SMPS

Environmental hazards

Article 1(3) of REACH). This principle is applicable to substances in whatever size or form and for all their identified uses. Thus, a registration of a nanomaterial has to include all relevant information on the nanomaterial as manufactured or imported, covering the properties, uses, effects and exposure related information as well as the relevant classification and labelling, safety assessment and any relevant exposure scenarios " (p. 6), and "For substances at nanoscale that are phase-in substances, the registration can be more complex, especially when the same substance exists in the nanoform as well as in the bulk form. In such a case not only the information of the substance in the bulk form should be included in the registration dossier, but also any information regarding intrinsic properties where the properties of a substance in the nanoform differs from the bulk form, any different classification and labelling, any different chemicals safety assessment as well as all identified uses (see also Annex VI.3 of REACH) and relevant exposure scenarios for the nanoform of the substance." (p.8). 1.2.3 Until more concrete guidance is provided by ECHA, it is suggested that the registrant follows this line. This has a direct influence on the generation of hazard data, e.g. any read-across from one form to the other (being from a bulk form to a nanoform or between nanoforms) should be scientifically justified. It also has influence on the information in the supply chain, which has to be appropriate to the 2 http://ec.europa.eu/environment/chemicals/reach/pdf/nanomaterials.pdf - 3 - form(s) passing down the supply chain and the Chemical Safety Assessment should support this. The suggested guidance updates from the RIP-oN 2 project need to be seen in this light. 1.3 PROJECT OBJECTIVES 1.3.1 The objectives of the RIP-oN 2 project were to:  Develop specific advice on how REACH information requirements on intrinsic properties of nanomaterials can be fulfilled. This should address and advise on the appropriateness of the relevant test methods (including dosimetry) for nanomaterials and outline, when relevant, possible specific testing strategies.  Develop advice on the information that is needed for safety evaluation and risk management of nanomaterials and in particular if information is needed beyond or in addition to the current information requirements listed in REACH Annexes VI-X. 1.4 THE PROJECT CONSORTIUM 1.4.1 The consortium awarded the tender for RIP-oN 2 comprises the Institute of Occupational Medicine (IOM) through its SAFENANO unit, the Nanotechnology Industries Association (NIA) and the European Chemicals Industry Council (CEFIC). 1.4.2 IOM/SAFENANO, with an established reputation for independent scientific work, led the consortium and carried out majority of the technical activities. NIA facilitated and provided a transparent interface between the project and the stakeholder group, as well direct access to industry and industrial knowledge. CEFIC contributed a breadth of experience and expertise on REACH activity as well direct access to industry and industrial knowledge. - 4 - 2 DESCRIPTION OF THE PROJECT 2.1 OVERVIEW 2.1.1 The project was implemented through a series of specified and linked tasks (A, B1- B5, and C). The relationship between each task is illustrated in the scheme below, with specific details of the task description provided in the table of Deliverables overleaf. Existing IRs B1 B1 Gap analysis A B3 B4 Final Project Evidence base B5 Report Strategies B2 B2 for Guidance update & recommendations Additional IRs and R&D C C Metrics 2.1.2 The project commenced with the identification and review of all relevant information sources (Task A) for carrying out the subsequent tasks (B1-B5 and C), which included: an analysis of the current REACH information requirements and testing and whether these requirements are appropriate for nanomaterials (Task B1); identification of additional relevant specific intrinsic properties for which an adaptation of the information requirements and testing and other information generation methods/strategies might be needed for nanomaterials (Tasks B2 & B3); identifying needs for further research and development of test methods and other information generation methods /strategies in regard to nanomaterials (Tasks B4 & B5); an outline of the needs and options for metrics/parameters in the hazard assessment compatible with the exposure assessment parameters/metrics in order to prepare a meaningful risk characterisation (Task C). Where relevant, additional information requirements beyond current REACH requirements have been identified when it is considered that they are needed to address adequately the properties of nanomaterials. - 5 - 2.1.3 The Final Project Report (Task D) compiles and summarises all the previous Deliverables in a single comprehensive document in such a form that advice on specific issues related to nanomaterials can be considered for integration into the REACH guidance documents and further research and development on relevant issues can be initiated. 2.2 DELIVERABLES 2.2.1 A series of reports were developed for the specified tasks, as summarised below. Task Deliverable A A Short Report containing: 1. A brief description of the approach/methodology used to identify relevant information sources; 2. A list of identified information sources with clear indications of which ones are relevant for the subsequent tasks; 3. For relevant information sources, a brief summary of relevant content and timelines for final outputs (in the case of on-going projects). B1 An Analysis Report making direct reference to the existing Guidance on IR & CSA for REACH (Part and (sub)Chapter) of the general information requirements and testing(/information generation) strategies for nanomaterials as applied today, based, among others, on the three case example materials. B2 A Scientific Report and table/grid on "Identification and overview of additional relevant specific intrinsic properties for nanomaterials". - 6 - Task Deliverable B3 A Summary Analysis Report containing sections on: 1. Practical advice on the relevance and applicability of the experience reported in finalised and on-going FP6/7 projects on nanomaterials characterisation and hazard identification and assessment for workers, consumers and environment into the REACH context; 2. Practical advice on the relevance and applicability of the experience reported in the scientific literature on nanomaterials characterisation and and risk assessment of nanomaterials and are therefore also listed as a possible source of information. Due to the present uncertainties regarding analytical characterisation (e.g. distinction between background concentration and nanomaterial), exposure quantifications, dose metrics and a generally low environmental concentration, field studies are not recommended. - 27 - 3.3.23 The solubility of a test substance is a key parameter in defining the test strategy for determining ecotoxicity and environmental fate for bulk material; for nanomaterials the stability of a dispersion is the equivalent key parameter. A soluble nanomaterial would loose its particle characteristics when dissolved and exposure conditions would not be discernible from dissolved bulk material. 3.3.24 Conclusions Concerning Classification, Labelling & Packaging (CLP) 3.3.25 The Regulation on Classification, Labelling and Packaging of substances and mixtures (CLP Regulation) (EC) No 1272/2008 does not contain specific provisions for nanomaterials nor for particulate materials but some guidance on classification of metal powders can be found in its Annex IV. However, nanomaterials are covered, as in REACH, by the definition of substance and therefore the provisions of CLP apply to nanomaterials as well. Moreover, the CLP recognises the potential impact a change in physicochemical properties might have on the intrinsic properties of a substance, see Articles 5 and 9.5. In doing so the CLP requires the manufacturer or importer to first ensure that the information used to classify relates to the forms or physical states in which the substance is placed on the market and in which it can reasonable be expected to be used. Secondly, CLP requires additional testing relating to physical hazards (explosivity, flammability, etc) to be performed if such information is missing or not adequate to conclude on a classification. 3.3.26 With regard to nanomaterials, if information only exists for coarser materials it should be assessed whether this information is also applicable to nanomaterials, due to the impact of the increased surface area on physicochemical properties. Information derived by registrants to fulfil the registration requirements in REACH, according to Annexes VII-IX and following the methods in the Test Methods Regulation (EC/440/2008), may not be sufficient to determine all physical hazards in accordance with CLP. Any evaluation of particulate (nano)materials in the context of CLP regulation should be conducted in accordance with the principle of using the worst case scenario where the finest relevant fraction of the form and physical states as placed on the market, should be used when testing for physicochemical Article 33.4.1.4.3.5 in the Manual of Test and Criteria (the Orange Book) (United Nations New York and Geneva, 2009). 3.3.27 Based on the opinions expressed by OECD (ENV/JM/MONO(2009)20, ENV/JM/MONO(2009)21, ENV/JM/MONO(2010)25) and the SCENIHR (2007), it is - 28 - considered that in general there are no significant differences in the applicability of the standard test methods themselves between nanomaterials and conventional substances. However, this report has highlighted a number of considerations in relation to sample preparation, dosing vehicle and the actual exposure of the test system and these consideration remains the same also for any test method preformed under CLP. Nevertheless, the impact of an increased surface area on physicochemical properties should be given thorough consideration when determining the physical hazards. - 29 - number TG123 TG121 - 30 - Guidance reference REACH R.7.1.9 R.7.1.10 R.7.1.11 R.7.1.12 R.7.1.13 R.7.1.3 R.7.1.2 R.7.1.5 R.7.1.4 R.7.1.6 R.7.1.8 R.7.1.7 R.7.1.14 R.7.1.15 R.7.1.16 R.7.1.17 R.7.1.18 stated stated stated stated stated stated stated stated methods methods methods methods methods methods methods methods methods TG108, TG112 - - - TG117, TG105 documents OECD TG107, Modify TG106, Modify Apply Apply Apply Apply Apply Apply Apply Apply Modify guidance and as- as- may to from + + t/y): t/y): Property change produced tested t/y): 10 REACH 100 1 above in relevant imported for not nanomaterials? as specifically relevant any O O O O O O O O - O + + + O O - or requirements substance or manufactured data manufactured - Property redundant for - + o manufactured substances physico-chemical data REACH n-octanol/water solvents of for screening for table with point requirements (required properties temperature properties tension coefficient (required organic constant Properties Annex Flash Flammability Explosive Self-ignition Oxidising Boiling Melting/freezing Vapour Relative Surface Partition Water Granulometry Annex Adsorption/desorption Annex Stability Dissociation Viscosity Summary VII point pressure point density VIII IX in solubility 3.3.28 Guidance number - 31 - R.7.12 R.7.4 R.7.2 REACH reference RNC/RIP-oN2/FPR/1/FINAL (Skin B.36: (Acute class – toxicity B.40 Electrical test) (Human Membrane Model B.44: B.45: bis) procedure) tris) toxic bis (EPISKIN™, Skin / skin) oral rat B.1 430/EU B.40 vitro Corrositex®) methods (EU guidelines (EU Acute (Acute EU Method) using Method) 417/EU tests (In (Human dose test EpiDerm™)) 427/ Vivo 428/EU Vitro Fixed – procedure) (TER 431/EU method, 439 OECD Absorption OECD Absorption OECD (Toxicokinetics) Oral OECD oral OECD oral method) OECD Up-and-down In OECD (Transcutaneous Resistance OECD Skin EpiDerm™)) OECD Barrier OECD tests Test In In TG 420 – 423 425 TG 435 documents OECD toxicity (EPISKIN™, TG TG Model TG test TG vitro: guidance as- as- may to and from t/y): 1) Property tested1) 1 REACH change produced above in or substance relevant imported for not solubility?) data nanomaterials? as specifically (increased requirements relevant any or + o O Property manufactured redundant for - o + substances toxicological data REACH of requirements (required for table with Properties Annex Toxicokinetics Acute Summary toxicity Irritation VII Skin 3.3.29 Guidance number - 32 - R.7.2 R.7.3 R.7.7 REACH reference RNC/RIP-oN2/FPR/1/FINAL eye corneal test Murine (endorsed pig and Node Bacterial test chicken (BCOP) chorio-allantoic test B.42: Guinea (GPMT) Lymph guidelines / (IRE) Bovine Assay B.6: B.13/14: methods Isolated Local test – eye test 437: test 429/EU Node 406/EU Test 438: permeability (HET-CAM) 442A: 442B: 471/EU OECD rabbit TG & egg TG Lymph TG test TG DA TG BrdU-ELISA TG mutation In Isolated OECD (ICE) OECD opacity Hen’s membrane OECD Local method) OECD maximisation Buehler OECD Assay OECD Assay OECD reverse test vitro: as- as- may to from t/y): 1) Property tested1) 1 change produced above or substance relevant imported for not nanomaterials? as specifically relevant any O or Property manufactured redundant for - o + substances gene data in REACH In mutation bacteria requirements (required for Toxicity vitro with Properties irritation Sensitisation VII Annex Eye Skin Genetic (Acute (“Acute Dose (“Acute Guidance Toxic number (Acute Dermal - 33 - VII): R.7.4 R.7.2 REACH reference Annex to in guidelines studies; (“Acute Procedure”) studies; Acute Toxicity: according B.2) 433 Fixed 436 Acute / mechanistic B.3) mechanistic 2004/73/EC, studies methods test Toxicity, species Dose studies; species 404, Directive Acute OECD requirements Method”) (EU compliant toxicokinetic OECD compliant toxicokinetic (EU TG studies; 434 Inhalation OECD inhalation Draft Inhalation Procedure”) Draft Inhalation Class ICH and non-rodent Dermal OECD dermal Draft Toxicity, ICH and non-rodent In OECD Irritation/Corrosion Commission Method Irritation/Corrosion Test toxicity) OECD toxicity) Fixed TG 403 402 TG TG B4, to vivo: as- as- addition may in from t/y, 1) to 1) Property change produced tested1) 10 above imported substance relevant or for not nanomaterials? as specifically relevant any or + O Property o + manufactured redundant for - substances depending on data based REACH with inhalation appropriate for requirements (required most or Properties Annex Acute (either on exposure) what toxicity Irritation VIII dermal is Skin Guidance number - 34 - VII): R.7.2 R.7.5 REACH reference Annex to Eye days; by according Acute 2004/73/EC, certified methods guidelines (5 / Toxicity: test B.7 B.9 B.8 2008; OECD requirements B.5, TG WPMN 407 410 412 inhalation et test 405, Directive Acute EU al., SG7) In OECD Irritation/Corrosion Commission EU Irritation/Corrosion Oral: OECD Dermal: OECD Inhalation: OECD Short-term Ma-Hock OECD / Test TG L to vivo: as- as- addition may in from t/y, 1) 1) to Property change produced tested1) 10 above imported substance relevant or for not nanomaterials? as specifically relevant any or O o + Property o + manufactured redundant for - substances short-term appropriate (28 data inhalation REACH most for or requirements (required dose with is toxicity, dermal what VIII oral, exposure) on Properties irritation on Annex Eye Repeated days) (either depending based Guidance number In marrow B.39: (UDS) cells - 35 - VII): R.7.7 REACH reference Annex to according vitro aberration mammalian vitro test mutation vitro test mutation vivo erythrocyte in rare very In / test liver B.10: B.17: assay B.11: B.12: methods test synthesis on 473/EU chromosome 476/EU cell 475/EU aberration 474/EU test DNA mammalian (only guidelines vitro test. bone 486/EU gene EU gene In 487: 476/ cell TG Test requirements TG test TG lymphoma cells: TG cells OECD vitro: micronucleus vivo: with TG In OECD mammalian test. OECD cell OECD mammalian – OECD mammalian – In Somatic OECD mammalian chromosome OECD mammalian micronucleus HPRT Mouse OECD Unscheduled test vivo Germ occasions) to as- as- addition may in from t/y, 1) to Property change produced tested1) 10 above imported substance relevant or for not nanomaterials? as specifically relevant any or o O Property o + manufactured redundant for - substances mutation damage repair data cell REACH chromosomal aberration mammalian gene chromosomal aberration DNA and/or for requirements (required with VIII Toxicity Properties Annex Genetic repeated Guidance number - 36 - VII): R.7.6 REACH reference Annex to according Reproductive reproductive methods guidelines Combined / OECD requirements TG 421: / test 422: Test TG study toxicity study OECD screening OECD dose screening to as- as- addition may in from t/y, 1) to Property change produced tested1) 10 above imported relevant substance relevant or for not nanomaterials? as specifically or relevant any O Property o + manufactured redundant for - substances data REACH for requirements (required reproduction with Properties Annex Toxicity VIII to and Guidance number - 37 - VII REACH reference R.7.5 Annexes R.7.6 to RNC/RIP-oN2/FPR/1/FINAL according in species, / toxicity / toxicity / toxicity generation TGs ‘F1- Prenatal B.26/B.27 study OECD study’) a or EU dose B.34 B.31: test methods B.28 B.29 OECD multi-) B.35, requirements 408/409 repeated screening 411/EU 422: repeated screening 413/EU 422: repeated screening one-generation 414/EU / EU addition Oral OECD rodent/non-rodent respectively OECD Combined reproductive Dermal OECD OECD Combined reproductive Inhalation OECD OECD Combined reproductive One- studies 415 extended (or as toxicity OECD developmental or 422: two- to TG (such 416, TG TG TG or in as- as- may to t/y, from 100 1) Property tested1) 1) change produced above imported substance relevant or for not nanomaterials? as specifically relevant any or o + O Property o + manufactured redundant for - substances sub-chronic appropriate of table the REACH for Summary VII toxicity VIII toxicity in behaviour with (required 3.3.30 Properties requirements Annex Aquatic Degradation Annex Aquatic Degradation Fate and Guidance number - VIII): 40 - and R.7.8 R.7.9 R.7.10 R.7.1.15 REACH reference VII RNC/RIP-oN2/FPR/1/FINAL Annexes for the to test on stage toxicity growth water testing high Annex on in toxicity (preferred already surface simulation sediment) VII stages testing potential a Identification fish in according unless of Annex early-life high short-term sac-fry juvenile with to in simulation a information depending required Long-term Fish test Fish and Fish Simulation Soil with Sediment adsorption specified] products Bioaccumulation preferably Further methods requirements TG on TG toxicity TG embryo TG TG TG substances TG (for apart TG species, TG of soil) study 211 invertebrates 210 212 215 309 307 to 308 substances 305 106 the Dahpnia) as degradation for method OECD OECD testing species provided requirements OECD (FELS) OECD test OECD OECD ultimate OECD (for adsorption OECD testing potential [No of OECD aquatic OECD adsorption/desorption results VII on test to degradation as- as- addition may to in from Property 1) tested1) change produced t/y, 1) 100 above substance imported for not nanomaterials? as specifically relevant any or relevant + O Property manufactured redundant for - o + data substances REACH for the in IX toxicity behaviour with (required Properties requirements Annex Aquatic Degradation Fate and REACH reference to Guidance number fate and/or already VIII, physicochemical relate characterisation - VIII): and tested 41 - and R.7.11 VII, R.7.9 R.7.10 R.7.1.15 R.7.11 the VII of Annexes to the to e.g, soil Biotic toxicity according term according substance unless agglormeration/disagglomeration) toxicity on toxicity Further IX Effects testing specified] environmental Long-term Annex particularly adequate 207 216/217 208 302/303/304/306 on products Short 220/222 invertebrates part Short-term which the of the of an possible methods requirements TG requirements TG simulation behaviour TG on tests, method as for to OECD test OECD vertebrates OECD micro-organisms OECD plants OECD further [No information and degradation OECD testing provided requirements account allowing to may addition toxicological as- addition into (e.g. 1) t/y, 1) to in taken system, in Property tested1) be For from all change produced t/y, 100 1000 to test above need vehicle. the given. of for not be redundant considerations actual known) substance imported nanomaterials? as specifically exposure should relevant the (if any or relevant dosing o o o O the or Property relevant to in for manufactured important including metric - o + dose substances endpoints nanomaterial, consideration the appropriate data REACH toxicological appropriate to for the organisms in organisms of the terrestrial Properties requirements behaviour with Annex Effect terrestrial Annex IX): Degradation (required Effect Concerning characteristics nanomaterial, consideration IX X on and on Fate 1) 3.4 ADDITIONAL RELEVANT SPECIFIC INTRINSIC PROPERTIES FOR NANOMATERIALS (TASK B2) 3.4.1 It is important to acknowledge that in identifying additional relevant specific intrinsic properties for nanomaterials, consideration has been given to how the existing Information Requirements included in REACH (considered in Task B1) could reflect any new or refined (i.e. improved or clarified description in the guidance) intrinsic properties specific for nanomaterials, without unnecessarily duplicating the effort and repeating content developed for Task B1. This has involved acknowledgement of the definitions provided in the legal text and guidance (and in nomenclature standards as necessary) and current regulatory status of some intrinsic properties, so that acceptable and pragmatic recommendations can be made regarding whether additional relevant specific intrinsic properties of nanomaterials could and should be addressed under the existing Information Requirements or as new ones. 3.4.2 It is arguable, from a technical/scientific definitions perspective, whether some of the candidate additional relevant specific intrinsic properties identified may be considered logically under the term ‘granulometry’ (used to describe an already existing Information Requirement (IR)). The only ISO definition of granulometry (sourced from the ISO Concept Database; http://cdb.iso.org) is a “measure of the particle (grain) content of irrigation water, as characterized by size dispersion and total amount of solids”. No definition of the term ‘granulometry’, including the one purported (but not explicitly stated) to be a definition in the current REACH Guidance (section R.7.1.14), extends beyond the consideration of a size distribution of grain sizes. Hence, the logic of including intrinsic properties technically unrelated to characterising particle size distribution under the existing Information Requirement ‘granulometry’ (even in the context of them being particle-associated properties), may be questionable and has led the Consortium to suggest the option of a limited amendment of the title of an existing Information Requirement. With a more appropriate choice of term (in a similar vein to those adopted for the toxicology and ecotoxicity Information Requirements), this could facilitate the inclusion of a range of particle-associated properties as subordinate IRs, with provisions in Column 2 for the introduction of specific rules for adaptation from Column 1 in Annexes VI to X, as appropriate. Alternatively, a robust definition of the term ‘granulometry’ is required to be developed and adopted to facilitate the inclusion of additional particle-associated subordinates to the existing Information Requirement for Granulometry. - 42 - 3.4.3 However, it is noted that Annex II to Regulation (EC) No 1907/2006 has been amended and now explicitly states in Section 9.1 that “The physical state (solid (including appropriate and available safety information on granulometry and specific surface area if not already specified elsewhere in this safety data sheet), liquid, gas) and the colour of the substance or mixture as supplied shall be indicated.” The inclusion of specific surface area as a separate term distinct from granulometry in the amendment of Annex II is observed. For specific surface area at least, confusion (or even a suggestion of heightened importance) may arise, albeit minor, when the property is seen to be stated separately from granulometry in Annex II but as subordinate in the guidance, if this were to be the case. This ambiguity is simply highlighted, for resolution by regulators in their future considerations. 