EFfCI Guidance Paper on Nanomaterials in Cosmetics Practical Recommendation for Particle Size Assessment

Transcription

1 15 July 2015 EFfCI Guidance Paper on Nanomaterials in Cosmetics Practical Recommendation for Particle Size Assessment 1. Introduction and Purpose 2 2. Definitions Definition of Nanomaterial Nanomaterial (Cosmetics Regulation (EC) No 1223/2009) Nanomaterial (EU Commission Recommendation 2011) Definition of Specific Terms proposed by EFfCI Insoluble Biopersistent Intentionally Manufactured Internal Structure 4 3. Decision Tree 4 4. Particle Sizing Methods General Remarks Sample Preparation Details on Selected Analytical Methods Obtaining a Number Weighted Particle Size Distribution Electron Microscopy (EM) Transmission Electron Microscopy (TEM) Scanning Electron Microscopy (SEM) Laser Diffraction (LD) Dynamic Light Scattering (DLS) Centrifugal Liquid Separation (CLS) Volume Specific Surface Area (VSSA) Assessed by the Brunauer, Emmet & Teller Method (BET) X-ray diffraction (XRD) Conclusion Glossary/Abbreviations Literature/References 17 1/18

2 1. Introduction and Purpose Within the last years several definitions of nanomaterials have been published showing slight differences concerning its scope 1. A common problem of all these definitions is the lack of analytical methods linked to the particle size distribution and other parameters included in each definition. Therefore in practice industry is free to use whatever method appears suitable to determine the particle size distribution of a substance. The differences between and within methods often lead to diverging results whether a substance is considered a nanomaterial or not. This means that the classification of a substance even under one definition depends strongly on the analytical method used for particle size distribution measurement and the sample preparation method. This can lead to diverging conclusions concerning regulatory status and compliance issues. The purpose of the Guidance Paper is to suggest appropriate analytical methods to decide whether the substance has to be considered as a nanomaterial acc. to the definition of the Cosmetics Regulation (EC) No. 1223/2009. The focus is on methods, which are readily available, easily applied and provide comparable and reproducible results. This EFfCI Guidance Paper is suggested as a recommendation for member companies and may be used as a reference. 2. Definitions 2.1. Definition of Nanomaterial Two of the various definitions of nanomaterial are particularly relevant for our industry. The Cosmetics Regulation (EC) No 1223/2009 introduced a definition of nanomaterial. This is the current legally binding definition for the cosmetics industry. The EC Recommendation of 18 October 2011 has currently no direct obligations for cosmetics but may be the basis for a revised definition to be adopted in the EU cosmetic legislation in the future Nanomaterial (Cosmetics Regulation (EC) No 1223/2009) According to Article 2 of the Regulation on Cosmetic Products a nanomaterial is defined as follows: 1 Cosmetic Regulation 1223/2009[Ref], EU Recommendation 2011, ISO, ICCR, JRC, and references therein 2/18

3 an 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. The Regulation does not define any terms used in the definition (e.g. insoluble, biopersistent, ). EFfCI therefore proposes for practical reasons and as a basis for common understanding working definitions for these specific terms in Chapter 2.2. of this Guidance Paper Nanomaterial (EU Commission Recommendation 2011) The definition of the EU Recommendation is currently under review by the EU Commission and is expected to be revised in The current wording is: 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 100 nm. This definition does not have a direct impact on any regulation or product. The definition however may serve in future as the basis for sectorial definitions, like food, cosmetics etc. and REACH Definition of Specific Terms proposed by EFfCI In view of a lacking legal definition EFfCI is proposing the following interpretation of specific terms used in the Regulation Insoluble In the context of the Cosmetic Regulation there is no applicable definition of insoluble since it requires knowledge of the solvent and the context in which solubility is being assessed. However, it is generally accepted to mean solubility in water and as defined in the EU REACH and Classification, Labelling and Packaging (CLP) Regulations a substance is considered insoluble if less than 1 mg/litre is soluble. 2 Solubility in other solvents or liquids typically used in cosmetic formulations (e.g. glycerol) may also be of relevance and should be considered under the definition. 2 Deviations are possible, e.g. SCCS opinions on ZnO and SiO2 (currently under assessment) 3/18

