:1455 OECD (Organisation for Economic Co-operation and Development) (2009) Preliminary Review of OECD Test Guidelines for their Applicability to Manufactured Nanomaterials. ENV/JM/MONO(2009)21 OECD (Organisation for Economic Co-operation and Development) (2010) Guidance Manual for the Testing of Manufactured Nanomaterials: OECD’s Sponsorship Programme; first revision. ENV/JM/MONO(2009)20/REV Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WAH, Seaton A, Stone V, Brown S, Macnee W, Donaldson K (2008) Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol 3:423–428 Roebben G, Linsinger TPJ, Lamberty A, Emons H (2010) Metrological traceability of the measured values of properties of engineering materials. Metrologia 47:S23–S31 Roebben G, Emons H, Reiners G (2011), Nanoscale Reference Materials, in Nanotechnology Standards, eds. V. Murashov, J. Howard, Springer Science?Business Media, New York/Heidelberg, pp 53–75 Scaravelli E, Brohe´e M, Marchelli R, van Hengel AJ (2009) The effect of heat treatment on the detection of peanut allergens
Page 13 of 13 as determined by ELISA and real-time PCR. Anal Bioanal Chem 395:127–137 Singh C, Friedrichs S, Levin M, Birkedal R, Jensen KA, Pojana G, Wohlleben W, Schulte S, Wiench K, Turney T, Koulaeva O, Marshall D, Hund-Rinke K, Ko¨rdel W, Van Doren E, De Temmerman PJ, Abi Daoud Francisco M, Mast J, Gibson N, Koeber R, Linsinger T, Klein CL. (2011), NMSeries of Representative Manufactured Nanomaterials, Zinc Oxide NM-110, NM-111, NM-112, NM-113, Characterisation and Test Item Preparation. EUR 25066 EN, ISBN 978-92-79-22215-3 UNODC (United Nations Office on Drugs and Crime) (2009) Glossary of terms for quality assurance and good laboratory practices, ST/NAR/26/Rev.1 Warheit DB (2010) Debunking some misconceptions about nanotoxicology. Nano Lett 10:4777–4782 WHO (World Health Organisation) International Biological reference Preparations, Geneva (2011), catalogue downloadable from http://www.who.int/bloodproducts/ref_ materials/
123