3.4.4 Identifying Potential Additional Relevant Specific Intrinsic Properties for Nanomaterials 3.4.5 The debate over which parameters to use to characterise nanomaterials has been ongoing for some years. The task report’s discussion highlights that in many instances the suggestions are generic (e.g. surface chemistry). Furthermore, they are made in the absence of a) any detailed understanding of the characteristic, b) the relevance and applicability of any interpretable data on the characteristic (should it be available), and c) the availability of a technique to gather such data. 3.4.6 Of the numerous sources considered and commented on in RNC/RIP- oN2/B2/2/FINAL (including reports from ILSI, ECETOC, SCENIHR, VCI, OECD, and RIVM amongst others and more generally in the literature), the ‘pathfinding’ body identifying, developing and establishing properties and endpoints for nanomaterials hazard assessment is the OECD WPMN and its Sponsorship Programme on the Testing of Manufactured Nanomaterials. In 2008, a list of endpoints was recommended by the OECD-WPMN (ENV/JM/MONO(2010)46) for the first phase of testing that is intended to take into account ‘human health and environmental safety’ and ‘ensure consistency between the various tests to be carried out on specific nanomaterials’. OECD stated that it should also lead to the development of dossiers for each selected nanomaterial describing basic characterization, fate, ecotoxicity and mammalian toxicity information. It was acknowledged that the list of endpoints should be refined based on the practical results obtained through the testing programme and as such, phase one testing was expected to be of an exploratory - 43 - nature, science-based and without any consequences for existing regulatory datasets. (It is anticipated that incorporation of such refined recommendations and methods into appropriate OECD Guidance Documents will be forthcoming.) 3.4.7 A particular complex issue identified, including by the OECD-WPMN, concerns the stability of nanomaterials in the context of i) sample preparation for the determination of properties and ii) the behaviour of nanomaterials in the environment. Information on these two aspects is absent from REACH Guidance, and has been determined to be of crucial importance in the context of gathering characterisation data for Information Requirement purposes. 3.4.8 A number of suggested physico-chemical ‘properties’ have been identified as having a greater bearing on the quality of determining a characteristic or (eco)toxicological test outcome, due to its influence on sample preparation considerations. These include dispersion stability and state of agglomeration, which may also be important phenomena, in a physico-chemical sense, which influence the behaviour of nanomaterials in environmental media. These have been highlighted before being subsequently addressed in recommendations for Guidance amendment as part of Task B5. We suggest that their importance as supplementary information to the data gathered for the Information Requirement is acknowledged in the Guidance, but not necessarily as additional Information Requirements in their own right, due to their supplementary nature (i.e. non-intrinsic), their dependence upon other primary properties, and the need for further research and development of applicable methods as well as the interpretation of the primary data for these properties (and how it may inform the interpretation of data on existing Information Requirements) in the context of risk assessment. Nevertheless, it is acknowledged that such in which the characterisation or testing is being done, and as a result can significantly impact on the determination, relevance and quality of other intrinsic properties or endpoints. These properties, indicated with an asterisk in the summary table below, have not been formally considered in the gap analysis conducted in Task B4, but are considered to warrant acknowledgement in updated guidance and recommendations have been made. It is also recognised that other aspects, such as assay interferences, also influence the determination, relevance and quality of intrinsic properties or endpoints, but as these cannot be defined as properties or be justified as a specific Information Requirement. Recommendations on issues related to sample preparation and assay interference were identified in Task B2 and subsequently have been further elaborated in the recommendations for Guidance update in Chapter 4. - 45 - Suggested IR ‘incorporation’ status Guidance Subordinate to an existing recommendation Possible (E) or (with or without New IR an update to the Updated: (U) appropriate IR Column 2 rule) Candidate property / endpoint Particle shape U/E (7.14) Surface area U/E (7.14) Surface energy U/E (7.14) Surface chemistry U/E (7.14) Surface charge U/E (7.14) Redox potential  Cell-free ROS/RNS production capacity  State of dispersion*  (7.14 & 9.3) State of agglomeration*  (7.14 & 9.3) Cell uptake*  (8.3, 8.4, 8.8) Cell viability  (8.1, 8.2) Oxidative stress  (8.4, 8.5, 8.6) Inflammation  (8.1, 8.2, 8.5, 8.6) Fibrosis  (8.6) Immunotoxicity (sensitisation)  (8.3) E Cardiovascular toxicity (8.6) Ventilation rate#  (9.1) Gill pathologies#  (9.1) Mucus secretion#  (9.1) - 46 - Suggested IR ‘incorporation’ status Guidance Subordinate to an existing recommendation Possible (E) or (with or without New IR an update to the Updated: (U) appropriate IR Column 2 rule) Candidate property / endpoint Brain pathology#  (9.1 & 9.6) Animal behaviour#  (9.1 & 9.6) Oxidative stress biomarkers (CAT,  SOD, GPX, GST)# (9.1 & 9.6) *Properties or endpoints recommended in the sources considered in Task B2 for nanomaterials testing are not strictly intrinsic properties or the proxy-effect of a single intrinsic property, but are influenced by the material’s ‘conditions’ or ‘environment’ in which the characterisation or testing is being done, and as a result can significantly impact on the determination, relevance and quality of other intrinsic properties or endpoints.

SECTION 2: Hazards identification

and assessment for workers, consumers and environment into the REACH context; 3. Practical advice on the use of information from e.g. the Organisation for Economic Co-operation and Development Working Party on Manufactured Nanomaterials (OECD-WPMN) and other sources on the appropriateness of existing testing methods and results from the sponsorship programme in fulfilling the REACH data requirements; 4. Practical advice on the basis of on-going work in ISO and CEN (and, as identified, other harmonization bodies) in relation to whether relevant methods for substance characterisation could be used in fulfilling REACH data requirements. B4 A Summary Analysis Report on the "Gap analysis of relevant intrinsic properties for nanomaterials, which may not be addressed by standard test guideline methods and for which further development of in vitro, in vivo or other methodologies is required". - 7 - Task Deliverable B5 Scientific Report on Test Methods and Strategies for Nanomaterials, including chapters on: 1. Advice on integrated testing strategies relevant to specific nanomaterials properties and how the specific intrinsic properties of nanomaterials might affect the need for adaptations to the testing regime; 2. Advice on the scientific basis for the categorisation of nanomaterials and application of in silico methods, read-across and category approaches for deriving hazard information for nanomaterials from the information on bulk substances or from comparison between nanomaterials; 3. Proposals for further amendment of the REACH guidance documents in regard to information requirements, test methods or testing strategies for nanomaterials, where appropriate, taking into account the provisions to minimise use of animals for testing. These should also consider whether

Additional information

requirements beyond the standard REACH requirements would be appropriate for nanomaterials e.g. for addressing the gaps identified in B4; 4. Proposal for further research and development of test methods and other data generation methods/strategies in regard to nanomaterials. C A working document on identification of critical items on dose descriptors and related parameters, outlining needs for adequate metrics/parameters as appropriate for hazard assessment compatible with the ones used for exposure assessment as well as for the read-across from bulk substances and from other nanomaterials. This document has been developed in close collaboration with RIP-oN 3. D A Compiled Summary Report on the overall project, describing: 1. Specific issues related to nanomaterials that can be integrated into the existing REACH Guidance on Information Requirements and Chemical Safety Assessment with reference to the corresponding Part and (sub)Chapter; 2. Needs for further research and development of test methods and other methods of information generation. 2.2.2 The present report, as already indicated in the introduction, constitutes the Final Project Report (Task D) and provides advice for updating the guidance and on research and development needs. This takes the form of specific recommendations or options for consideration by the Commission. For issues which are not currently - 8 - technically/scientifically mature for developing detailed guidance, the need for further research and development is indicated. 2.2.3 The focus of the RIP-oN 2 has been on nanomaterial relevant issues. Nevertheless, due to the nature of some nanomaterials, some proposals may have implications for other substances that are not nanomaterials. These would have to be considered if reshaping of the REACH guidance would take place. 2.3 REVIEW AND CONSULTATION 2.3.1 All Task Reports were subject to review by the project’s Steering Group (constituting representatives of JRC, DG Environment, DG Enterprise and ECHA) and a Stakeholder Consultation Group (SCG) consisting of the members of the REACH Competent Authorities Sub-Group on Nanomaterials (CASG-Nano) and other relevant experts from Member States, industry and NGOs nominated by the REACH and CLP Competent Authorities (CARACAL). The draft Task Reports were opened for consultation with the above mentioned groups, discussed at meetings of the SCG, revised by the Project Consortium and re-opened for comment before being finalised. 2.4 TECHNICAL APPROACH 2.4.1 The project has been performed as an objective review of the existing guidance and available scientific evidence pertinent to the specified tasks. Existing Information Requirements across all tonnage levels have been considered and potential Requirements for nanomaterials identified, prior to conducting the gap analysis of properties and methods to facilitate the identification of recommendations and research & development requirements. 2.4.2 The conduct of this scientific review is based on an informed, objective and systematic gathering and consideration of evidence by experts who have used their knowledge and professional judgement when considering the impact and contribution of a source document to the task objective. It is important to note the inherent limitations of a review activity. Reviews are conducted at a fixed point in time which precludes the inclusion of information becoming available after a cut-off date. Information sourced may be incomplete or, on closer inspection, the content of a source document bears no relevance to the issues being considered. Information may also change in revisions of the sources considered. - 9 - 2.4.3 Based on the objective and informed assessment of published reports constituting the evidence-base available, a synthesis of findings, implications and/or issues distilled from the sources has been developed and integrated into the task reports. The review of source reports has identified the key findings and gaps to establish a technical basis facilitating the development of advice pertinent to the project. 2.4.4 Identification and review of information sources (Task A) 2.4.5 Relevant information was collected, assessed, categorised and made available to the project team. There is a range of relevant information and information types. The information included background information from organisations such as CASG Nano, OECD WPMN, SCENIHR, Standards organisation such as ISO and CEN, FP6/7 projects, other ongoing national projects, other international regulatory organisations such as NIOSH and EPA and from the peer reviewed literature. Reports and papers were assessed for specific relevance to the project. 2.4.6 The report from Task A comprises a brief description of the approach/methodology used to identify relevant information sources, the list of identified information sources with clear indications of their relevance to the respective tasks and comment of the relevant content. 2.4.7 REACH Information requirements for nanomaterials (Task B) 2.4.8 Task B comprised five sub-tasks (B1-B5). Each sub-task was conducted using data available from the public domain, in the broadest context of nanomaterials, and used case-example materials (MWCNT, Ag, TiO2 and ZnO for ecotoxicology) as appropriate, to exemplify specific aspects necessary for these types of nanomaterials. Each of the five sub-task reports built on the previous task(s) and its associated report, and comprise multiple components addressing physico-chemical properties, toxicology, and eco-toxicology aspects, as well as cross-cutting issues. 2.4.9 For Task B1, a thorough analysis was carried out of the endpoint specific guidance, R7a-c. Chapter-by-chapter, the current guidance text has been analysed to establish if there are any differences in application between what could be called conventional substances and those at nanoscale. The report is organised by REACH Guidance headings or groups of headings (in bold and labelled with their - 10 - corresponding section number from the current REACH Guidance document) and comments provided in the context of the task objective. 2.4.10 The approach undertaken to identify candidate additional relevant specific intrinsic properties for nanomaterials in Task B2 utilised key reports from different organisations, scientific opinions and working documents of OECD and ISO/CEN. Previously suggested properties and endpoints from these reports for use in nanomaterials risk assessment are highlighted. The task report (RNC/RIP- oN2/B2/2/FINAL) identifies candidate additional relevant specific intrinsic properties and provides a contextual or principle-based overview of their relevance to nanomaterials and REACH. 2.4.11 In Task B3, a comprehensive review of information sources identified in Task A was carried out. Detailed consideration of supporting information pertaining to intrinsic properties was undertaken in Task B3 in the context of i) the relevance and applicability of the experience reported in the scientific literature and gained in several finalised and on-going FP6/7 projects; ii) the use of information from OECD- WPMN; and iii) the basis of on-going work in ISO and CEN. The review of information sources provides a basis for the gap analysis in Task B4 of relevant intrinsic properties which may not be addressed by standard test guideline methods and for which further development of in vitro, in vivo or other methodologies is required. The associated Task report (RNC/RIP-oN2/B3/2/FINAL) is organised first into Sections in accordance with the sequence of objectives (the peer-reviewed literature is however reported separately from FP6/7 projects), and then by relevance to physico-chemical information,

TOXICOLOGICAL INFORMATION

and Where possible, it has been identified where OECD test guidelines and ISO/CEN (or equivalent) standards have been utilised on the basis of the information provided in the publications reviewed. Thus, in the absence of any statement to the contrary, the protocols utilised in the FP7/FP6 projects and scientific literature are either non-standardised or there is insufficient information provided by the publication’s authors to determine whether a standardised test method has been used. Insufficient information was often stated to describe the detail of the non-standardised methods and protocols used. - 11 - 2.4.12 Gap analysis of relevant intrinsic properties for nanomaterials possibly not addressed by standard test guideline methods and requiring further development of in vitro, in vivo or other methodologies (Task B4) 2.4.13 The gap analysis table provided in the task report (RNC/RIP-oN2/B4/2/FINAL) is the outcome of an informed, objective and systematic consideration of the evidence by the Project Consortium, who have used their knowledge and professional judgement when considering the relevance of the existing and additional intrinsic properties and the applicability of methods for nanomaterials. 2.4.14 The gap analysis has assembled and further developed the findings from the examination of existing REACH Guidance related to information requirements and testing (information generation) strategies (Task B1), the identification of additional relevant specific intrinsic properties for nanomaterials (Task B2), the assessment of relevance and applicability of testing, endpoints and methods described in the scientific literature and on-going international work relevant to the fulfilment of the data requirements under REACH (Task B3). 2.4.15 The structural framework used for the gap analysis considers physico-chemical properties and toxicological and ecotoxicological endpoints, and integrates the existing and additional properties/endpoints to identify those which may and may not be addressed by standard test guideline methods and where further development of in vitro, in vivo or other methodologies is required. 2.4.16 The gap analysis tables are structured by property / endpoint, with tables presenting systematically an assessment of methods using the following format:  Method name;  Supporting information from conclusions of the preceding B1, B2, and B3 reports;  A judgement on whether the property / endpoint is applicable to nanomaterials and the need for guidance amendment, according to the following categories outlined in the table below: - 12 - Category Judgement Outcome 1 Property / endpoint not No change to guidance. applicable to nanomaterials. 2 Property / endpoint applicable No change to guidance. to nanomaterials, but no difference between nano and non-nano in terms of the applicability of methods. 3 Property / endpoint applicable Change(s) to guidance to to nanomaterials, with be suggested. differences between nano and non-nano in terms of the applicability of methods 4 Property / endpoint applicable No change to guidance to nanomaterials, with proposed at this time, but suspected important R&D requirements to be differences between nano and stated as needed. non-nano in terms of the applicability of methods, but an insufficient basis for guidance to be provided. 5 Property / endpoint applicable No change to guidance at to nanomaterials, with no this point in time. suspected important differences between nano and non-nano, but an insufficient evidence basis to warrant acknowledgement in guidance.  Where specific methods for a relevant property are considered, a prioritisation based on whether the method is applicable to nanomaterials and the need for guidance amendment was assessed according to the following categories outlined in the table below: - 13 - Category Judgement Outcome 1 Method not applicable to No change to guidance. nanomaterials. 2a Existing method will work with No change to guidance. nanomaterials, with evidence of no difference of applicability between nano and non-nano. 2b New method will work with Change(s) to guidance to nanomaterials, with evidence be suggested. of no difference of applicability between nano and non-nano. 3a Existing method will work with Change(s) to guidance to nanomaterials, but with be suggested. evidence of differences in applicability between nano and non-nano. 3b New method will work with Change(s) to guidance to nanomaterials, but with be suggested. evidence of differences in applicability between nano and non-nano. 4a Existing method will work with No change to guidance, but nanomaterials, but with R&D requirements to be suspected important stated as needed. differences in applicability between nano and non-nano, but an insufficient evidence basis for guidance to be provided. 4b New method will work with No change to guidance, but nanomaterials, but with R&D requirements to be suspected important stated as needed. differences in applicability between nano and non-nano, but an insufficient evidence basis for guidance to be provided. 5b New method will work with No change to guidance, but nanomaterials, but with no R&D requirements to be suspected important stated as needed. differences in applicability between nano and non-nano but an insufficient evidence basis to warrant acknowledgement in guidance.  (For applicable methods (i.e. categories 2-5), a differentiation is made between existing methods (category suffixed with ‘a’) and new methods (category suffixed with ‘b’). The absence of a category 5a is self-evident, - 14 - as an ‘existing method that will work with nanomaterials, with no suspected serious differences in effectiveness between nano and non- nano’ is equivalent to category 2a and does not have an insufficient evidence basis to warrant acknowledgement in guidance.)  A comment on the method type, according to the following categories: • Standard (e.g. ISO, OECD TG method); • Non-standard method; • Widely-accepted R&D method.  