4 Biopersistent (Bio)Persistence is the property of the continuation of the existence of a chemical/ substance in the body but neither the Cosmetic Regulation nor the EU Chemical legislation (REACH) define biopersistence. Toxicologically it is accepted to mean the potential risk that a substance might pose due to its potential to maintain its effects in mammalian systems i.e. it is not metabolised. For nanomaterials this means that the particulate structure is maintained in the body. The biopersistence of the molecular structure or metabolites is substance-specific and does not depend on the size scale of the product Intentionally Manufactured Intentionally manufactured nanomaterial is a material engineered by physical and/or chemical processes in order to have one or more external dimensions or an internal structure (aggregate / agglomerate) on the scale of 1 to 100 nm engineered to perform a specific purpose Internal Structure According to the Commission Recommendation of 18 October 2011 materials with internal structures in general are not in the focus of the definition nanomaterial only those forming aggregates / agglomerates are explicitly included. Materials with an internal structure with dimensions smaller than 100 nm should be considered as nanomaterial if they show typical properties of a nanomaterial, e.g. in connection with a large surface area. 3. Decision Tree A decision tree (Figure 1) illustrates the process how to investigate a material in question and how to make decisions based on results of investigations. Figure 1 serves as a comprehensive tool to decide whether a chemical substance is considered a nanomaterial under the Cosmetics Regulation (EC) No 1223/2009 (Article 2.1(k)) considering the 50 number percent threshold of the EC Recommendation of 18 October 2011 on the definition of nanomaterial (2011/696/EU). Making a valid decision according to the process lined out in Figure 1 requires an expert skilled in the state of art measurements and their limitations and potential pitfalls. The recommended methods are described in more detail in the following chapters of this document. 4/18

5 Potential (nano)-material Not (nano) No Solid or solid-inliquid dispersion Liquid in liquid dispersions have nano-size dimensions, which on evaporation or exposure to humans most likely are not persisting. The nano-size droplets would simple coalesces and disappear as nanostructure. A similar reasoning can be used for nano-sized polymeric dispersions. (review EPDLA position paper polymer dispersions 2013). Polymer particles may fuse also quickly. In case of doubt evaluate by EM. Not (nano) Yes Solubility Try to dissolve material in water at 1 mg/l (OECD 105) or other solvents commonly used in cosmetic formulations in the intended concentration (e.g. glycerine) Not (nano) No No Biopersistent Yes or no Info Decision requires independent information about destiny in biological systems (e.g. blood, plasma, urine). Can be literature based. If solubility and biopersistence (remaining a solid nanoparticle under biological conditions) cannot be reliably assessed the workflow has to continue. Not (nano) No Intentionally manufactured as nano Yes or in case of doubt Intentionally manufactured to perform a specific product function or purpose based on nano sized structures (1 nm 100 nm) of the product (Nanoporous materials will not be discussed in this paper.) Not (nano) (nano) Special process and additional analytical technology needed, e.g. Techniques that distinguish the three dimensions, e.g. quantitative TEM for fibers Techniques that can differentiate between primary particles and agglomerates/ aggregates, e.g. quantitative TEM VSSA Not (nano) No nano structures Yes, majority is nano & intentionally made No spheres (aspect ratio > 3:1) Incomplete dispersion Limit criteria not reached Qualitative TEM x Not intended or unclear origin of nanosized structures Aspect ratio (length : width measurement) Spherical (aspect ratio < 3:1) Sample preparation LD or DLS or CLS Complete dispersion Size distribution Limit criteria exceeded Detection of intentionally manufactured nano-sized structural elements (primary particles nm). High resolution FE-SEM is also appropriate (but 1 nm boundary cannot be assessed by SEM) Could be performed also after particle size distribution and is necessary if DLS or CLS does not detect (nano) EM measurements might be omitted, if product is well known, has spherical particles and is not mainly aggregated/agglomerated Powders: disperse into primary particles in solvent, try to model consumer product manufacturing technology and/or consumer application (e.g. avoid creating nano particles by preparation method). Check plausibility of results with e.g. EM investigations. Guiding dispersion protocols have not yet been proposed. Obtain quantitative particle size distribution: If DLS detects nano, earlier TEM measurements can be omitted LD and DLS not suitable for broad PSD CLS or EM Limit criteria depend on the relevant regulation Nano-sized particles between nm > 50 % by number (nano) Figure 1 Guiding (nano)-decision tree 5/18

6 4. Particle Sizing Methods 4.1. General Remarks This chapter describes the most commonly used particle size measurement techniques in the industry. A thorough discussion of the applicability of these and other analytical techniques for the assessment, if a material should be considered as nanomaterial can be found in the JRC reports [JRC reports] Sample Preparation Many measurement techniques e.g. laser diffraction (LD), dynamic light scattering (DLS), centrifugal liquid sedimentation (CLS) require the generation of a stable dispersion of the solid material in a liquid. Solvent selection during the sample preparation process should be representative of the intended final application (e.g. water, cosmetic oil). The sample dispersion is the most critical step of the particle size determination. It has to be optimized for each individual product and the intended analytical method. With respect to the EC Recommendation of a nanomaterial a complete break up of all agglomerates is necessary and a stable dispersion of the constituent particles must be achieved, as LD, DLS and CLS cannot differentiate between constituent particles and agglomerates. The energy input from the dispersion equipment (e.g. an ultrasonic bath or ultrasonic finger) must be optimized to ensure complete de-agglomeration of all agglomerates but avoid partial re-agglomeration by excessive energy input as well as introduction of artefacts by particulate contamination or destruction of the primary particles. The plausibility of the results, including the question, if a complete dispersion into the constituent particles was achieved, should be checked by an independent method like electron microscopy (EM). Other techniques like Small-angle X-ray scattering (SAXS), X-ray diffraction (XRD) and Volume-specific surface area (VSSA) may also be applicable in selected cases. LD, DLS and CLS assume spherical particle shapes for the calculation of the particle size distribution and are therefore limited to particles that deviate not too much from a spherical or cubical shape. Furthermore, LD, DLS and CLS cannot differentiate between constituent particles and agglomerates/aggregates. LD, DLS and CLS are therefore limited to materials that can be 6/18

7 completely dispersed into the primary particles. In case of incomplete dispersion into the primary particles or larger deviations from the spherical particle shapes, other methods have to be used for the assessment, if a material should be considered as nanomaterial, e.g. electron microscopy or VSSA Details on Selected Analytical Methods According to the proposed definition of a nanomaterial, a size range of nm has to be assessed. Laser diffraction, centrifugal liquid sedimentation and scanning electron microscopy are not able to measure particles at the lower end of this size range. The absence of a significant number of particles in this low size range has to be assessed by other methods like transmission electron microscopy, if a material should not be considered as nanomaterial Obtaining a Number Weighted Particle Size Distribution Laser diffraction, dynamic light scattering and centrifugal liquid separation obtain raw data that are based on the volume, scattering intensity or light extinction respectively. These results have to be converted to a number distribution using mathematical algorithms that involve a number of assumptions. This can often result in large errors Electron Microscopy (EM) Electron microscopy allows the direct imaging and counting of individual particles in dried powders after suitable sample preparation (e.g. direct deposition on a support). Sometimes also primary particles within agglomerates/aggregates can be characterized. Commonly used are transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Both techniques deliver a 2D projection of the analysed object. Qualitative EM with magnifications of x or above gives a good indication if intentionally manufactured nano-sized structural elements are present. In case that no nano structural elements are detected, further analysis may be omitted and the material can be considered as non-nano. In case that the EM images unambiguously show that the majority of structures is in the nano range, the material may be considered as nano material and further analysis may be omitted. Furthermore, qualitative EM often allows the assessment of the agglomeration state, the particle shape and aspect ratio. Only in case of nearly spherical particle shapes (aspect ratio < 3:1) and non-agglomerated particles methods like LD, DLS and CLS can give meaningful results concerning the question, if a material should be considered as nanomaterial. 7/18