A commentary, providing further detail on the applicability, limitations, and R&D needs, where considered necessary;  A comment on whether the property / endpoint is applicable to substances, particles, or nanomaterials only;  Information on the type of data (kind of information) provided by the method;  Suggested Guidance amendments, where appropriate, identifying the relevant Guidance documents, sections, and figures and the basis of the change to be suggested. 2.4.17 Where a consensus opinion could not be reached within the Project Consortium on any aspect of the data considered, the different positions are stated. 2.4.18 On the basis of the gap analysis, the relevant intrinsic properties for nanomaterials which may not be addressed by standard test guideline methods (and others) and for which further development of in vitro, in vivo or other methodologies is required have been identified. 2.4.19 Scientific Report on Test Methods and Strategies for Nanomaterials (Task B5) 2.4.20 The Scientific Report on Test Methods and Strategies for Nanomaterials (RNC/RIP- oN2/B5/2/FINAL) comprises four aspects: i) integrated testing strategies relevant to specific nanomaterials properties; ii) categorisation of nanomaterials for deriving - 15 - hazard information for nanomaterials; iii) proposals for further amendment of the REACH guidance documents for nanomaterials; and iv) proposal for further research and development of test methods and other data generation methods/strategies in regard to nanomaterials. 2.4.21 The assessment of the integrated testing strategies relevant to specific nanomaterials properties, reported in full in RNC/RIP-oN2/B5/2/FINAL, presents the current ITS for each of the existing properties and endpoints in the REACH guidance, its relevance and applicability for nanomaterials, and any indicated recommendations for alteration. 2.4.22 The latter two objectives (amendment of the REACH guidance documents for nanomaterials, and proposal for further research and development) have been developed from the comprehensive series of tables that constitute the gap analysis undertaken in Task B4, in which the available evidence from Tasks B1, B2 and B3 has been gathered together and analysed. Where consensus has not been reached on any aspect, this is indicated in the report and requires further consideration by the European Commission. 2.4.23 The philosophy adopted for the development of specific recommendations for guidance updates and for research & development related to nanomaterials is based on the following aspects:  The content of a recommendation for a specific update to guidance is consistent with the focus of current REACH Guidance document, its level, and language, such that:  where the need is for ‘strategic-level’ guidance applicable to nanomaterials (i.e. high-level or overarching principles), succinct contextual information and reference(s) to primary sources of information are provided;  where the need is for updated detailed pragmatic information on, for example methods, a synopsis of specific guidance with appropriate reference(s) are provided; - 16 -  where there is simply a need identified to acknowledge an important relevance or limitation in existing guidance to nanomaterials, a simple wording clarification may be proposed.  Recommendations for updates to Guidance are made on the basis of the findings of the RIP-oN 2 tasks, and where there is a recognised case for doing so. Wide-scale acknowledgement confirming the general applicability of Guidance to nanomaterials has not been made. 2.4.24 Metric(s) to compare in the risk characterisation (Task C) 2.4.25 Critical aspects concerning descriptors and related parameters, outlining available and adequate metrics and needs have been identified based on consideration of the published positions of the OECD-WPMN, SCENIHR, and peer-reviewed scientific literature. A key issue considered is the possibility of using metrics which link toxicological effects and exposure assessment, based on a relationship between measured parameters (e.g. number of particles, alone or in combination) and existing metrics used for dosages (mass of substance per kg bodyweight). 2.4.26 A perspective of metrics used historically and currently in risk assessment and the positions of OECD-WPMN and SCENIHR are presented, followed by discussion of toxicological (in the context of inhalation, dermal and ingestion exposure routes) and ecotoxicological aspects. The report discusses metrics, measurement methods and epidemiological aspects in occupational and environmental settings, and the conversion between metrics. - 17 - 3 SUMMARY OF FINDINGS 3.1 PREAMBLE 3.1.1 A comprehensive discussion of the findings is provided in the individual Task Reports, which were refined using input from consultation with the project’s Steering Group and the Stakeholder Consultation Group, and from knowledge gained from preceding tasks. 3.1.2 This Final Project Report compiles findings from the previous deliverables into a single document, summarising the key specific issues related to nanomaterials in a REACH context and a form compatible with the possible future integration into the existing REACH Guidance on Information Requirements and Chemical Safety Assessment, with clear reference to the existing REACH Guidance Part and Chapter and subchapter. 3.1.3 The Summary of Findings is presented, with cross-referencing, according to the key outcomes from each task undertaken. 3.1.4 List of Task Reports:  Final Report on Task A: Identification and Review of Information Sources (RNC/RIP-oN2/A/1/FINAL)  Final Report on Task B1: Evaluation of the applicability of existing information requirements under REACH for Nanomaterials (RNC/RIP-oN2/B1/2/FINAL)  Final Report on Task B2: Identification and Overview of Additional Relevant Specific Intrinsic Properties for Nanomaterials (RNC/RIP-oN2/B2/2/FINAL)  Final Report on Task B3: Practical advice on relevance and applicability of existing information in fulfilling REACH information requirements (RNC/RIP- oN2/B3/2/FINAL)  Final Report on Task B4: Gap analysis of relevant intrinsic properties for nanomaterials possibly not addressed by standard test guideline methods and requiring further development of in vitro, in vivo or other methodologies ((RNC/RIP-oN2/B4/2/FINAL)  Final Report on Task B5: Scientific report on test methods and strategies for nanomaterials (RNC/RIP-oN2/B5/2/FINAL)  Joint Final Report on RIP-oN2 Task C & RIP-oN3 Task D: Metric(s) to compare in the risk characterisation (RNC/RIP-oN2/C/2/FINAL) - 18 - 3.2 IDENTIFICATION & REVIEW OF INFORMATION SOURCES (TASK A) 3.2.1 The identification and review of information sources (Task A) in RIP-oN 2 have identified screened (for relevance) and then categorised the sources of information to compile a resource for use in the subsequent tasks of the project. 3.2.2 Key organisations, FP6/7 projects and other national projects of relevance to the scope of the project were identified by the project team and through consultation with the European Commission, via JRC. Publically-available reports and outputs of relevance for the project from these sources were then identified and obtained directly from their associated websites and/or through web-based searching. 3.2.3 In relation to the OECD WPMN, it is recognised that there are three levels of accessible documents which can be used and referenced:  Published documents available on the public OECD WPMN website;  Documents approved for declassification but not yet published;  OECD documents developed by the Steering Groups and presented at meetings of the WPMN. 3.2.4 With regard to ISO and CEN publications, only published documents and those classified as being at Final Draft International Standard (FDIS) or Draft International Standard (DIS) stage were assessed and utilised where appropriate. Documents in development but not possible to be cited at the time of carrying out the RIP-oN 2 project have been identified; recommendations for them to be considered as soon as they become available have been made. 3.2.5 A substantial resource of peer-reviewed literature references was constructed. Literature from the recently completed FP7 Coordination & Support Action entitled Engineered Nanoparticles – Review of Health & Environmental Safety (ENRHES) (Stone et al., 2009), provided an initial comprehensive listing of literature published up to 31st December 2008. The ENRHES literature search was updated for the period 1st January 2009- 3rd March 2010 and supplemented with additional literature of specific relevance to the RIP-oN 2 project through a non-date-limited Boolean search strategy similar to that of ENRHES using PubMed and Web of Knowledge. In cases where excessively large numbers of references were - 19 - obtained, the searches were refined by incorporating material-specific terms (e.g. silver, titanium dioxide, zinc oxide). 3.2.6 This search strategy provided a comprehensive bibliography of references across the topic areas of physico-chemical characterisation, production, use and exposure, toxicology, epidemiology, ecotoxicology, and environmental fate and behaviour. 3.2.7 The criteria upon which judgements were made for tagging a reference as relevant for a task are outlined in the table below: Task Task Name Criterion for Inclusion B1 Analysis of current REACH Reports and publications highlighting or information requirements and commenting generally or specifically on the testing and information- existing information requirements and generation strategies for testing approaches for nanomaterials or nanomaterials. other relevant substances. B2 Identification and scientific Reports and publications which discuss overview of additional relevant physico-chemical, toxicological and specific intrinsic properties of concerning nanomaterials. nanomaterials or other relevant substances, which may propose or highlight or identify potential additional relevant specific intrinsic properties. B3 Advice on relevance and Reports and publications which discuss applicability of existing physico-chemical, toxicological and information in fulfilling REACH ecotoxicological properties of nanomaterials information requirements. or other relevant substances. B4 Gap analysis of relevant Reports and publications which themselves intrinsic properties for provide a property-test gap analysis for nanomaterials, which may not nanomaterials or extensive discussion of be addressed by standard test knowledge gaps. guidelines methods and for which further development of in vitro, in vivo or other methodologies is required. - 20 - Task Task Name Criterion for Inclusion B5 Scientific advice and proposals Reports and publications which themselves for incorporation of make specific recommendations for dealing (categorised) nanomaterial- with nanomaterials under REACH. specific requirements into REACH: (research on) integrated testing/(information generation) strategies. C Metric(s) to compare in the risk Reports and publications dealing characterisation. specifically with metrics relevant to risk characterisation. 3.2.8 The number of information sources identified and categorised for RIP-oN 2 are as follows:  89 published reports and standards from key organisations;  54 reports and standards under development from key organisations;  161 reports and publications from EU FP6/7 and other relevant international projects;  557 reports and publications reviewed in the ENRHES report;  931 additional publications from the peer-reviewed literature. 3.2.9 An appendix in the corresponding task report provides the complete listing of the sources of information. - 21 - 3.3 APPLICABILITY OF EXISTING INFORMATION REQUIREMENTS (TASK B1) 3.3.1 The applicability of the existing Information Requirements to nanomaterials under REACH has been considered and summarised for physico-chemical properties, toxicological endpoints and ecotoxicological endpoints below. Tabular summaries of the assessment are also provided. 3.3.2 Conclusions Concerning Physico-chemical Properties 3.3.3 Of the physico-chemical properties, the guidance to assess melting/freezing point (R.7.1.2), boiling point (R.7.1.3), relative density (R.7.1.4) and vapour pressure (R.7.1.5), flammability (R.7.1.10), explosive properties (R.7.1.11), self-ignition temperature (R.7.1.12) and oxidising properties (R.7.1.13) are all considered to be applicable to nanomaterials. Stability in organic solvent and degradation products (R.7.1.16) and dissociation constant (R.7.1.17) are also applicable to nanomaterials. Dissociation constant is indicated to be likely applicable to nanomaterials in OECD_15 Preliminary Review of OECD Test Guidelines for their Applicability to Manufactured Nanomaterials, ENV/JM/MONO(2009)21). Stability in organic solvent is not covered by the OECD review. 3.3.4 Surface tension (R.7.1.6) is not in general relevant for nanomaterials, except for the special sub-classes of Janus particles which may exhibit domains of differing hydrophilicity. Flash point (R.7.1.9) is not considered relevant for nanomaterials; the current Guidance states appropriately that “Flash Point is only a relevant property for liquids, thus it does not need to be done for substances that are solids or gases at room temperature.” Viscosity (R.7.1.18) is also not considered relevant for nanomaterials; the current Guidance states appropriately that “Viscosity is relevant only to liquids, therefore for many substances this determination is not required.” 3.3.5 Water solubility (R.7.1.7) is a significant relevant property for nanomaterials, and in many cases its determination is compatible with the determination of the state of agglomeration, ideally by the method of simple sedimentation. At strong agglomeration, the material would be considered 'not dispersible', which is the nanomaterial analogue of 'not soluble'. The state of agglomeration is determined by the surface modification / functionalisation shell, not necessarily by the particulate core of the nanomaterial. The same applies to the water solubility in the sense of dispersability. Water Solubility may change between as-produced and as-tested - 22 - materials (which could incorporate an as dosed / as exposed stage, and at the point(s) of interaction with the organism), so in situ characterisation is required. REACH guidance takes that into account already. We noted that SCENIHR (Risk Assessment of Products of Nanotechnologies; 28th plenary on 19 January 2009) devotes a major part of the 2009 report to the spontaneous changes of properties and strongly supports the need for in-situ characterisation, in the sense of characterisation in the biological test environment and indicated that currently available standard methods for measuring dissolution may not be applicable. However, OECD concluded that the test guideline relevant to characterising the water solubility (OECD TG 105) might be applicable under some circumstance or to some classes of manufactured nanomaterials. It stated that this TG is applicable to solutions but it is not known how the results might be impacted by the presence of a colloidal suspension, which might be present if the sample manufactured nanomaterial does not completely dissolve. Hence, further work is required to determine this and to modify the TGs, if necessary (ENV/JM/MONO(2009)21). 3.3.6 For the methods to determine the partition coefficient n-octanol/water (R.7.1.8) to be applicable to nanomaterials, the OECD methods acknowledged in the Guidance would need to be revised. SCENIHR (Risk Assessment of Products of Nanotechnologies; 28th plenary on 19 January 2009) finds that the octanol-water partition coefficient Kow is likely to have a limited role in predicting water-solids partitioning. All OECD guidelines on this property are marked as applicable under some circumstances or to some classes of manufactured nanomaterials in OECD_15 Preliminary Review of OECD Test Guidelines for their Applicability to Manufactured Nanomaterials, ENV/JM/MONO(2009)21). The property is determined by the presence of the substance in an oil-water interface. The possibility of a particle’s surface changing with age and exhibiting different partitioning behaviour may result in the measurement being somewhat artificial with limited predictive value for environmental fate. 3.3.7 Granulometry (R.7.1.14) is, without doubt, the central issue for any nanomaterial. This property may change from as-produced, hence as-tested (or in situ) characterisation is required. Consideration of the adequacy of the definition of granulometry used in the Guidance and the appropriateness of incorporating properties distinct from purely size distribution characterisation, has been undertaken as part of Task B2. Methods specifically mentioned (Cascade impaction - 23 - "A well established techniques to measure the distribution of particles of respirable or inhalable size particles of all kind, size range: 0.1-20 and 0.5-80 microns", Laser scattering/diffraction "The method is suitable to determine the distribution of particles of respirable and inhalable size. Particles of all kind Size range: 0.1-100 microns"; Rotating drum method (BS EN 15051) do not cover the range below 100nm. Hence they are not adequate to generate data on nanomaterials. The important properties of surface area, state of agglomeration, shape, size & length (particle / platelet / fibre etc) are already acknowledged in the REACH Guidance under granulometry (Table R.7.1-30 Methods to determine particle size distribution of the material as it is), but there is a need of explicit guidance for granulometry of nanomaterials. 3.3.8 For adsorption/desorption (R.7.1.15), the definitions used can be applied with minimal changes also to a dispersed (not dissolved) substance such as a non- soluble nanomaterial. However, the guidance document states that the methods may not be suitable for: i) substances that react with the column, ii) solvent or other test system components, iii) surface active substances; iv) substances that interact in a specific way with inorganic soil components such as clay minerals; v) inorganic compounds; and vi) moderate to strong acids and bases. Nanomaterials may be close in their properties to clay minerals, surface active substances and inorganic compounds. All methods require a quantitative analytical method for the substance, reliable over the range of test concentrations. This presents an issue for many nanomaterials, if identification both by chemical nature and physical structure is to be performed. A practical solution for most cases is the elemental analysis, e.g. by ICP-MS of fractions, since most nanomaterials contain inorganic elements. Similar techniques (FFF-ICP-MS) have been developed for ecotoxicology sample characterisation, but are far from ISO or OECD standardisation (compare Hasselöv et al, Ecotoxicology (2008) 17:344–361). 3.3.9 Conclusions Concerning Toxicological Information 3.3.10 The toxicological data requirements under REACH that are further described in the corresponding guidance documents R.7a and R.7c are also relevant for the assessment of nanomaterials (summarised in Annex 2 of RNC/RIP- oN2/B1/2/FINAL). According to OECD WPMN and SCENIHR, the described and preferred OECD test guidelines to fulfil these data requirements are basically also - 24 - applicable for the assessment of nanomaterials (SCENIHR, 2007 and 2009; OECD WPMN, ENV/JM/MONO(2009)21). 3.3.11 Concerning toxicological tests, special attention needs to be given to measuring, dosing, delivery and tracking of nanomaterials in the test system. Furthermore, concerning toxicological endpoints it is also important to consider the physicochemical characteristics of the nanomaterial, including in the dosing vehicle. Therefore, there is a need for guidance on sample preparation and in situ characterisation for the toxicological assessment of nanomaterials. 3.3.12 For all toxicological tests, an adequate characterisation of the tested nanomaterial should be carried out and appropriate consideration of the actual exposure of the test system (e.g. allowing for possible agglomeration/disagglomeration) and the appropriate dose metric should be given. 3.3.13 With regard to non-testing data, it should be noted that non-testing approaches such as (Q)SAR, read-across etc are currently not applicable to nanomaterials, because available models and understanding of issues such as similarity are not trained/developed to properly address nanomaterials. As such their use will require scientific justification. However, the possibility to extrapolate certain information from studies conducted with bulk forms of the substance or modifications of the respective nanoforms which could influence the decision on testing or the testing strategy of the nanomaterial should be addressed. Guidance on such a grouping approach for nanomaterials based on toxicological test results is currently not available and therefore needed. Research is needed on these issues so that appropriate guidance can be developed. 3.3.14 According to OECD WPMN, studies on toxicokinetics of nanomaterials are important for the assessment of their potential health effects (OECD WPMN, ENV/JM/MONO(2009)21). Due to the current lack of knowledge concerning toxicokinetic behaviour of nanomaterials, especially concerning absorption, distribution and excretion, we propose to include newly developed methods in the guidance on toxicokinetics, e.g. barrier transfer methods (ENRHES review, Stone et al., 2009), to assess, if appropriate, the toxicokinetic behaviour of certain nanomaterials. - 25 - 3.3.15 Conclusions Concerning Ecotoxicological Information 3.3.16 The current ECHA guidance document on Chemical Safety Assessment ECHA (2008) refers specifically to a number of OECD Test guidelines. The appropriateness of these OECD Test guidelines as well as other guidelines have been reviewed by the OECD project ‘Safety Testing of a Representative Set of Manufactured Nanomaterials’ (ENV/JM/MONO(2009)21). OECD stated that: “For 24 OECD ecotoxicity test guidelines, the subgroup for biotic effects section concluded that the guidance on preparation, delivery, measurement, and metrology is currently insufficient for testing of manufactured nanomaterials” (ENV/JM/MONO(2009)21, p. 13). 3.3.17 Currently 31 OECD guidelines exist for the determination of potential ecotoxicological effects of test substances in relevant environmental compartments (aquatic, terrestrial, sediment) after acute or chronic exposure. Endpoints determined in these studies, with a diverse set of species representing different taxa and environmental compartments, are mortality and non-lethal endpoints like growth, respiration, reproduction and development. The parameters determined in these studies generally reflect responses of complete organisms covering several modes of toxicity and often also several routes of exposure. Thus, the basic toxicological properties as well the endpoints described and determined in these guidelines are adequate and relevant also for nanomaterials. 3.3.18 At the moment, 18 OECD test guidelines exist to determine environmental fate covering methods evaluating e.g. ready biodegradability (OECD 301 A-F) to further guidelines on physico-chemical properties which will influence substance behaviour in the environment (e.g. OECD 105 Water Solubility, OECD 107/117/123 Octanol- water Partition Coefficient; OECD 111 Hydrolysis; OECD 106/121 Adsorption to soil or sediment). OECD test guidelines also exist for bioconcentration (OECD 305) and bioaccumulation methods (OECD 315, 317). For the environmental fate assessment, a pre-requisite for biodegradation is that the test material is based on organic carbon chemistry (for bulk chemicals as well as for nanomaterials). Furthermore, the test strategy and studies used in this assessment may need to be adapted to the particulate nature of nanomaterials dispersed in water or the test- specific test media. - 26 - 3.3.19 Within the RNC/RIP-oN2/B1/2/FINAL report, and in the conclusions of the OECD WPMN document ENV/JM/MONO(2009)21, it was noted that many of the majority of the OECD test guidelines are applicable (in some cases with conditions). However, the guidance given on preparation, delivery of test substances to test system, exposure quantifications, dose metrics, measurement, and metrology in all of these test guidelines is currently insufficient for testing of nanomaterials. Therefore preliminary guidance notes have been developed in the OECD-WPMN for practical testing (ENV/JM/MONO/201025 Preliminary Guidance Notes on Sample Preparation and Dosimetry for the Safety Testing of Manufactured Nanomaterials). 3.3.20 Currently, research also suggests that particle number, size distribution, surface area, charge and other surface characteristics might be better predictors of toxicity, and more accurate metrics to be used in statistical determinations of dose-response relationships than (mass) concentration. Given the historical and established use of (mass) concentration as a dose metric, it continues to be important in assessing dose-response relationships and modelling toxic effects, however, its usefulness in particle toxicology depends not least on knowledge of the nature of the substance under test. Sample preparation and delivery issues are complicated by the stability and consistency of the properties of nanomaterials in the various exposure media used. Due to the current uncertain situation, a combination of dose metrics is recommended for nanomaterial testing. In general, the current test guidelines do not provide adequate direction for monitoring the characteristics of nanomaterials over the duration of tests. (ENV/JM/MONO(2009)21 Preliminary Review of OECD Test Guidelines for their Applicability to Manufactured Nanomaterials). 3.3.21 (Q)SAR analyses are mentioned in the guidance documents R.7b and R.7c as another way to fulfil the information requirements, but models currently used are not applicable because they were not designed for specific nanomaterial characteristics. 3.3.22 Field studies in ecotoxicity and environmental fate can contribute significantly to the