8 If LD, DLS or CLS are not applicable for the assessment, if a material should be considered as nanomaterial, often quantitative EM can be applied. In case of well-distributed and isolated primary particles an automated image detection and analysis may be possible. Otherwise, the particles must be detected and analysed manually, which may introduce a large operator bias. To obtain a statistically relevant quantitative particle size distribution, a large number of particles has to be analysed, dependent on the width of the particle size distribution and the homogeneity of the shape and size of the analysed particles. Electron microscopy is not widely used in industry for routine measurement of particle size distributions due to high purchase and running costs, technical complexity and slow throughput Transmission Electron Microscopy (TEM) Typical size range: 1 nm 10 µm (depending on instrument and sample) Raw data: image analysis yields number based particle distribution Investment cost: high Operating cost: high Operator time: high Number of analysed particles: < 1000 Agglomerates/aggregates: tolerable Availability: limited Sample preparation: powders or dried dispersions Strengths: - Measurement of primary particles in agglomerates/aggregates - Not much affected by broad particle size distribution Limitations: - Good sample preparation required. The particles should be dispersed to minimize overlaps for automated image analysis - Operator bias (finding representative particles, interpretation, artefacts) - Stability of sample in electron beam and under high vacuum - Problem with plates (2D representation of a 3D particle) 8/18

9 Scanning Electron Microscopy (SEM) Typical size range: 10 nm 100 µm (depending on instrument and sample) Raw data: image analysis yields number based particle distribution Investment cost: medium Operating cost: medium Operator time: high Number of analysed particles: < 1000 Agglomerates/aggregates: tolerable Availability: medium Sample preparation: powders or dried dispersions Strengths: - Details of particle structure, in particular 3-D effects - Less operator skill necessary in comparison to TEM - Possibility to analyse raw powder - Possible to investigate formulations (cryo-sem) Limitations: - One order of magnitude lower resolution than TEM - Good sample preparation required. The particles should be dispersed to minimize overlaps for automated image analysis - Harder to automate particle counting in comparison to TEM - Operator bias (finding representative particles, interpretation, artefacts) - Stability of sample in electron beam and under high vacuum - Problem with plates (2D representation of a 3D particle) 9/18

10 4.6. Laser Diffraction (LD) Laser diffraction [ISO 13320] is the most widespread particle-sizing technology in the industry due to its ability to deliver quick and easy particle size distributions over a broad measurement range from a single measurement. Typical size range: 50 nm 2 mm (depending on instrument and sample) Raw data: volume distribution has to be converted to number distribution Investment cost: low Operating cost: low Operator time: low Number of analysed particles: high Agglomerates/aggregates: not tolerable Availability: high Sample: liquid dispersion or dry dispersion Strengths: - Fast method for obtaining particle size distribution Limitations: - Evaluation assumes particles are spherical, i.e. the particle size obtained is the diameter of an equivalent sphere - Refractive index of liquid and particles have to be known for MIE evaluation - Size distribution will only show the upper part of the nanoparticle range - Technique is blind to fraction of small particles if coarse particles are present (overestimates coarser particles) 10/18

11 4.7. Dynamic Light Scattering (DLS) DLS [ISO 13321, ISO 22412] measures the hydrodynamic diameter of suspended particles in a liquid medium. It is a widely used measurement technique for sub-µm particles which nicely complements LD. As the results are strongly biased by a presence of a few large particles (the intensity of the scattered light is proportional to the sixth power of the particle radius), any fraction of large particles as well as agglomerates/aggregates have to be removed before measurement. DLS is thus mainly suitable for monodisperse non-agglomerated/non-aggregated spherical particles. Typical size range: 1 nm 1 µm (depending on instrument and sample) Raw data: scattering intensity has to be converted to number distribution Investment cost: low Operating cost: low Operator time: mean (strongly dependent on effort for sample preparation) Number of analysed particles: high Agglomerates/aggregates: not tolerable Availability: high Sample: liquid dispersion Strengths: - Fast method for obtaining particle size distribution in the sub-µm range Limitations: - No fraction of large particles is tolerated - Evaluation assumes particles are spherical, i.e. the particle size obtained is the diameter of an equivalent sphere. - Refractive index of particles and medium have to be known 11/18

12 4.8. Centrifugal Liquid Separation (CLS) CLS [ISO , ISO , ISO ] combines a size separation (by application of a centrifugal force in a liquid medium) with particle detection. This leads to a better resolution in comparison to LD and DLS and the dominance of the measured signal from the large particles is much less pronounced. Typical size range: 20 nm 10 µm (depending on instrument, density difference between particles and liquid and particle scattering properties) Raw data: Light extinction has to be converted to number distribution Investment cost: medium Operating cost: low Operator time: low Number of analysed particles: high Agglomerates/aggregates: not tolerable Availability: high Sample: Liquid dispersion Strengths: - Good repeatability if method follows a standardized protocol - Good resolution Limitations: - Measurable size range will depend on density and optical properties - Only spherical particles - Long measuring time - Particles > 1 µm sediment too quickly (only small fraction can of the sample is thus analysed) 12/18

13 4.9. Volume Specific Surface Area (VSSA) Assessed by the Brunauer, Emmet & Teller Method (BET) The BET method allows the measurement of the specific surface area of dry powders [ISO 9277]. It may be used as a proxy measurement of the median particle size within the EC definition. If a VSSA > 60 m 2 /cm 3 is determined for a material consisting of non-porous, not surface-treated particles, the material can often be classified as nanomaterial according to the EC definition. The 2 nd JRC report [JRC 2014] introduced a shape-specific evaluation, which was validated by FP7 project NanoDefine [NanoDefine 2015], and provides a screening strategy for nearspherical, platelet and fiber shapes, which uses VSSA to classify a material as non-nano according to the EC definition, even in the presence of aggregation, non-spherical particle shape and polydisperse samples. Typical size range: - Raw data: Absorption isotherm. Has to be converted to a VSSA which can be a proxy of the median particle size. Investment cost: low Operating cost: low Operator time: low Number of analysed particles: high Agglomerates/aggregates: tolerable Availability: high Sample: Dry powders Strengths: - No sample dispersion necessary Limitations: - Density required - Conversion of VSSA to median particle size mainly reliable for non-porous, monodisperse spherical particles 13/18

14 4.10. X-ray diffraction (XRD) From XRD measurements, the mean primary crystallite size may be obtained. Typical size range: 1 nm 100 nm Raw data: scattering intensity has to be converted to number distribution Investment cost: mean Operating cost: low Operator time: low Number of analysed particles: high Agglomerates/aggregates: tolerable Availability: high Sample: Powder or dried dispersion Strengths: - Fast method for obtaining crystallite size in the sub-µm range - Measurement can be highly automated Limitations: - Only fully crystalline materials (insensitive to amorphous matrix) - Crystal structure and morphology should be known beforehand 14/18

15 5. Conclusion This Guidance Paper suggests appropriate analytical methods to decide whether a substance has to be considered as a nanomaterial according to the definition of the Cosmetics Regulation (EC) No. 1223/2009. Furthermore, interpretation of specific terms used in the Regulation were given as a basis for common understanding. The decision tree provided in this document is the key element how to investigate a material in question and how to make decisions based on results of these investigations. The focus of this paper is on analytical methods that are readily available and easily applied. It may be used as a reference in absence of concrete analytical methods linked to the Regulation. 15/18