Teratogenic Effects

data inhalation REACH most requirements dose on exposure) for reproduction or with toxicity, dermal Toxicity / is (required what IX oral, to Annex VIII): Repeated (90 (either depending based Properties days) Toxicity on Developmental VIII, REACH reference Annexes according toxicity Combined studies Guidance Combined physicochemical relate characterisation agglomeration/disagglomeration) R.7.5 R.7.7 the - tested 38 - number VII, of to the to study methods Chronic B.33: B.30: Carcinogenicity carcinogenicity particularly adequate possible OECD requirements 452/EU 453/EU 870.4200: which an for / Carcinogenicity / to 451: tests, addition OECD OECD chronic US-EPA studies account TG toxicity TG toxicity TG allowing toxicological OECD study OECD chronic into (e.g. may to in taken all system, as- as- t/y, Property 1000 1) tested1) 1) For test given. from the be be to change produced above need vehicle. of should relevant imported known) substance indicated) considerations in actual (if for not dosing exposure nanomaterials? as specifically important including relevant any or the redundant relevant the metric o Property o + manufactured if dose for (only to - endpoints consideration appropriate substances appropriate nanomaterial, data inhalation REACH toxicological of appropriate the requirements for (required dose most on exposure) the to long-term consideration or is with toxicity, dermal what Properties Annex and Repeated (either depending based Carcinogenicity X oral, IX): on Concerning 1) characteristics nanomaterial, and Guidance number - 39 - R.7.8 R7.9 R.7.8 R.7.9 R.7.1.15 REACH reference VII): Annex toxicity (preferred toxicity respiration Biotic desorption a study RNC/RIP-oN2/FPR/1/FINAL to as documents Short-term invertebrates Growth (algae Biotic biodegradability Short-term inhibition preferred) 302/303/305/306 111 Abiotic. according Absorption/ sludge Hydrolysis methods TG on Daphnia) TG plants TG requirements TG on 209 TG TG of TG 202 201 301 Ready 203 fish Activated testing pH 121 310 guidance OECD OECD testing species OECD aquatic OECD A-F OECD testing OECD inhibition OECD OECD function OECD screening to and as- as- addition may to in from + REACH + 1) tested1) t/y): 1) 10 change produced t/y, 1) 1 in Property above requirements relevant substance imported for not nanomaterials? as specifically or relevant data relevant any or O O or O o + manufactured Property manufactured redundant for ecotoxicological - o + data substances

Supplemental label elements

may importantly advance and facilitate the use of responsible waiving, read-across, and QSARs, and the ability to select appropriate metrics that in turn widens the relevance and applicability of test methods to nanomaterials. 3.4.9 Furthermore, many biological effects may be only indirectly or non-causatively associated with a property or properties being measured. The potential for confounding in the assessment of a nanomaterial’s hazard and exposure is particularly pertinent in the context of sample preparation and the limitations of - 44 - experimental techniques for characterisation and assessment of hazard and exposure. 3.4.10 Conclusions Concerning Additional Relevant Specific Intrinsic Properties For Nanomaterials 3.4.11 Potential additional relevant specific intrinsic properties have been identified on the basis of an objective review of published scientific sources of information, and a pragmatic and rationalised approach to their incorporation into REACH has been suggested. By virtue of their inclusion in this final report they are considered to be relevant to nanomaterials. For more in-depth considerations and details of the additional relevant specific intrinsic properties and how they have been identified in Task B2 and considered further in subsequent Tasks, the reader is referred to the relevant reports for Task B2, B3, B4 and B5. 3.4.12 Moreover, some properties or endpoints recommended in the sources considered in Task B2 for nanomaterials testing are not strictly intrinsic properties or the proxy- effect of a single intrinsic property, but are influenced by the material’s ‘conditions’ or

Health hazards

testing of nanomaterials, citations for and details from which are of relevance for incorporation into the updated REACH Guidance (ECHA, 2008. Chapter R.7a), specifically: Chapter 6 of the ENRHES review (Stone et al., 2009);  Nanocare review on the in vivo and in vitro assessment of nanotoxicology (Kroll, 2009)  Nanocare review on the issues related to genotoxicity assessment of NMs (Landsiedel et al, 2009).  Review by Gwinn & Tran (2009). 3.5.63 The outputs and recommendations of the FP6/7 projects indicate that both in vitro and in vivo findings of projects have identified clear variations in fate and biological effect according to nanomaterial type. 3.5.64 In addition, it is notable that for many NM, any toxicity exhibited may result from multiple mechanisms. For example, the ENRHES project notes that metal oxide nanoparticle toxicity is thought to be inflammogenic, oxidative, and genotoxic in mechanism; with all endpoints considered to be inherently linked. Testing for NM toxicity within REACH should therefore consider this complexity of mechanism in the experimental approach adopted. 3.5.65 In addition, there also exists a strong consensus from projects that thorough and accurate particle characterisation of nanoparticles prior to commencing toxicological testing is an essential component of understanding their potential toxicity. 3.5.66 R.7.2 Skin- and eye irritation/corrosion and respiratory irritation, & R.7.3 Skin and respiratory sensitisation 3.5.67 To date, no information of specific relevance to this information requirement has been identified within the outputs of those FP 6&7 projects reviewed. However, the Nanoderm project, which aimed to develop new techniques and methodologies to complement high resolution transmission microscopy (HRTEM), undertook a number of studies to examine dermal penetration by NMs, and in vitro testing to investigate damage at the cellular level. This included monitoring for cell proliferation, apoptosis, necrosis, expression of adhesion molecules, and differentiation). - 61 - 3.5.68 The project concluded that there was no indication that diffusive transport of nanoparticles occurred, thus suggesting that existing techniques such as static Franz-diffusion cell (OECD 428) might not be suitable to study nanomaterial permeation into the skin. Moreover, the project consortium suggested that standardised protocols to study mechanical flexion need to be developed to study dermal penetration of nanomaterials. 3.5.69 Within the NANOSH project, exposure in healthy and immune-compromised mice (asthmatic model) using a 28 day repeated-dose testing assay (discussed in detail within the repeated-dose IR summary), it was found healthy animals expressed an immune and cytokine medicated response to TiO2, whereas compromised (asthmatic) animals showed suppression of most mediators of allergic asthma when exposed. Its results suggest that TiO2 modulates airway inflammation depending on allergic status and coating of the nanoparticles. 3.5.70 R.7.4 Acute toxicity 3.5.71 A number of FP6/7 projects undertook short term exposure studies both in vivo and in vitro. Although the exposure periods were not within a 24hour period, their results are nonetheless which are relevant to the acute toxicity IR. Notable examples are detailed below: 3.5.72 In vivo 3.5.73 The Nanocare developed a short term inhalation study in the rat (short inhalation exposures over 5 days, followed or not by a recovery period of 21 days), in order to provide an earlier screening of particle toxicity compared to the typical 90-days study. Endpoints of cytotoxicity & inflammation were monitored, via analysis of broncho alveolar lavage fluid (BALF). The results of this study indicated that the effects observed in the lung with TiO2 were similar in this short term inhalation study as in typical sub-chronic inhalation studies (such as that outlined within OECD TG412). From this pilot study, the authors recommended that analysis be carried out at day 3 of exposure, and at 21 days after the end of exposure. Their proposed minimal selection of endpoints for inclusion was BALF differential cell count, total protein, LDH, GGT and glucosaminidase activities. The authors also suggested that haematology and respiratory tract histopathology, which are required in OECD studies, were not very sensitive in this short-term inhalation study. - 62 - 3.5.74 The NANOSH project undertook 5 day inhalation exposure testing in mice using TiO2, silica coated TiO2, SWCNT, MWCNT and ZnO NPs in order to investigate endpoints of genotoxicity & pulmonary inflammation. 3.5.75 The PARTICLE_RISK project undertook an acute in vivo assessment of inflammation and genotoxicity was via intra-tracheal instillation in compromised ApoE-/- mice (Jacobsen et al. 2009). 3.5.76 The NanoKem project undertook work to develop an in vivo murine model for screening lung inflammation following intra-tracheal instillation (Roursgaard et al., 2010). Acute inflammation was assessed by BALF analysis. The authors also suggested that at 3 months BALF analysis and histopathology were sufficient to assess sub chronic lung inflammation. 3.5.77 It is notable that in vivo acute inhalation exposures are not generally encouraged within REACH due to their animal welfare implications, and the general move towards weight-of-evidence approaches utilising pre-existing information and supported by QSAR and other non-animal data sources. However, as there is little pre-existing information on nanomaterial toxicity any information generated by projects to date it is likely that the results of those in vivo investigations outlined above are of relevance to their evaluation within the context of REACH. 3.5.78 In vitro 3.5.79 Work is underway in a number of projects to develop in vitro assays to study relevant endpoints of acute toxicity. For example, the NANOSH project demonstrated that TiO2, silica coated TiO2, SWCNT, MWCNT and ZnO NPs were dose-dependently cytotoxic in macrophages and dendritic cells, and that the most significant induction of inflammatory mediators was in macrophages following nanoparticle exposure. The PARTICLE_RISK project also investigated pulmonary inflammation and genotoxicity within pulmonary, endothelial and immune cell lines. 3.5.80 Although at the current time no validated tests exist for in vitro acute toxicity, it is recognised that in vitro approaches would be important for the replacement of in vivo acute toxicity testing. Therefore, assays such as those utilised here may be both relevant and applicable after they will have gone through the appropriate validation and regulatory adoption processes. In addition, such approaches will - 63 - support the 3Rs principle in relation to the number of animals required for acute toxicology testing. 3.5.81 R.7.5 Repeated dose toxicity 3.5.82 In relation to repeated dose toxicity, the ENRHES project observed that in many published peer-reviewed studies a single dose was administered and toxicity assessed at a number of post exposure time points. However, it notes that repeated dose studies over a long time period are likely to be of greater relevance in consideration of the potential risk of fullerenes within occupational or consumer settings. 3.5.83 Of those projects completed to date, the following outcomes are of particular relevance to consideration of repeated dose toxicity testing within REACH. 3.5.84 In vivo 3.5.85 The Nanocare project undertook a 28 day inhalation study within rats (followed by a 90 day recovery period) using a protocol similar to that of OECD TG412 (Pauluhn, 2009). Endpoints of cytotoxicity & inflammation were monitored. The protocol produced findings similar to those produced when carrying out repeated dose testing according to OECD TG413. ). The authors stressed the importance of having at least 3 months recovery post exposure in the study design, to allow assessment of pulmonary toxicity related to biopersistence of nanomaterials. In particular, the recovery period is essential in order to avoid misinterpretation of the results about retention-specific effect due to lung overloading. 3.5.86 The NANOSH project also developed a (non-standard) 28 day repeated dose protocol. Following inhalation exposure in healthy and immune-compromised mice (asthmatic model), the consortium found that particles accumulate in alveolar macrophages, and that of the nanomaterials tested, silica-coated TiO2 nanoparticles elicited a clear-cut pulmonary neutrophilia in healthy mice. Healthy animals expressed an immune and cytokine medicated response to TiO2, whereas compromised (asthmatic) animals showed suppression of most mediators of allergic asthma when exposed. Its results suggest that TiO2 modulates airway inflammation depending on allergic status and coating of the nanoparticles. The project therefore provides a repeated-dose testing protocol which may be of - 64 - relevance to NM toxicity within the context of REACH, and also information which is relevant to considering immune-modulated sensitisation. 3.5.87 Projects underway with relevance to both acute and repeated toxicity testing 3.5.88 Several of those FP6/7 project reviewed which are underway but yet to provide significant outputs include investigations which, once completed, are likely to be of relevance to both acute and repeated dose toxicity testing considerations for nanomaterials within REACH. 3.5.89 Of those FP6/7 projects reviewed, the NANOMMUNE project is the only to focus solely on the immune aspects of NM toxicity. The project is undertaking an array of in vitro (murine and human cell lines, as well as primary and more complex co- cultures of cell lines and/or primary cells) and in vivo (following exposure via the lung and skin) investigations, as well as transcriptomic and oxidative lipidomic profiling strategies to determine specific nanotoxic profiles (signatures) of these materials. 3.5.90 In addition, as part of the NANOTEST project, investigations of immune toxicity are also underway. These include monitoring of lymphocyte proliferation, phagocytic activity, adhesion molecules and interleukins/cytokines. 3.5.91 The outcomes of this work, once completed and published, should provide relevant information to developing testing strategies for consideration of immune toxicity within acute and repeated-dose testing paradigms. 3.5.92 Also of relevance to both acute and repeated dose toxicity IRs, the NANOTEST project is working to develop alternative testing strategies and high-throughput toxicity-testing protocols using in vitro and in silico methods. In vitro screening tests being undertaken by the consortium are intended to provide alternatives to animal testing to a number of major target organs (blood, vascular system, live, lung, placenta, digestive system and central nervous system), via three main exposure routes (intravenous, respiratory and digestive). In vivo, validation of in vitro testing protocols which investigate toxicity to the heart/aorta, liver, lung, brain, blood, spleen, and bone marrow, following a maximum of three doses by either intravenous injection, intratracheal instillation or per os administration route will be selected according to the medical use of the nanoparticles under consideration.. In - 65 - addition, in silico, development of quantitative structure-activity relationships (QSARs) and physiologically-based pharmacokinetic (PBPK) modelling for nanomaterials is underway. The outcomes of this work, once completed and published, should provide relevant information to further developing NM testing strategies within the context of REACH. 3.5.93 R.7.6 Reproductive and developmental toxicity 3.5.94 To date, there exists a limited body of work on the reproductive and developmental toxicity of nanomaterials. Of the preliminary work undertaken, the following experiments and results may be of use when considering testing of nanomaterials within the context of REACH. 3.5.95 A study undertaken as part of the NanoInteract project (Park et al, 2009), examined the in vitro effects of silica nanoparticles on stem cell differentiation, with a focus on the uptake of particles by the cells and at their cytotoxic effect, using the WST-1 assay. One of the important features of this study is that it tested for potential interference of the material tested in the WST-1 assay. Based on the results, the authors suggested that this in vitro embryonic stem cell differentiation test might be a valuable tool to test embryotoxicity of nanomaterials. 3.5.96 In vivo work undertaken within the NanoKem project used a non-standard protocol to assess the effect of prenatal exposure to TiO2 nanoparticles in mice via inhalation (Hougaard et al. 2010). The study focused both on maternal and embryonic effects, and investigated endpoints including lung inflammation, quantification of Titanium in tissue and milk, and several behavioural tests. 3.5.97 One published output of the NANOTEST project considers reproductive toxicity in vitro. Bhabra et al (2009), details results of in vitro work on the translocation and potential for genotoxic effects to be elicited by nanoparticles across the placental barrier. However, whilst the results of this work are interesting to the field from a mechanistic perspective, no results are of direct relevance to the consideration of NM within the context of REACH. 3.5.98 R.7.7 Mutagenicity & Carcinogenicity 3.5.99 The endpoint of mutagenicity/genotoxicity was frequently studied within the FP6 & 7 projects using a variety of different assays and techniques. Amongst these were: - 66 -  The Comet assay – NanoInteract, NANOSH, PARTICLE_RISK, NANOTEST  micronucleus assay – NanoInteract, NANOSH, NANOTEST  lacZ assay – NanoInteract  FISH (fluorescence in situ hybridisation) – NANOSH 3.5.100 Of the in vitro test methods outlined, the micronucleus assay (OECD test guideline 474), comet assay, and chromosome aberration tests are specifically recommended as Information Requirements under REACH guidance. However it is recognised within the guidance that many protocols for mutagenicity are modified following expert judgement, or alternatives used as appropriate. 3.5.101 As part of the Nanocare project, Landsiedel et al. (2009) published a review focusing on the issues related to genotoxicity assessment of NMs. This recommended that as the mechanisms triggering genotoxicity are numerous, the use of several assays in order to study the potential genotoxicity of any substance should be considered. It also highlighted that the most commonly used assay to test genotoxicity of nanomaterial was the comet assay, which at this time is not an OECD test guideline, but is undergoing validation by ECVAM. This was followed by the micronucleus assay, which was recently included in the OECD test guidelines collection. 3.5.102 R.7.12 Guidance on Toxicokinetics 3.5.103 With reference to toxicokinetics, the ENRHES project considered the toxicokinetics of uptake (ingestion, inhalation, dermal adsorption and injection), distribution, metabolism and excretion of for four key types of manufactured unfixed nanoparticles and nanotubes in and by the body (CNT, fullerenes, metals and metal oxides). Toxicokinetic aspects specifically considered included persistence and bioaccumulation potential of nanoparticles and nanotubes in the body; differences in toxicokinetics and any subsequent toxicity posed by variations in nanoparticle size, physical structure, chemical composition; mechanisms of interaction of nanoparticles with cells and their components, and partitioning within and between tissues in organisms. - 67 - 3.5.104 Although the project did not itself generate any novel data, the body of evidence reviewed in ENRHES summarises a wealth of knowledge relating to the toxicokinetics of NM within the body, and draws conclusions on this. In particular, the review focussed on in vitro and in vivo studies using the lungs, skin, gastrointestinal tract (GIT), or blood as routes of entry, with the inclusion of sub- sections which examine toxicity at a number of target organs following the realisation that nanomaterials can distribute from their exposure site within the blood or even nerves. As such, ENRHES represents one of the most comprehensive reviews of toxicokinetic data available to date, and is thus valuable to the consideration of NM within the context of REACH. 3.5.105 The Nanocare project developed new in vitro models to assess the translocation of particles well as their interactions with the cells, both of which provide relevant input to the building of a toxicokinetic profile for nanomaterials. 3.5.106 Using a range of cell types, the INOS project undertook in vitro studies which considered cell-particle interactions and translocation into the cellular compartments, outcomes of which may be of relevance to establishing toxicokinetic profiles of NM within the context of REACH. 3.5.107 The NANOTEST project is undertaking in vitro investigations of uptake and intracellular distribution of NPs Uptake and intracellular distribution of NPs (using labelled NPs and endpoints of Phagocytic activity), and Transport of NPs across biological barriers (using TEER and diffusion). This work is underway thus there are no significant results to date. 3.5.108 The PARTICLE_RISK project undertook investigation of translocation and effects of nanoparticles in vivo via three different routes of administration: (1) intratracheal instillation into the lungs (IT) or (2) intravenous injection into the tail vein or (3) intra- oesophageal instillation (gavage) into the gastro-intestinal-tract (GI-tract). The results indicated that the pattern of nanoparticles biodistribution differed according to both administration route and physico-chemical characteristics. 3.5.109 To investigate the potential neurotoxicity of NPs, the Neuronano project is undertaking a series of investigations relating to the physico-chemical properties of NM dictating their translocational potential and fate in vivo. To date, in vivo dosimetry studies have suggested that route of exposure, size and protein corona - 68 - around the nanoparticle are all influencing factors. However, to date, no significant translocation of nanoparticles into the brain has been shown. In vitro results show that transcytosis of nanoparticles is an energy-dependent mechanism which is a function of the cell polarisation of the system, and depends on many parameters such as the surface, size and protein corona. 3.5.110 It is worth noting that some of the projects stress that the concept of “protein corona” associated with NMs forms one of the major differences between chemical and particle toxicity. According to these projects (NanoInteract, PARTICLE_RISK), such nanoparticle-protein is likely to be important in determining the “identity” of the nanomaterial and therefore its subsequent interaction with the environment, such as cellular uptake. 3.5.111 Additional endpoints of specific relevance to NMs 3.5.112 The following endpoints, although not specific Information Requirements, are considered to be of particular relevance to establishing toxicity of NMs and as a result are included in many of the FP6/7 projects reviewed. Relevant testing outcomes for each are discussed in detail within the B2 report. However, commonly utilised assays for each are highlighted below. 3.5.113 Cell Viability 3.5.114 The endpoint of cell viability was frequently studied within the FP6 & 7 projects using a variety of different non-standardized assays and techniques, and forms an important endpoint which should be further considered in relation to recommended updates to the REACH Guidance for toxicity testing (ECHA, 2008. Chapter R.7). Amongst those assays most commonly utilised were:  Cell Morphology - NANOTEST  BALF cell count & protein analysis (in vivo) - Nanocare  Cell metabolic activity (MTT) – Nanocare, Nanoderm, PARTICLE_RISK, NANOTEST  Cellular membrane integrity (LDH release) – Nanocare , PARTICLE_RISK  Lung cell damage (gamma glutamyl transferase; GGT) - Nanocare  TransEpithelial Electrical Resistance (TEER) - Nanocare - 69 -  Apoptosis – NanoCare, Nanoderm  Necrosis - NANOTEST 3.5.115 Oxidative Stress 3.5.116 The initiation of oxidative stress has been frequently studied within the FP6/7 project outputs, and forms an important endpoint which should be further considered in relation to recommended updates to the REACH Guidance for toxicity testing (ECHA, 2008. Chapter R.7). Amongst the non-standardised assays most commonly utilised were:  ROS Production – Nanocare, NANOTEST  Glutathione Status – Nanocare, PARTICLE_RISK, NANOTEST  NO Generation – NANOTEST 3.5.117 Within the ENRHES project, the mechanism of toxicity associated with metal oxides was thought to be inflammogenic, oxidative, and genotoxic in nature; with all endpoints considered to be inherently linked. In addition, ENRHES highlighted that it appears fullerene toxicity also involves an oxidant driven response, thus suggesting that toxicity evaluations should evaluate the potential of fullerenes to cause oxidative stress and related consequences such as inflammation or genotoxicity (Johnson et al. 2010). CNT pathogenicity was noted to be most likely linked to their ability to elicit oxidative stress and inflammation. 3.5.118 The INOS project undertook in vitro assays for oxidative stress, the outputs of which may be of relevance to supporting consideration of this outcome in its recommendation as a specific endpoint for consideration of nanomaterials within the context of REACH (justification is outlined within the B2 report). 3.5.119 Pro-Inflammatory effects in vitro 3.5.120 The investigation of inflammation has been commonly studied within the FP6/7 project outputs, and forms an important endpoint which should be further considered in relation to recommended updates to the REACH Guidance (ECHA, 2008. Chapter R.7). Amongst those assays most commonly utilised were: - 70 -  TNF-ɑ – Nanocare, PARTICLE_RISK  Trypan Blue assay – NANOSH  Cytokine production – NANOTEST, Nanocare, PARTICLE_RISK  Signalling pathways (NFκB, AP-1 etc) - NANOTEST 3.5.121 The PARTICLE_RISK project also investigated pulmonary inflammation and genotoxicity within pulmonary, endothelial and immune cell lines. 3.5.122 Within the ENRHES project, the mechanism of toxicity associated with metal oxides was thought to be inflammogenic, oxidative, and genotoxic in nature; with all endpoints considered to be inherently linked. CNT pathogenicity was also noted to be most likely linked to their ability to elicit oxidative stress and inflammation. 3.5.123 The Nanocare project utilised a co-culture system to study pro-inflammatory response in vitro (via monitoring for IL-8 release). Its results indicate that in vitro the use of a co-culture system as well as the use of lipopolysaccharide stimulation increases the sensitivity of the assay, in detecting pro-inflammatory potential of a nanomaterial. 3.5.124 Mechanistic Aspects / Experimental Design 3.5.125 All projects were in consensus that the physico-chemical characterisation of NMs to be tested for human health endpoints was imperative to enable a full interpretation of test results. This is discussed in detail within the physico-chemical characterisation section of RNC/RIP-oN2/B3/2/FINAL. 3.5.126 In relation to study design, the ENRHES review noted that exposure method, dose administered, species used, cell type under investigations and light conditions also have the potential to impact on the toxicity of metal oxide particles. In addition, the reviewers highlighted that experimental quality (including the concentrations used & model selected), of conducted studies is of vital importance when considering the risk associated with metal oxide exposure. 3.5.127 The INOS project emphasised the importance of preliminary characterisation of the materials tested, in the media used, when assessing the hazard in vitro (Meissner - 71 - et al., 2009b). It also stressed that comparing toxicity test results from the salts of nanoparticles that contained metals to the particles themselves to establish whether the effect produced was linked to the NP or its salt. 3.5.128 The Nanocare project developed several standard operating protocols (SOP) for bioassays, dispersion of the nanomaterial tested in relevant media and for cell exposure. This need to develop SOPs highlights the fact that current standard guidelines need to be adjusted for the testing of nanomaterials and/or that additional tests have to be carried out to avoid misinterpretation of results. 3.5.129 In addition, the Nanocare project (Kroll, 2009) published a number of reviews on the in vivo and in vitro assessment of nanotoxicology, which highlights specific properties of nanomaterials which make them more prone to interfere with assays, and suggests that new standardised in vitro methods might need to be developed to assess the toxicity of nanomaterials which overcome such drawbacks. 3.5.130 Finally, the IMPART project highlighted undertook a large review of the literature about nanotoxicity and highlighted several key issues related to the assessment of nanomaterial hazard. Specifically in relation to experimental design and conduction, the project highlighted the need to consider interference of nanomaterials with toxicity assays, the importance of verification of oxidative stress as a marker for potential toxicity, modification of dosimetrics by particle aggregation, development of new strategies to determine the mechanisms of action for toxicity, and formation of models to predict potential impacts over their whole life-cycle (McCormack et al., 2008). All of these considerations are particularly relevant to hazard identification and characterisation within REACH. 3.5.131 ECOTOXICOLOGICAL INFORMATION 3.5.132 The key experience relevant to REACH from the FP6/7 projects is summarised below. The advantages, limitations and applicability of which are being considered further within the gap analysis (Task B4) with a view to informing the updated REACH guidance. 3.5.133 Only a few FP6/7 projects i.e. ENRHES, INOS and Nanointeract have reported results and/or used methods (i.e. fish cytotoxicity, growth inhibition of algae and - 72 - acute toxicity of crustaceans and fish embryos) that are relevant in regard to providing practical advice in the REACH context. 3.5.134 As noted previously, it is evident from the FP6/7 projects with relevance for the ecotoxicity of NM that a thorough and accurate particle characterisation is an essential component of assessing the potential ecotoxicity of nanomaterials. 3.5.135 Considered specifically, ENRHES, INOS and Nanointeract have underlined the importance of considering agglomeration behaviour over time in various form of medium and hence underline the importance of measuring the state of agglomeration both at the beginning and the end of the experiment (Kuhnel et al. 2009, van Hoecke et al., 2009, Stone et al. 2009). 3.5.136 An FP6/7 funded review of the scientific literature (ENRHES) has highlighted that changes in the metal speciation (association of a nanomaterial with a molecular or ionic dissolved chemical substance) can occur depending on redox conditions, salt content, etc. and this need to be determined and reported in order to enable to interpretation of the reported studies in a REACH context (Stone et al. 2009). 3.5.137 It has furthermore been highlighted how the use of any solvents (for instance THF which was used in some of the first ecotoxicological studies on C60) should be avoided since, as for the mammalian toxicology studies, it has been demonstrated that not only nanoparticle/solvent interactions may affect ecotoxicity, but also solvent degradation products may be responsible for some of the observed effects. Instead of using solvents, extensive stirring and sonication can be used as alternatives to bring some nanoparticles in dispersion. - 73 - 3.5.138 B3 Sub-Task II: Practical advice on the Relevance and Applicability of Experience Reported in the Scientific Literature on Nanomaterial Characterisation, Hazard Identification and Assessment for Workers, Consumers and Environment in the REACH Context 3.5.139 PHYSICO-CHEMICAL PROPERTY INFORMATION 3.5.140 Key characterisation considerations 3.5.141 Based on a review of the scientific literature, it is evident that it is now widely acknowledged that adequate characterisation of a nanomaterial is necessary to accompany any toxicity study, particularly in cases where nanomaterials (e.g. carbon nanotubes) can be produced by different processes yielding notionally the same material, but which exhibit quite different properties (Zuin et al., 2007; Boverhof & David, 2010). 3.5.142 Within the scientific literature, several publications have been identified which provide useful overviews and comparisons of the most common methods for the physico-chemical characterisation of nanomaterials, further details from which are of relevance for consideration in relation to the updated REACH Guidance (ECHA, 2008. Chapter R.7a), specifically Nanoforum (2006) and Tiede et al. (2008) 3.5.143 A number of key general points have been noted from the review of the scientific literature for further consideration in the gap analysis and subsequent guidance amendments: o Whilst characterisation of nanomaterials as-produced or as-supplied is the most direct and currently realistic approach to obtaining physico-chemical information about the material being studied, this data may not appropriately represent the properties of the material when in contact with the environment in which it is being observed, for example in air or physiological environments of in vivo or in vitro assays; o It has been suggested that adequate particle characterisation should be performed in three distinct phases, primary, secondary, and tertiary, where: primary characterisation is performed on particles as-synthesised or as- received in its dry native state; secondary characterisation is performed on particles in the wet phase as a solution or suspension in aqueous media; - 74 - tertiary characterisations are performed on particles following interactions with cells under in vivo or in vitro conditions (Sayes and Warheit, 2009). o Characterisation after administration is particularly advantageous where the possibility of physico-chemical changes in the material before and after administration exists; o It is recognised that in many cases characterisation at the point of administration will remain to be essential for the comparison of studies; o The limitations of each analytical method for nanoparticle characterisation can lead to inconsistent results and, therefore, to inaccurate predictions of material properties and structure (Carter et al., 2005); o Nanoparticle sizing standards, as well as standardised methods for sampling and measurement, are urgently required to overcome the problem of inconsistent data (Borm et al., 2006); o The lack of consistent reference materials and standards further exacerbates this problem (Lead and Wilkinson 2006). Some progress has been made recently regarding reference materials for characterisation, but standardised nanoparticles are not yet widely available and researchers have to rely on commercially available, often not well-characterised, nanoparticles. 3.5.144 Sample preparation 3.5.145 Within the review of the scientific literature, sample preparation has been highlighted as one of the most critical steps towards successful characterisation of nanoparticles, in which there are many variables to consider when designing a method for preparation. This issue is of key importance for further consideration in the gap analysis (Task B4) and proposals for amendments to the guidance. In relation to this issue, key points to note are: o the need to have “reliable” sampling, such that a test alliquot is collected from a defined sample of particulate material that can be considered to be representative of the entire sample (NIST 960-1); - 75 - o Powder sampling is more difficult than sampling from suspension, but some general guidelines on powder sampling are available (Allen, 2001a and 2001b); o Steps in the sample preparation will be governed largely by the requirements of individual measurement methods; o Ideally, samples for analysis should be free from the inherent aggregation problems associated with nanoparticles and other contaminants not associated with the said nanoparticles. However, to achieve such goals are not trivial; o The use of sonication to disperse nanoparticles in solution has the potential to change the size distribution of the nanotubes and introduce defects (Islam et al., 2001); o It is important to establish the ‘state’ of the sample required for analysis i.e. whether the nanoparticles should be fixed on to a solid substrate, suspended in liquid media or aerosolised (solid or liquid aerosols). 3.5.146 A number sample preparation protocols and guidance for nanomaterials such as carbon nanotubes (Decker et al., 2009) have been emerging in the scientific literature. However, these procedures cannot be universally applied to all nanomaterials and certain modifications to the procedures may be required for different nanomaterials. It is therefore important to stress that such procedures should be carefully examined to determine if they are adequate for the test material under consideration. 3.5.147 Particle size/size distribution 3.5.148 The current REACH guidance on particle size measurement (albeit with a focus on micron-sized particles) refers to the TG110 document published in 1981, with limited recognition of more modern particle sizing techniques. The literature review has identified that there are many methods available for detecting and accurately characterising the size/size distribution of nanomaterials in powder form, suspension and aerosols. - 76 - 3.5.149 A number of important points have been noted and will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the guidance, specifically: o It has been highlighted that different methods, based on different measurement principles, may yield different results when measuring the same nano-object or structural feature (Lövestam et al., 2010); o It has been recommended that measured particle size values should be regarded as ‘method-dependent’, and should be reported with sufficient detail on the analytical technique used to acquire the data and the applied protocol used to deduce the size from the measured raw data (Lövestam et al., 2010). This also applies to other physico-chemical properties in addition to particle size/size distribution measurement; o It is noted that no single technique can be considered to be without artefacts or can be employed in all cases when determining nanoparticle sizes, and it is thus recommended that the multi-analytical techniques and/or multiple preparation techniques should be used when characterising nanoparticles (Domingoes et al., 2009); o It is noted that Dynamic Light Scattering (DLS) does not provide a full particle size distribution. DLS measures fluctuations in the intensity of scattered light caused by Brownian motion, from which the hydrodynamic diameter is calculated, enabling estimation of the particle size distribution. Thus, even though DLS does not measure particle size distribution directly, this method provides a good background for the estimation of the full particle size distribution. The method also provides a number (the ‘polydispersity index’) indicating the polydispersity of the particle population. There are software routines available that facilitate the calculation of a particle size distribution from DLS data, but the adequacy and the comparability of these routines needs to be further evaluated (Lövestam et al., 2010). o Drying samples under vacuum for analysis using electron microscopy may alter the size and shape of the particles being characterised. Plasma sputter- coating the surface-adhered particles with a layer of a conducting material may modify the sample being characterised; - 77 - o The analysis of particles in solution has been advanced through the development of Environmental Scanning Electron Microscopy; this offers the potential for dispersed samples prepared for exposure/toxicological experiments to be characterised as well as limiting the need to dry samples which may influence the observed size distribution; o The quality of the images to be analysed using electron microscopy techniques is of critical importance and it should also be noted that electron microscopy normally provides only two-dimensional images, so care must be taken to avoid bias introduced by orientation effects. High-resolution microscopy may be subject to artefacts caused by sample preparation or special analysis conditions; o In aerosol physics, the most commonly used methods for particle sizing are differential electrical mobility (from about 10 – 1000 nm) and light scattering aerosol spectrometry (from 60 nm – 45 µm). These methods allow determination of the equivalent aerodynamic diameter which may be different from the geometric diameter measured with microscopy techniques (Lövestam et al., 2010); o Relatively few toxicological studies published in the scientific literature have directly compared nanoparticulate and microparticulate forms of metals, which may limit establishing, at this time, the validity of read-across in the REACH context; o Overall, the size at which genuinely nanoscale properties are observed depends strongly on the material, and although most of these effects appear at sizes of 30 nm and below, no general limit can be given (Lövestam et al., 2010). As such, case-by-case studies are necessary for every material, since there is no direct, material-independent relationship between size and novel effects or functions. 3.5.150 Aggregation/agglomeration state 3.5.151 The aggregation/agglomeration state of nanoparticles affects the stability of nanoparticle dispersions prepared for (eco)toxicological experiments. The size of aggregate particles can be determined using many of the same methods described - 78 - above for ‘particle size/size distribution’ analysis, an overview of which is provided earlier in the “particle size/size distribution” and “aggregation/agglomeration state” sub-section of chapter 4.1 of RNC/RIP-oN2/B3/2/FINAL. 