16 6. Glossary/Abbreviations BET Method of Brunauer, Emmet & Teller CLP Classification, Labelling and Packaging CLS Centrifugal Liquid Separation EC European Commission EFfCI European Federation for Cosmetic Ingredients EM Electron Microscopy EU European Union FE-SEM High resolution SEM using a Field Emission cathode DLS Dynamic Light Scattering JRC Joint Research Centre, the European Commission in-house science service LD Laser Diffraction PSD Particle Size Distribution REACH - 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 SAXS Small-Angle X-ray Scattering SCCS Scientific Committee on Consumer Safety SEM Scanning Electron Microscopy TEM Transmission Electron Microscopy VSSA Volume-Specific Surface Area XRD X-Ray Diffraction 16/18

17 7. Literature/References Scientific Committee on Consumer Safety (SCCS): Guidance on the Safety Assessment of Nanomaterials in Cosmetics adopted on 26th-27th July 2012 (SCCS/1484/12) Scientific Committee on Consumer Safety (SCCS): MEMORANDUM on "Relevance, Adequacy and Quality of Data in Safety Dossiers on Nanomaterials" adopted on 12th December 2013 (SCCS/1524/13) H. G. Merkus, Particle Size Measurements: Fundamentals, Practice, Quality (Particle Technology Series), Springer, ISO 13320:2009, Particle size analysis Laser diffraction methods, International Organization for Standardization, Geneva. ISO 13321:1996, Particle size analysis Photon Correlation Spectroscopy, International Organization for Standardization, Geneva. ISO 22412:2008, Particle size analysis Dynamic light scattering, International Organization for Standardization, Geneva. ISO :2001, Determination of particle size distribution by centrifugal liquid sedimentation methods Part 1: General principles and guidelines, International Organization for Standardization, Geneva. ISO :2001, Determination of particle size distribution by centrifugal liquid sedimentation methods - Part 2: Photocentrifuge method, International Organization for Standardization, Geneva. ISO :2001, Determination of particle size distribution by centrifugal liquid sedimentation methods - Part 3: Centrifugal X-ray method, International Organization for Standardization, Geneva. ISO 9277:2010, Determination of the specific surface area of solids by gas adsorption - BET method, International Organization for Standardization, Geneva. H. Rauscher, G. Roebben, V. Amenta, A. Boix Sanfeliu, L. Calzolai, H. Emons, C. Gaillard, N. Gibson, T. Linsinger, A.Mech, L. Quiros Pesudo, K. Rasmussen, J. Riego Sintes, B. Sokull-Kluttgen, H. Stamm: Towards a review of the EC Recommendation for a definition of the term "nanomaterial" Part 1: Compilation of information concerning the experience with the definition, JRC Scientific and Policy Report, EUR EN, /18

18 G. Roebben, H. Rauscher, V. Amenta, K. Aschberger, A. Boix Sanfeliu, L.Calzolai, H. Emons, C.Gaillard, N.Gibson, U.Holzwarth, R. Koeber, T. Linsinger, K.Rasmussen, B.Sokull-Klüttgen, Hermann Stamm: Towards a review of the EC Recommendation for a definition of the term "nanomaterial" Part 2: Assessment of collected information concerning the experience with the definition, JRC Scientific and Policy Report, EUR EN, J. B. V. Pena, K. Kund, U. Hempelmann, W. Wohlleben, T. Koch, A. Burke, G. McNulty, A. Hartl- Gunselmann, S. Knobl, M. Reisinger, D. Gilliland, N. Gibson, B. Sokull-Klüttgen, H. Stamm, H. Liewald: Basic comparison of particle size distribution measurements of pigments and fillers using commonly available industrial methods, JRC Technical Report, EUR EN, NanoDefine public Deliverable 3.5 (2015) Evaluation report on the applicability ranges of the volume specific surface area (VSSA) method and the quantitative relation to number based particle size distribution for real-world samples T. Linsinger, G. Roebben, D. Gilliland, L. Calzolai, F. Rossi, N. Gibson, C. Klein: Requirements on measurements for the implementation of the European Commission definition of the term nanomaterial, JRC Reference Reports, EUR EN, W. D. Pyrz and D. J. Buttrey Particle Size Determination Using TEM: A Discussion of Image Acquisition and Analysis for the Novice Microscopist, Langmuir, 24, (2008). 18/18