3.5.152 In addition, a number of other important points have been noted and will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the guidance, specifically: o The state of dispersion (i.e. level of (de)agglomeration) is typically estimated using comparative particle size measurements, with shaking, sonication, and/or surfactants commonly used to disperse nanoparticles in solution. However, these tools may damage cells and interfere with toxicity testing if used in living systems (Powers et al., 2006); o The combined use of dielectrophoretic assembly and Raman spectroscopy has been suggested to be a more sensitive measure of the aggregation state of carbon nanotubes than either one of these methods alone (Kumatani and Warburton, 2008); o Surface energy, charge and solvation have been suggested to be relevant parameters to consider in relation to nanoparticle-nanoparticle interactions (SCENHIR, 2006). o The propensity of particles to aggregate has prompted researchers to prevent against its occurrence through the use of sonication, or inclusion of dispersants within particle suspensions, particularly in the case of CNTs. However, the relevance of considering monodispered CNT also requires consideration, since, if this is so difficult to achieve experimentally, the relevance to human and environmental exposure may be questionable. It might be more useful to achieve a CNT suspension with limited or controlled aggregation, promoting uniform, more easily characterisable exposure conditions for the model under investigation. In addition, the exposure route is likely to impact on the aggregation and agglomeration of CNT. For example, limiting the aggregation of CNT when generating aerosols is difficult, whereas a number of dispersants can be employed to improve the dispersion of CNT suspensions; - 79 - o The use of grinding to increase dispersion has been demonstrated to impact on both length and surface properties, and therefore toxicity of the MWCNT (Muller et al., 2005). o There is a difficulty in testing CNT toxicity due to their high propensity to aggregate and achieving a suspension or aerosol where individual CNT are contained is difficult. It is therefore important to consider how interactions between CNT, which promote the formation of larger structures, impact on the toxicity of CNT; o Preliminary studies on the inclusion of commonly used surfactants (namely Pluronic L61, Pluronic L92, Pluronic F127, Tween 20, and Tween 60, at concentrations of 0.1-10%) in dispersions of MWCNT has illustrated that all surfactants (with the exception of Pluronic F127) reduced cell viability at all concentrations, and were therefore deemed inappropriate to use despite their ability to reduce CNT aggregation (Monteiro-Riviere et al., 2005); o Filtering can be used as a technique to reduce the presence of aggregates within the dispersing solution of SWCNT (Raja et al., 2007); o It has been suggested that the degree of particle aggregation and agglomeration associated with metal oxide administration is also likely influencing the resultant toxicity of these particle types, in addition to particle size. However the nanoparticles that make up the agglomerates are within the nano size range, and this appears to be fundamental to driving their toxicity; o Nanoparticle aggregation is also general issue that has been reported in a wide range of the published environmental studies. The extent of particle aggregation has been reported to be influenced by particle type and differences in test media as well as procedures to prepare test suspensions as note before; o Only a few studies have systematically explored the influence of aggregation on the ecotoxicity of various nanomaterials; o From the research published to date, it is not yet clear how various levels of aggregation of nanoparticles influence their ecotoxicity, and the derivation of LC50, EC50 and NOEC also remains uncertain. Adding to the complexity of - 80 - this issue, it has recently been found that aggregation behaviour in the media might follow a non-linear concentration-aggregation relationship (Baalousha et al., 2009; Baun et al., 2009). 3.5.153 Surface area 3.5.154 The reduction in size to the nanoscale is accompanied by an inherent increase in the surface-to-volume ratio, and therefore a greater proportion of entities at the surface compared to the bulk (non-nanoscale) material. For particle-based substances, the surface plays an important role in influencing the physical and chemical interactions between the substance and the receptor (i.e. cell, tissue, organism, media etc). The influence of surface area on toxicity is obviously intrinsically linked to particle size. Powers et al. (2006) highlight that it has been established in several toxicity studies that effects correlate with surface area (Powers et al. reference Brown et al., 2001; Donaldson et al., 1998, 2002; Oberdorster et al., 1992; Tran et al., 2000) to a greater extent than mass as a dose metric. 3.5.155 Several methods have been identified for measuring the surface area of nanoparticles. A description of the techniques identified has been provided along with their advantages and limitations. Emerging methods such as diffusion charging have begun to provide a more viable approach to measuring aerosol surface area in situ. Implications of a number of issues, however, remain to be considered including the effect of initial aerosol charge, the composition of the material, presence of aggregates and the effect of particle shape. The advantages and disadvantages of measuring deposited particle surface area, rather than aerosol surface area, also need to be considered further. - 81 - 3.5.156 Shape/Aspect ratio 3.5.157 Length has been accepted to influence fibre clearance, because it dictates the ability of phagocytic cells to completely internalise CNT. Longer fibres promote the development of frustrated phagocytosis, reduced clearance and hence the potential to persist to increase their propensity for damage (Brown et al., 2007; Poland et al., 2008). The shape of nanoparticles is also likely to influence their uptake by cells, as has been demonstrated for metal nanoparticles (Chithrani et al., 2006; Chithrani and Chan, 2007; Pal et al., 2007). Only a few studies have documented links between physico-chemical characteristics and ecotoxicity. In regards to shape, only three published studies have been identified. It is evident that much more research is needed before specific properties, or combinations of properties, can be linked to the effects observed in ecotoxicity tests (Stone et al., 2009). 3.5.158 A number of methods have been identified for measuring the surface area of nanoparticles, primarily microscopy based. A description and comparison of the techniques identified is readily accessible in the “shape/aspect ratio” sub-section of Chapter 4.1 of RNC/RIP-oN2/B3/2/FINAL. 3.5.159 In addition, a number of other important points have been noted and will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the guidance, specifically: o A method for processing SPM images in order to estimate nano-object positions and dimensions, using dictated fits based on the least-squares method and the matrix operations has been published (Silly, 2009); o UV-visible spectroscopy, commonly used to confirm the presence of nanoparticles in a liquid, can be used to indicate the shape of the nanoparticles, but confirmation is usually performed using imaging methods such as TEM (Weir et al., 2008); o An extension of thermogravimetic analysis (TGA), a method principally used for determining chemical composition and purity, to analyse the specific properties of SWCNT including length and diameter has recently been reported. However additional characterisation methods (such as SEM or - 82 - Raman Spectroscopy) may be needed to support and fully interpret the TGA results (Mansfield et al., 2010). 3.5.160 Surface Charge 3.5.161 In relation to toxicity testing, the major influence of surface charge is on the stability of the administered dispersion (see, for example, Jiang et al. (2009)). However, size and charge can also influence the adsorption of ions, contaminants, and biomolecules, and the way cells react when exposed to them (e.g. Goodman et al., 2004). Particle uptake by cells has also been observed to be influenced by the particle's charge, particularly for metal oxides (Hankin et al., 2008). It is evident that much more research is needed before specific properties, such as surface charge, can be related to the effects observed in ecotoxicity tests (Stone et al., 2009). 3.5.162 A number of methods have been identified for determining the zeta potential of nanoparticles, as described in the “surface charge” sub-section of chapter 4.1 of RNC/RIP-oN2/B3/2/FINAL. 3.5.163 Surface chemistry 3.5.164 The term surface chemistry is often used in the context of surface chemical composition, and is somewhat a broad and non-specific term which does not predispose itself to ‘quantitative’ characterisation according to a single comparable metric or measurand. Surface chemistry includes elements of solubility equilibrium, catalytic properties, surface charge, and surface adsorption and desorption of molecules from solution, amongst others. Most of these properties are functions of the atomic or molecular composition of the surface and the physical surface structure. Chemical purity, functionalisation and surface coating are also important aspects to take into account. 3.5.165 Modification of the surface of CNTs has been demonstrated to both enhance and reduce toxicity. It is therefore unreasonable to make definite conclusions about how the modification of CNT affects their toxicity, as it driven by the particular modification employed, which is generally undertaken for a specific purpose. The modification of the surface of TiO2 particles has also been demonstrated to influence its toxicity. However, this is likely to be dependent on the modification, and cell type in question. The impact of functionalisation on fullerene toxicity has also been the - 83 - focus of investigation since functionalisation of the surface of fullerenes is often completed for a specific purpose, such as improving water solubility. Fullerene functionalisation has also been observed to promote the appearance beneficial properties such as antioxidant, or anti-inflammatory activity. The influence of surface attachments on fullerene toxicity may be dependent upon the target cell/organ and/or the fullerene type under investigation. 3.5.166 Although some evidence is available in the literature to suggest that surface functionalisation may influence the ecotoxicity of nanomaterials, the number of studies is, at present, too limited to draw general conclusions on the influence of functionalisation on ecotoxicity, speciation, and accumulation. 3.5.167 A number of methods have been identified for analysing the surface properties of nanoparticles, as described in the “surface chemistry” sub-section of chapter 4.1 of RNC/RIP-oN2/B3/2/FINAL. 3.5.168 In addition, a number of other important points have been noted and will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the guidance, specifically: o Atomic Force Microscopy (AFM) can provide 3D imaging/visualisation of nanoparticles distributed on a flat surface, allowing access to qualitative and/or quantitative information about the physical properties of nanoparticles including size, morphology, surface texture, and roughness. However, the influence of the AFM tip size and shape on the acquired images must be properly accounted and corrected for; o Another property related to surface chemistry that may be of relevance to characterise is porosity. It has been suggested that there is no all- encompassing analytical technique available to study porosity and a synergistic approach involving application of a combination of various techniques is necessary (Heo et al., 2006); o The nano scale chemical and structural environment, including factors such as surface roughness, may also be important properties to consider in relation to bacterial activity. - 84 - 3.5.169 Redox activity 3.5.170 Redox potential may be useful in determining how active a given nanomaterial (and in principal any chemical substance) would be in human and environmental oxidation-reduction processes, potentially also generating reactive oxygen/nitrogen species (ROS, RNS). Redox reactions can occur abiotically or biologically, and may alter a nanomaterial’s physico-chemical properties including surface area, surface charge, and chemical composition, which in turn can affect the material’s potential to aggregate, size, toxicity and mobility. Redox reactions are the basis of chemical transformations of inorganic and organic species and the precipitation and dissolution of inorganic substances that influences their sequestration and mobility. Hence measurement of the redox potential would be potentially meaningful for nanomaterials which can participate in electron transfer or uptake. 3.5.171 It has been suggested that chemically stable inorganic nanomaterials in physiological redox conditions do not appear to exhibit cytotoxicity in vitro, whereas nanomaterials with strong oxidative (e.g. CeO2, Mn3O4 and Co3O4) or reductive powers (e.g. Fe0, Fe3O4, Ag0 and Cu0) can be cytotoxic and genotoxic towards biological targets in vitro (Auffan et al., 2009). Standard electrochemical methods, such as cyclic voltammetry, may be used to study the redox activity of nanomaterials. 3.5.172 ROS generation potential 3.5.173 The ability to generate ROS and oxidant injury is one paradigm that may be used to compare the toxic potential of nanomaterials (Xia et al., 2006; Auffan et al., 2009). However, the relationship between ROS generation and ecotoxicity has been studied to a lesser extent. 3.5.174 Whilst it has been demonstrated that ROS generation and oxidative stress can be used as a paradigm to assess nanomaterial toxicity, not all nanomaterials exhibit the electronic configurations or surface properties that allow spontaneous or acellular ROS generation; particle interactions with cellular components could generate ROS during these interactions. - 85 - 3.5.175 A number of methods have been identified for detecting ROS generation from nanomaterials, under both abiotic conditions and in cells, as described in the “ROS generation potential” sub-section of chapter 4.1 of RNC/RIP-oN2/B3/2/FINAL. 3.5.176 Photocatalytic activity 3.5.177 The ability of UVA or visible light to increase the toxic potency of metal oxide particles, through increased ROS production, has been a focus of a number of toxicology studies (e.g. Dunford et al., 1997; Zhang and Sun, 2004; Dufour et al., 2006), but does not always transpire (Linnainmaa et al., 1997; Theogaraj et al., 2007). Results of ecotoxicology studies to date also do not allow any firm conclusions to be drawn regarding the influence of photocatalytic activity on ecotoxicity. 3.5.178 A number of methods for studying the photocatalytic activity have been described in the “Photocatalytic activity” sub-section of Chapter 4.1 (RNC/RIP-oN2/B3/2/FINAL). However, it is currently unclear what distinct measurand for photocatalytic activity could be standardised to the extent that would be meaningful to risk assessment. The lack of clarity in the relationship between photoactivty and (eco)toxicity, combined with the lack of a clear measurand, may not provide sufficient justification for the inclusion of photocatalytic activity as a new Information Requirement. 3.5.179 Dustiness 3.5.180 Measurement of the ‘dustiness’ of a material provides information relating to the propensity of that material to produce airborne dust, and is important to consider from an exposure perspective. Dustiness is also a key parameter for assessing the risk of dust explosions. This property is covered to some extent under the current REACH guidance chapter on Granulometry. 3.5.181 Rotating drum and continuous drop methods are currently suggested in the REACH Guidance chapter for granulometry for the measurement of airborne dispersed or nebulised particles (ECHA, 2008. R.7.1.14, Table R 7.1-31). However, rotating drum dustiness tests are usually performed as three replicate tests and need quite large amounts of test material, typically 300–600 g. The EN 15051 continuous single-drop method requires a total amount of 500 g for the required five single-test runs. It has been highlighted that such large amounts of test material may not be - 86 - practical if very toxic and/or costly materials are to be tested and there is a need for test systems that can be operated under controlled atmospheric environments using much smaller amounts of material (Schneider & Jensen, 2008). Several advances towards this have been reported in the scientific literature, specifically: o A fluidisation (vortex shaker) method has been developed for testing nanosize powders (Maynard, 2002; Baron et al., 2003; Maynard et al., 2004). This method is included in ISO’s forthcoming technical document entitled “Nanomaterials – General framework for determining nano-object release from powdered nanomaterials by generation of aerosols”. However, sufficient methodological detail has yet to be incorporated into the document being developed; o A dustiness test that uses only 6 g of material per test run and that characterises the test material by both a single-drop and a rotating drum type of challenge. The test apparatus is based on a downscaled version of the EN 15051 rotating drum, whilst maintaining important test parameters, and was demonstrated to provide very reproducible results both in terms of amount and size distribution of the generated particles (Schneider & Jensen, 2008). 3.5.182 Explosive properties 3.5.183 No studies have been identified in the literature relating to the explosive properties (an existing REACH Information Requirement) of nanomaterials, over and above the outputs from the NANOSAFE2 project discussed in the FP6/7 section of RNC/RIP- oN2/B3/2/FINAL, therefore the ability to prepare practical advice in the context of REACH is limited. 3.5.184 However, it has been suggested that read-across of explosivity data from bulk materials (non-nanoscale) to nanomaterials is not possible, since nanomaterials may have explosive properties which are solely due to the small particle size (RIVM, 2009). This issue will be considered further in the gap analysis (Task B4) and used to inform any subsequent amendments to guidance. - 87 - 3.5.185 Boiling/Melting/Freezing Point 3.5.186 No studies have been identified amongst the extensive literature sourced during Task A in which the boiling, melting or freezing point (existing REACH Information Requirements) of nanomaterials is specifically addressed, therefore the ability to prepare practical advice in the context of REACH is limited. 3.5.187 However, in undertaking a hypothetical registration of nanosilver under REACH, RIVM (2009) concluded that these properties will be similar to those of metallic bulk (non-nanoscale) silver. Data for these parameters was effectively read across from the bulk form in their hypothetical registration of nanosilver. However, they conclude that “the ‘sameness’ analysis can, as yet, not be properly tackled under REACH, and thus the read-across from data on the bulk form of a substance to its nanoform will be very troublesome”. Furthermore, they concluded that “a nanomaterial cannot be properly characterized with the data normally required under REACH, and it is unclear how to address different sizes of a nanomaterial in substance identification”. 3.5.188 Relative Density 3.5.189 No studies have been identified amongst the extensive literature sourced during Task A in which the relative density (an existing REACH Information Requirement) of nanomaterials is specifically addressed, therefore the ability to prepare practical advice in the context of REACH is limited. 3.5.190 However, RIVM (2009) concluded that the relative density of nanosilver will be similar to that of metallic bulk (non-nanoscale) silver. Data for this parameter was effectively read across from the bulk form in their hypothetical registration of nanosilver. However, RIVM (2009) highlight that, for relative density, caution needs to be taken when performing read-across of data from the bulk form. Overall, they conclude that “the ‘sameness’ analysis can, as yet, not be properly tackled under REACH, and thus the read-across from data on the bulk form of a substance to its nanoform will be very troublesome”. Furthermore, they concluded that “a nanomaterial cannot be properly characterized with the data normally required under REACH, and it is unclear how to address different sizes of a nanomaterial in substance identification”. - 88 - 3.5.191 Solubility & release of metal ions into solution 3.5.192 As stated in the Task B1 report, the property of water solubility is considered to be very relevant and applicable to nanomaterials. The release of ions into solution (including through an active electrochemical ‘corrosion’ process) may confound the interpretation of results perceived to be the result of solubilised substances. It is anticipated that the release of silver ions from nanoparticulate silver is a realistic prospect, responsible for their antibacterial properties and potentially linked to the observed toxicity. It is postulated that Ag+ mediates these effects, although the mechanism by which this occurs is unknown at this time but likely to involve particle oxidation to enable their release. Further investigations are necessary to confirm the contribution of particles and/or ion release to their toxicity. In relation to ecotoxicity of nanosilver, the issue of dissolution may also be crucial to understanding the mechanisms involved. A number of studies have reported that the observed ecotoxicity of these nanomaterials towards various organisms may be partly or fully attributed to the release of dissolved metal ions, covering zinc oxide as well as nanosilver. However, the influence of dissolved metal ions on ecotoxicity is not clear based on current literature and requires further investigation. 3.5.193 In undertaking a hypothetical registration of metallic silver (in bulk and nanoform) under REACH, RIVM (2009) highlighted that there is no information available on the kinetics of dissolution in dependence of nanosilver particle properties (such as (time- dependent shifts in) size distribution, shape) and properties of the medium (like pH, dissolved organic carbon, silver-complexing ions and recommended the inclusion of ‘Dissolution Kinetics’ as a sub-information requirement under the existing REACH Information Requirement for ‘Solubility’. 3.5.194 TOXICOLOGICAL INFORMATION 3.5.195 Considerations for study design 3.5.196 Investigations undertaken and reported to date have highlighted a number of key issues or gaps in existing testing strategies which may influence the outcome of studies, and thus should be observed closely in the consideration of nanomaterials within the context of REACH. - 89 - 3.5.197 In relation to the manner in which experiments are set up, factors such as the exposure method, dose selected, species used, cell type under investigation and in the case of photo-reactive nanomaterials such as some metal oxides, light conditions (Warheit et al. 2005) all have the potential to impact on the toxicity of nanoparticles, indicating that the experimental set up is very influential. 3.5.198 A summary and discussion of those issues considered to be of importance in this respect is provided below. 3.5.199 Dispersion 3.5.200 As discussed at various points within this B3 report, it is clear that dispersion impacts upon the potential toxicity of NMs in animals or cells. A multitude of dispersing processes have been utilised by investigators to improve the dispersion including solvents (such as acetone), surfactants (such as pluronic), proteins (albumin and serum) or mechanical processes (such as centrifugation, or sonication). At this time it is not possible to conclude which techniques are most appropriate, but in designing experimental protocols for consideration of nanomaterials in the context of REACH, it is essential that such techniques should try to mimic realistic exposure scenarios, routes and avoid interference by dispersants. 3.5.201 Selection of Dose 3.5.202 Many investigations reported have used very high doses, raising the question of whether the toxic effects observed are likely to derive from dose used or the material. In particular, at high doses, the aggregation of CNT is promoted, and so the toxicity that transpires in vivo is potentially a result of the blockage of airways and blood vessels, rather than to a specific toxic effect. 3.5.203 In order to inform selection of relevant nanomaterial exposure concentrations to be used within in vitro and in vivo experiments, information regarding the human exposure levels is required as a reference. However, at the current time this is severely lacking and thus exposure assessment is of key importance to developing sound dose selection for future studies 3.5.204 In relation to dosing regime, many investigations of NM toxicity have used a single dose administered to animals or cells. Within occupational or consumer settings, it is - 90 - more likely that normal NM exposures (non-accidental) will occur over a period of time, depending on their application. 3.5.205 Selection of exposure route and duration 3.5.206 Testing methods for initial in vivo toxicity assessment normally use the oral route of exposure. However for the testing of nanomaterials, this may not be the ideal first candidate. As a general observation, repeated dose studies with lower doses over a long time period and use of a route of exposure appropriate to the end use of the nanomaterial in question, are likely to be of greater relevance in consideration of the potential risk of nanomaterials within occupational or consumer settings, than extremely short exposures at high doses. In addition, the use of chronic studies will also allow for the more relevant identification of the potential carcinogenic consequences of NM exposure. 3.5.207 Interaction of nanoparticles with biological molecules 3.5.208 Attention should be directed to consideration of the role that interaction of NPs with biological molecules plays in altering their behaviour within biological systems. It is known that on entering the body, particles immediately become coated in biological molecules, including proteins. It is hypothesized that this coating can influence particle behaviour and toxicity, with different particles having different capacities to bind different molecules. Furthermore, there is a possibility that the particles can alter the protein structure and function (and thus behaviour), which again may contribute to toxicity. Further research is required to generate a greater understanding of this complex area, and it is advised that consideration should be given in future experimentation to the role that such interactions may play in toxicity. 3.5.209 Use physico-chemical data to inform experimental design 3.5.210 It has been highlighted within this report, but also more fully within the toxicology and physico-chemical sections of RNC/RIP-oN2/B3/2/FINAL that the physico- chemical characteristics of particles used such as size, crystallinity, functionalisation, contamination, solubility etc. can impact on their toxicity. Thus, as - 91 - these factors are able to influence the findings obtained, a thorough physico- chemical characterisation should be undertaken and used as justification of the relevancy of the experimental approach used in future toxicological investigations undertaken. 3.5.211 In addition, a specific consideration in relation to metal nanomaterials is determination of whether any toxicity observed derives from their small size, is mediated through the release of ions from particles, or perhaps a combination of both. Elucidation of this is essential for the hazard characterisation of metal NPs within the context of REACH 3.5.212 Target Organ Toxicity considerations 3.5.213 At the current time, there is a paucity of data relating to the systemic transfer of particles following exposure via the lungs, skin and gut, and this should be a focus of future experiments. Studies have focused on dermal and pulmonary toxicity of particles, but there is an absence of data on the consequences of exposure to the gastrointestinal tract, and within damaged/diseased skin. Other relevant target organs include the liver, kidney, cardiovascular system and brain which are necessary due to the fact that nanoparticles are likely to become systemically or neuronally available. The liver could be highlighted as a priority due to the propensity of particles to accumulate in this organ. Based on this, it has been suggested that toxicokinetics and certain additional target organ specific effects be considered as specific information requirements for hazard identification and characterisation of nanomaterials within the context of REACH 3.5.214 Adoption of standardised ‘controls’ to assist assessment of toxicity 3.5.215 The use of both nanoparticulate and non-particulate controls (such as carbon black or asbestos within CNT studies, or zymosan within inflammation studies) has been reported on numerous occasions. The choice of controls can, to some extent be driven by the hypothesis being tested (and links to determination of the physico- chemical characteristics responsible for toxicity). The use of such benchmark controls (those for which extensive background information is available) provides a useful indication of the relative toxicity of the nanomaterial under investigation versus other particles or reagents of known toxicity (see for example, Warheit et al. 2004, Shvedova et al. 2005). Thus, it should be encouraged as standard practise - 92 - for the hazard identification and characterisation of nanomaterials within the context of REACH. 3.5.216 Interference of nanomaterials with toxicity assays 3.5.217 Nanomaterials have been on occasion found to interfere with some of the assays utilised to determine their cellular or toxic effects. For example, some nanoparticles may contribute to the absorbance or fluorescence of colorimetric or fluorometric assays. In addition, due to their large surface area, nanoparticles may bind to assay components including the substrates (such as CNT with the reagent in MTT assays; Belyanskaya et al. 2007) or the biomarker being measured, (such as LDH and cytokine proteins, see for example Davoren et al. 2007). Kroll et al. (2009) undertook a review of cytotoxicity assays commonly used to investigate nanoparticle toxicity, and provided a discussion of interference reported for each. 3.5.218 All of these factors can contribute to the production of inaccurate, misleading results that make nanoparticles appear more or less toxic than they actually are. In many of the studies reported it is not possible to ascertain whether the assays were adequately controlled to assess for interference. Thus, as a general precaution, it is advisable to use more than one assay to assess the endpoint or effect in question. Further investigation of this area is required, as is integration of known interferences into any guidance developed for the toxicology testing of nanomaterials under REACH. 3.5.219 ECOTOXICOLOGICAL INFORMATION 3.5.220 Given the current state of the published science, it has not been possible to provide specific practical advice with regard to the ecotoxicological Information Requirements in REACH, but a number of factors have been found to influence the ecotoxicologic responses observed in the study of nanomaterials. These include: 1) particle impurities, 2) suspension preparation methods, 3) release of free metal ions, and 4) particle aggregation and 5) relevance of dose [concentration] - response for ecotoxicological studies of nanomaterials. The extent of influence of these factors on the ecotoxicological impact of nanomaterials is unknown and, even, the scientific evidence for them have an influence in the first place is contradictory and varies - 93 - from nanoparticle to nanoparticle (Baun et al., 2009). This impedes the reliability and interpretation of the available ecotoxicity data as well as the direct use of the reported LC50, EC50 and NOEC for PNEC derivation. 3.5.221 As noted by Stone et al. (2009) traditional predictions of fate and transport are based on inherent properties such as phase transfer properties (e.g. boiling point, vapour pressure, partition coefficients), reactivity (e.g. photo-reactivity and hydrolysis) and biological degradation behaviour (Mackay and Hendry 2009). Many of these inherent properties are reported on for regular chemicals under REACH. However, we know at this point that these properties are not adequate to understand and predict the fate and behaviour of nanomaterials. This is further complicated by our current lack of understanding of the novel physico-chemical properties exhibited by many nanomaterials and the effect these have on particle behaviour. In addition, it is most likely that those nanomaterials released into the environment will also exist as modified forms of their primary counterpart (SCENIHR. 2009). 3.5.222 The fate and behaviour of nanomaterials in the environment is dependent on type, form and physico-chemical characteristics of the nanomaterial in question, as well as those of the receiving environment (Chen et al., 2008; Chen and Elimelech 2008; Saleh et al., 2008). Nanomaterial transport and distribution are influenced by a number of factors, such as Brownian diffusion, inertia effects, gravitational influences, thermal influences, pH, ionisation, and presence/absence of Natural Organic Matter (NOM). These interactions ultimately affect the processes the nanomaterial consequently undergoes in its transport and subsequent fate. 3.5.223 Nevertheless, the lack of actual measured data in the available public domain in relation to the environmental fate and behaviour of nanomaterials in water represents a major gap in developing realistic prediction of fate and transport of nanomaterials in the aquatic environment. 3.5.224 Most of our current knowledge stems from colloid science, which provides preliminary information, but there is a need for systematic studies on different types of nanomaterials within aquatic bodies using a range of physico-chemical parameters (e.g. size, shape, form, surface area) to generate data and to support development of reliable models. Predictive modelling of emission scenarios and - 94 - subsequent transport pathways will also play an important role in furthering understanding in this area (Stone et al., 2009). 3.5.225 Knowledge to date indicates that in many cases rather than remaining intact, nanomaterials will tend to aggregate, agglomerate or become associated with other dissolved, colloidal or particulate matter present in the environment (SCHENIR 2009). However, the novel physico-chemical characteristics which make nanomaterials desirable also present a challenge for determining how they interact with the environment, how, when and where they are distributed, and in what form they ultimately end (Darlington et al., 2009). 3.5.226 Appropriate metrics for measuring engineered nanomaterials in the environment are still subject to much discussion, and in particular those pertaining to exposure concentrations, or dose, are considered to be of high importance. Differences in behaviour across different physical and chemical species of the same nanoparticulate material must also be considered. In addition, their tendency to aggregate/agglomerate, adsorb to NOM and, in the case of wastewater treatment, potentially associate with the solid phase, must be taken in consideration, as all of these processes could lead to environmental ‘hot spots’ where concentrations of nano-particulates are particularly high. - 95 - 3.5.227 B3 Sub-Task III: Summary of practical advice on the use of information from OECD-WPMN in fulfilling REACH data requirements 3.5.228 PHYSICO-CHEMICAL PROPERTY INFORMATION 3.5.229 The results from the OECD-WPMN sponsorship programme for exploratory testing of nanomaterials are expected to become available in 2012 and will provide further information for the further development of OECD test guidelines and ISO standards. It is anticipated that this work will contribute to the REACH implementation for nanomaterials and to the assessment of the REACH Information Requirements. The forthcoming results from OECD-WPMN Sponsorship Programme are expected to shed further light on practical testing issues and provide a common knowledge basis on key nanomaterials. However, at the time of writing this report, no testing results have yet emerged from the Sponsorship Programme. It is important to acknowledge that this limits the ability to identify and critically assess the appropriateness of existing test methods and results from the sponsorship programme in fulfilling the REACH data requirements for physico-chemical properties at this time. However, any practical advice that can be extracted from the information currently available from OECD-WPMN is summarised below. 3.5.230 Octanol-water partition coefficient (Kow) 3.5.231 OECD has concluded that the three TGs relevant to characterising the partition coefficient (OECD TG 107, 117, 123) might be applicable under some circumstances or to some classes of manufactured nanomaterials, although further work is required to determine this and modify the TGs, if necessary (ENV/JM/MONO(2009)21). This issue has been considered further in the Task B1 report, and will be taken forward into the gap analysis (B4). 3.5.232 Water solubility 3.5.233 The important points noted from the OECD publications in relation to water solubility which will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the guidance, are: o It has been suggested that the measurand of interest (beginning with a pre-determined unit of particles in a standardised solution and temperature) is to measure the mass proportion of nanomaterials which - 96 - are held in solution, and whether this mass diminishes after a set period of time, or; determine the amount of time required for mass to diminish by X% (ENV/JM/MONO(2009)20/REV); o OECD concluded that the test guideline relevant to characterising the water solubility (OECD TG 105) might be applicable under some circumstance or to some classes of manufactured nanomaterials. It stated that this TG is applicable to solutions but it is not known how the results might be impacted by the presence of a colloidal suspension, which might be present if the sample manufactured nanomaterial does not completely dissolve. Hence, further work is required to determine this and to modify the TGs, if necessary (ENV/JM/MONO(2009)21). 3.5.234 Particle size/size distribution 3.5.235 The important points noted from the OECD publications in relation to particle size/size distribution which will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the REACH guidance, are: o It has been suggested that the conditions to which a substance is subjected may affect the size of the discrete form of a substance (ENV/JM/MONO(2009)20/REV); o It has been highlighted that the measured size of a particle is always dependent on the particular method that is being used to examine, measure or visualize it such that the size of a particle reported by one technique might not be the same as the size when measured with another technique (ENV/JM/MONO(2009)20/REV); o The appropriate measurands have been suggested to be, for a representative sample of nanoparticles, both the average size of individual particles and the size distribution of the sample of particles (ENV/JM/MONO(2009)20/REV); o OECD concluded that method A (designed to provide information on the transportation and sedimentation of insoluble particles in water and air) of the test guideline for determining particle size distribution/fibre length and diameter distributions (OECD TG 110) "is not applicable to nanomaterials" whilst method B (used in the special case of materials which can form fibres, - 97 - involving microscopic examination) "would, with some modification (the inclusion of fibres of less than 5 microns in length and less than 100 nm in diameter), be applicable to nanoparticles as well as nanotubes and nano fibers”. It is suggested that studies should be carried out in order to extend its range of applicability to fibres with nano-scale dimensions. It is known that alternative methods for (nano)particle size distribution already exist, which OECD suggest should be taken into account if such studies are undertaken (ENV/JM/MONO(2009)21). 3.5.236 The alternative methods suggested by OECD to be used in the Sponsorship Programme are clearly detailed in the sub-section “Particle size/size distribution” in chapter 5.1 of RNC/RIP-oN2/B3/2/FINAL. 3.5.237 Agglomeration/aggregation 3.5.238 The important points noted from the OECD publications in relation to agglomeration / aggregation which will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the REACH guidance, are: o The measurands of interest, beginning with a pre-determine unit of particles, have been suggested to be (ENV/JM/MONO(2009)20/REV): a) The effective mean particle size in a given medium and its evolvement over time (including the standard deviation); and/or; b) Qualitative assessment of state of aggregation and estimation of the primary particle size by TEM pictures; and/or c) Indirect confirmation of the estimated primary particle size by BET measurements, for materials with low/no internal porosity as is typical for pyrogenic oxides. o Predictions of agglomeration in natural waters will be limited to homo- aggregation of the particles since the data needed to predict the deposition with a heterogeneous set of natural surfaces (e.g. Hamaker constants and zeta potentials) is often not available (ENV/JM/MONO(2010)25). - 98 - 3.5.239 The methods suggested by OECD to be used in the Sponsorship Programme for the determination of aggregation/agglomeration state are clearly detailed in the sub- section “Agglomeration/aggregation” in chapter 5.1 of RNC/RIP-oN2/B3/2/FINAL. 3.5.240 Crystallite and grain size 3.5.241 Crystallite size is included in OECD's list of testing endpoints of importance for (eco)toxicological evaluation (ENV/JM/MONO(2010)46), although no specific reason/evidence is provided for this. This limits our ability to provide practical advice on this property and its test methods in the REACH context. 3.5.242 Specific surface area 3.5.243 The important points noted from the OECD publications in relation to specific surface area which will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the REACH guidance, are: o It has been suggested that the specific surface area will dictate the surface charge density in cases where nanomaterials are surface functionalized and that this has direct consequences on (ENV/JM/MONO(2009)20/REV): (a) nanomaterial interaction (i.e., agglomeration) with other naturally occurring particulate matter (i.e., contaminant vectors); (b) route of exposure as a function of surface ligand-biological interface (i.e., bioaccumulation pathway, bioavailability); and (c) mechanisms of toxicity (e.g., dose response curves normalized for surface area may indicate different results compared to results presented on a per mass basis) o It has been highlighted that in many cases specific surface area measurements are derived quantities that depend on the nature of the probe molecule. Nevertheless, in comparison with some of the other characterisation procedures, measurement of the specific surface area of a given sample is relatively straightforward" (ENV/JM/MONO(2010)25); o It has been suggested that it may be appropriate to evaluate whether the particle size distributions (and surface areas) of sparingly soluble - 99 - manufactured nanomaterials are altered through ripening and/or phase alteration phenomena (ENV/JM/MONO(2010)25); o It has also been highlighted that the measurement of the specific surface area might most efficiently be conducted concurrently with measurements of pore size, pore size distribution, porosity and perhaps even particle density as these properties will most probably have an important influence on the (eco)toxicological properties of the material (ENV/JM/MONO(2010)25). 3.5.244 OECD propose the Brunauer, Emmett, and Teller method as a possible method for determining the specific surface area of nanomaterials within the Sponsorship Programme (ENV/JM/MONO(2009)20/REV). 3.5.245 Surface charge 3.5.246 The important points noted from the OECD publications in relation to surface charge which will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the REACH guidance, are: o It has been noted that the zeta potential is not measurable directly but can be calculated using theoretical models and an experimentally-determined electrophoretic mobility or dynamic electrophoretic mobility (ENV/JM/MONO(2009)20/REV); o OECD highlighted that the significance of zeta potential is that its value can be related to the stability of nanoparticle dispersions, in that (ENV/JM/MONO(2009)20/REV): a) The zeta potential indicates the degree of repulsion between adjacent, similarly charged particles in a dispersion; b) For molecules and particles that are small enough, a high zeta potential will confer stability, i.e. the solution or dispersion will resist aggregation; c) When the potential is low, attraction exceeds repulsion and the dispersion will break and flocculate. This has serious implications for - 100 - the interpretation of toxicological affects with a particle’s physical or chemical characteristics no longer in effect. 3.5.247 Surface chemistry 3.5.248 The important points noted from the OECD publications in relation to surface chemistry which will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the REACH guidance, are: o It has been highlighted that various modifications of the surfaces of nanomaterials will lead to numerous potential interactions and will play a key role in determining: i) fate in natural aqueous systems; ii) colloidal stability; iii) exposure (ENV/JM/MONO(2009)20/REV); o It has been suggested that a given modification to surface chemistry can affect other physical-chemical properties, such as agglomeration, dustiness, zeta potential, surface area, water solubility (ENV/JM/MONO(2009)20/REV). 3.5.249 Dustiness 3.5.250 The methods suggested by OECD to be used in the Sponsorship Programme for the determination of dustiness of nanomaterials are clearly detailed in the sub-section “Dustiness” in chapter 5.1 of RNC/RIP-oN2/B3/2/FINAL. The advantages and limitations of these methods will be further compared and assessed in Task B4 (gap analysis), and information on the most appropriate methods will be recommended to be added to the REACH Guidance accordingly in Task B5. 3.5.251 Porosity 3.5.252 The important points have been noted from the OECD publications in relation to porosity which will be taken forward for further consideration in the gap analysis and used to inform proposed amendments to the REACH guidance, are: o It has been suggested that porosity data should be taken into account as it is relevant to the indirect confirmation of the estimated primary particle size by BET measurements, for materials with low/no internal porosity (ENV/JM/MONO(2009)20/REV); - 101 - o An additional consideration while exploring this endpoint is that, in addition to the basic large surface area provided by nanomaterials, a high porosity may permit the nanomaterials to act as vectors for other contaminants, such as heavy metals (ENV/JM/MONO(2009)20/REV). 3.5.253 The methods suggested by OECD to be used in the Sponsorship Programme for the determination of porosity of nanomaterials are clearly detailed in the sub-section “Porosity” in chapter 5.1 of RNC/RIP-oN2/B3/2/FINAL. 3.5.254 Pour density 3.5.255 On the basis of the limited information identified in OECD-WPMN publications pertaining to pour density, a conclusion on the relevance of characterising this property for nanomaterials and its inclusion under REACH cannot be determined at this time. 3.5.256 Photo-catalytic activity 3.5.257 The important points noted from the OECD publications in relation to photo-catalytic activity, are: o It is suggested that photocatalytic activity may also lead to the generation of excited state species on a material’s surface, which have the potential to directly and indirectly lead to potential toxicity; o OECD states that measuring photocatalytic activity will give an indication of the potential for transformations in the environment which in turn represents an important point of concern when evaluating the full life-cycle of the nanomaterial (ENV/JM/MONO(2009)20/REV); o It has been highlighted that, due to absorption of UV-B radiation by water, impurities and substances in the environment, and the different masses of these at different locations, evaluation of UV activation in the environment is likely to be very difficult (ENV/JM/MONO(2009)20/REV). 3.5.258 However, as highlighted in the literature section of RNC/RIP-oN2/B3/2/FINAL, the current lack of clarity in the relationship between photoactivty and (eco)toxicity, combined with the lack of a clear measurand, may not provide sufficient justification for the inclusion of photocatalytic activity as a new Information Requirement. - 102 - 3.5.259 Radical formation potential 3.5.260 OECD WPMN have highlighted that the potential to induce free radicals in organisms has been demonstrated for a number of nanomaterials and may have relevance to the toxicity of a manufactured nanomaterial (ENV/JM/MONO(2009)20/REV). 3.5.261 However, whilst it has been demonstrated that ROS generation and oxidative stress can be used as a paradigm to assess nanomaterial toxicity, not all nanomaterials exhibit the electronic configurations or surface properties that allow spontaneous or acellular ROS generation; particle interactions with cellular components could generate ROS during these interactions. 3.5.262 OECD WPMN have not suggested any specific methods for the determination of radical potential, noting that it can be measured by various means for different biological systems. 3.5.263 Redox potential 3.5.264 OECD WPMN have highlighted that redox reactions are the basis of chemical transformations of inorganic and organic species and the precipitation and dissolution of inorganic substances that influences their sequestration and mobility (ENV/JM/MONO(2010)46). Hence, OECD suggest that measurement of the redox potential would be potentially meaningful for nanomaterials which can participate in electron transfer or uptake. 3.5.265 Flash point 3.5.266 OECD concluded that the test guideline relevant to characterising flashpoint (OECD TG 113) is considered applicable to nanomaterials (ENV/JM/MONO(2009)21). This TG is not currently referenced in the REACH Guidance (ECHA, 2008. R.7.1.9) and its inclusion will be considered as part of the gap analysis (Task B4). 3.5.267 Boiling Point 3.5.268 OECD has concluded that the test guideline relevant to characterising boiling point (OECD TG 103), though applicable for determining the boiling point of manufactured nanomaterials, is probably not relevant to existing solid nanomaterials (ENV/JM/MONO(2009)21). This guideline is currently referenced in the REACH - 103 - Guidance although not explicitly cited in the text and a number of other methods have been suggested (ECHA, 2008. R.7.1.3). 3.5.269 Melting/freezing point 3.5.270 OECD has concluded that the test guideline relevant to characterising melting point/melting range (OECD TG 102) is considered to be applicable to nanomaterials (ENV/JM/MONO(2009)21),. 3.5.271 Relative density 3.5.272 OECD has concluded that the test guideline relevant to characterising relative density is (OECD TG 109) might be applicable under some circumstances or to some classes of manufactured nanomaterials, although further work is required to determine this and modify the TG, if necessary (ENV/JM/MONO(2009)21). This guideline is currently referenced in the REACH Guidance although not explicitly cited in the text (ECHA, 2008. R.7.1.4). 3.5.273 Surface tension 3.5.274 In its preliminary review of OECD Test Guidelines and their applicability to nanomaterials OECD has concluded that the test guideline relevant to characterising surface tension (OECD TG 115) might be applicable under some circumstance or to some classes of manufactured nanomaterials. It stated that this TG is applicable to solutions but it is not known how the results might be impacted by the presence of a colloidal suspension, which might be present if the sample manufactured nanomaterial does not completely dissolve. Hence, further work is required to determine this and to modify the TGs, if necessary (ENV/JM/MONO(2009)21). However, as stated in the Task B1 report, surface tension is not in general a relevant property for nanomaterials. 3.5.275 Adsorption/desorption screening 3.5.276 OECD has concluded that the three test guidelines relevant for adsorption/desorption screening (OECD TG 106, 108, 121) might be applicable under some circumstances or to some classes of manufactured nanomaterials. It stated that this TG is applicable to solutions but it is not known how the results might be impacted by the presence of a colloidal suspension, which might be present if the - 104 - sample manufactured nanomaterial does not completely dissolve. Hence, further work is required to determine this and to modify the TGs, if necessary. 3.5.277 Dissociation constant 3.5.278 OECD have highlighted that surface acidity (related to dissociation constants of surface ionisable sites) is an aspect of surface chemistry that may be particularly relevant, noting that: o ionisable sites may influence the surface charge which has been considered significant in toxicological studies; and o surface ionisation may also play a major role in colloidal particle stability and may even inhibit migration into hydrophobic phases (e.g., octanol/water partition coefficients). 3.5.279 OECD has concluded that the test guideline relevant to characterising dissociation constant (OECD TG 112) might be applicable under some circumstances or to some classes of manufactured nanomaterials. It stated that this TG is applicable to solutions but it is not known how the results might be impacted by the presence of a colloidal suspension, which might be present if the sample manufactured nanomaterial does not completely dissolve. Hence, further work is required to determine this and to modify the TGs, if necessary (ENV/JM/MONO(2009)21). This test guideline is currently referenced in the REACH Guidance (ECHA, 2008. R.7.1.17), however a number of other methods are also suggested. 3.5.280 Viscosity 3.5.281 OECD has concluded that the test guideline relevant to characterising viscosity (OECD TG 114) is only applicable to liquids and does not refer to solutions, suspensions or emulsions (ENV/JM/MONO(2009)21). Although the viscosity of a solution can be measured, standardised preparation procedures would need to be included but are not given in TG 114. Additionally, it is not known what impact a colloidal suspension would have on the results. It is not clear yet what the importance of this property might be for the behaviour of nanomaterials, both in the environment and in living organisms. At the same time, there would be the need to define the medium. - 105 - 3.5.282 Sample preparation 3.5.283 OECD has published a report focusing on providing guidance for nanomaterial sample preparation and dosimetry, of high value for further consideration in the context of the updated REACH Guidance (ENV/JM/MONO(2010)25). It highlighted that: o Due to the wide variety of nanomaterials, it is difficult to develop specific or detailed advice applicable to all nanomaterials; o The Guidance Notes should be used with some degree of expert judgement on a case-by-case basis; o The Guidance Notes refer and apply to the water insoluble manufactured nanomaterials as the OECD considered that soluble nanomaterials are unlikely to need different sample preparation techniques than other chemicals, other than precautions dictated by the specific reactivity of each material. However their size will still affect where they are being deposited e.g. in the lung; o As few, if any, standard testing approaches have been developed for nanomaterials, the OECD stress such guidance cannot be considered a “cookbook” for preparing samples and administering doses, but rather an outline (often in a general or descriptive manner) of considerations based on early results with nanomaterials or other experience with chemicals and particulates. 3.5.284 Common issues regarding sample preparation and dosimetry have been outlined in the document of key importance for further consideration in Task B4 and B5, including: o storage and stability of the test material; o the chemical composition of the test media, with the following parameters recommended to be measured for ecotoxicology studies or salines used in mammalian studies: a) ionic strength, calcium concentration and hardness, pH, dissolved organic matter, alkalinity, dispersing agents; - 106 - o characterisation of stock dispersions; characterisation of samples (prepared from stock dispersions) prior to administration/testing. 3.5.285 It is also recommended that, when a procedure for generating nanomaterial preparations intended for (eco)toxicological studies is employed, attention should be paid to minimising any alteration of the physical, chemical or (eco)toxicological properties of the substrate. 3.5.286 OECD has also outlined a number of media considerations for both airborne particles and particles in aqueous solutions (ENV/JM/MONO(2009)20/REV). 3.5.287 TOXICOLOGICAL INFORMATION 3.5.288 As previously mentioned, the results from the OECD-WPMN sponsorship programme for exploratory testing of nanomaterials have yet to be made available, which limits the ability to identify and critically assess the appropriateness of existing test methods and results from the Sponsorship Programme in fulfilling the toxicological data requirements for REACH at this time. 3.5.289 It is however notable that ‘ENV/JM/MONO(2009)10: EHS Research Strategies on Manufactured Nanomaterials: Compilation of Output’ provides a summary of research status documents, and includes: i) a list of research themes relevant to EHS of nanomaterials; and ii) an overview on completed, current or planned research activities as well as urgent and medium/long term research priorities. As with the opening statement to this summary, the document also recognises that current available information on existing research projects and research strategies is too limited and heterogeneous to adequately address identifying common research needs and undertaking to meet those research needs. 3.5.290 One set of publications from the OECD sponsorship programme which are likely to be of particular importance are the Draft Dossier Development Plans. These are the output of a sponsorship programme for testing a set of manufactured nanomaterials using appropriate test methods (which include OECD Test Guidelines or other internationally agreed methods). No testing results have yet emerged from the Sponsorship Programme, however a number of draft dossier development plans (DDP) have been published, namely for Zinc Oxide, Cerium Oxide, Silicon Dioxide, Single Walled Carbon Nanotubes, Multi-Walled Carbon Nanotubes and Fullerenes. - 107 - This is of particular value, as many of the FP6/7 studies and much of the published literature do not (unless specifically stated) utilise OECD or ISO standardised methods. Thus, upon publication, their results are likely to be of particular relevance to consideration of nanomaterial human toxicity within the context of REACH. 3.5.291 ECOTOXICOLOGICAL INFORMATION 3.5.292 The current ECHA guidance document on Chemical Safety Assessment ECHA (2008) refers specifically to a number of OECD Test guidelines. The appropriateness of these OECD Test guidelines as well as other guidelines have been review by the OECD project Safety Testing of a Representative Set of Manufactured Nanomaterials (ENV/JM/MONO(2009)21). The reviewers stated that: “For 24 OECD ecotoxicity test guidelines, the subgroup for biotic effects section concluded that the guidance on preparation, delivery, measurement, and metrology is currently insufficient for testing of manufactured nanomaterials”. (ENV/JM/MONO(2009)21, p. 13). 3.5.293 As previously mentioned, the results from the OECD-WPMN Sponsorship Programme for exploratory testing of nanomaterials have yet to be made available, which limits the ability to identify and further critically assess the appropriateness of existing test methods and data generated in fulfilling the ecotoxicological data requirements under REACH at this time. - 108 - 3.5.294 B3 Sub-Task IV: Summary of practical advice in relation to whether relevant ISO/CEN methods for substance characterisation could be used In fulfilling REACH data requirements 3.5.295 PHYSICO-CHEMICAL PROPERTIES INFORMATION 3.5.296 Water solubility 3.5.297 Although no published standards of relevance for assessing the water solubility of nanomaterials have been identified, ISO/AWI TR 13014 (currently at Committee Draft stage), which aims to provide guidance on physico-chemical characterisation for manufactured nano-objects for toxicological testing, should be reviewed in further detail when the document reaches FDIS stage in order to assess its relevance to determining water solubility in the REACH context. 3.5.298 Particle size/ size distribution 3.5.299 CEN EN 481:1994, which currently forms the backbone of the existing REACH Guidance on Granulometry (ECHA, 2008. R7.1.14), defines sampling conventions for particle size fractions which are to be used in assessing the possible health effects resulting from inhalation of airborne particles in the workplace. A number of key issues have been highlighted: o For particles of aerodynamic diameter less than 0.5 µm (i.e. including nanoparticles < 100 nm), CEN EN 481:1994 states that the particle diffusion diameter should be used instead of the particle aerodynamic diameter, defined as "the diameter of a sphere with the same diffusion coefficient as the particle under the prevailing conditions of temperature, pressure and relative humidity"; o Thus, the common methods used to determine particle size ranges of aerosols may not be appropriate for nanoparticles. 3.5.300 These issues are acknowledged briefly in the current REACH Guidance chapter on Granulometry (ECHA, 2008. R7.1.14) but no information on the availability of alternative methods for handling these issues is currently provided. This has been addressed through recommendations to update guidance in Task B5. - 109 - 3.5.301 In ISO/TS 27687:2008 several key issues are highlighted in relation to particle size measurement, including: o the measured size of a particle is always dependent on the particular method that is being used to examine, measure or visualise the particle; o interaction with the environment will differ between particle types, and thus the particle size reported by one technique may not be the same as that reported with another technique; o even with a single detection method, the results depend upon how the information is processed and length parameters used for particle size characterisation should always be indicated; o when reporting particle size measurement results, the method used to determine the particle size should be reported. 3.5.302 ISO/TR 27628:2007 contains guidelines on characterising occupational nanoaerosol exposures against a range of metrics (mass, number, surface area) with a view to forming a basis for extending knowledge on how occupational exposure to nanoaerosols should be most appropriately measured. Specific information is provided within this technical report on methods for aerosol characterisation and single particle analysis, of relevance to consider in relation to the REACH Guidance chapter on Granulometry. ISO/TR 27628:2007 outlines the principles, advantages and disadvantages of each of these methods, of relevance for incorporation into the amended REACH Guidance chapter on Granulometry. In addition, valuable guidance on aerosol sample collection and preparation for SEM and TEM analysis is also provided. However, this technical report does not provide specific methodology or protocols for undertaking nanomaterials characterisation using these methods. 3.5.303 ISO 10808:2010 highlights that: o measurement of number-weighted particle size distribution and measurement of total particle mass concentration are essential parameters to characterise for inhalation toxicity testing of nanoparticles; o conventional methods of fine or coarse particle monitoring (such as weight based mass dose monitoring) are considered insufficient for nanoparticles, - 110 - since nano-specific parameters such as particle surface area, particle number etc. might be critical determinants and thus should also be monitored. 3.5.304 The FDIS currently suggests a battery of inhalation toxicity testing chamber monitoring to include a Differential Mobility Analyzing System (DMAS) to measure particle size, distribution, number, surface area and estimated mass dose, as well as morphological examination using Transmission Electron Microscopy (TEM) or Scanning Electron Microscopy (SEM) equipped with an Energy Dispersive X-ray Analyzer (TEM-EDXA) for chemical composition. This method thus allows evaluation of nanoparticle distribution, surface area, mass dose, composition and dispersion to support effective analysis of inhalation toxicity testing results. 3.5.305 For the purposes of inhalation toxicity testing, measurement with DMAS is the only currently available method that meets all of the following requirements in the size range below 100 nm: o measurement of particle size distribution during particle exposures in a continuous manner with time resolution appropriate to check stability of particle size distribution and concentration; o measurement range of particle sizes and concentrations covers those of the nanoparticle aerosols exposed to the test system during the toxicity test; o particle size and concentration measurements are sufficiently accurate for nanoparticle toxicity testing and can be validated by ways such as calibration against appropriate reference standards; o resolution of particle sizing is sufficiently accurate to allow conversion from number-weighted distribution to surface area-weighted or volume- weighted distribution. 3.5.306 However, it is noted within the FDIS that, for non-spherical particles (e.g. carbon nanotubes), estimation of diameter and mass concentration by DMAS can result in significant error. - 111 - 3.5.307 In relation to this, published standard ISO/15900:2009 provides guidelines on the determination of aerosol particle size distribution using a DMAS. This standard is not currently referenced in the existing REACH Guidance (ECHA, 2008. R.7a). It is highlighted in the standard that it is important to know the stability of the source, since rapid changes of the size distribution, particle concentration, or both, can affect measurement of the size distribution. This is relevant to consider for nanomaterials, which have a high tendency to agglomerate in the atmosphere. 3.5.308 ISO 28439:2011 provides further supplementary information regarding the use of DMAS to determine the particle size distribution on nanomaterial aerosols. It is noted that: o the size range 3-1000 nm in electrical mobility diameter can be partly covered by other instruments (e.g. nanometer aerosol differential mobility analyser). However, DEMC has the advantage that the electrical mobility diameter is approximately equivalent to the project-diameter of the particles (defined as the diameter of a sphere with the same projected area as the particles being sized) with compact geometries; o particle size calibration is possible with the use of polystyrene reference particles, available in the size range 20-900 nm. However, the smallest polystyrene particle that can be used for DMAS is approximately 100 nm. This means that the performance of a DMAS cannot be easily tested by the user in the size range of ultrafine and nanoparticles smaller than 100 nm. 3.5.309 Thus the DMAS method and associated standards are of importance for consideration in relation to the updated REACH Guidance on Granulometry, and will be further assessed in the gap analysis (Task B4). 3.5.310 ISO/21501-1:2009 specifies characteristics of a light scattering aerosol spectrometer (LSAS) which is used for measuring the size, number concentration and number/size distribution of particles suspended in a gas. Due to its coverage of the nano-size range, the LSAS method outlined in ISO/21501-1:2009 may be relevant to consider in relation to informing the characterisation of particle size distribution under the REACH Granulometry Information Requirement. However, - 112 - based on the reviewed literature, there is limited evidence of the application of this method for characterising nanomaterials at present. 3.5.311 ISO/13318-1:2001 outlines general methods for determining the particle size distribution of particulate materials, typically in the size range 0.1 - 5 µm by centrifugal sedimentation in a liquid. The methods described are applicable to slurries, particulate materials that can be dispersed in liquids and some emulsions. It is acknowledged in the standard that no single method of size analysis can be specified to cover the many different types of materials encountered, but that general procedures can be recommended that are applicable to the majority of cases. 3.5.312 ISO/13322-1:2004 provides a standardised description of static image analysis methods for particle size analysis. An exact standard method is not provided by ISO due to the wide variety of applications of the technique. ISO/13322-1:2004 highlights that this method is essentially limited to narrow size distributions of less than an order of magnitude, requiring over 6, 000 particles to be measured in order to obtain a repeatable volume-mean diameter heightening to 61, 000 particles for the mass median diameter. The second part of ISO 13322 (ISO/13322-2:2006) provides guidance for measuring and describing particle size distribution, using image analysis where particles are in motion (dynamic image analysis) i.e. in a gas or a liquid. Neither standard comments on its applicability to nanomaterials, however ISO has listed these standards as potentially relevant to nanoscale measurement or observation (NIST, 2008). There is also evidence in the peer-reviewed literature and FP6/7 outputs of the application of image analysis alongside electron microscopy methods (such as TEM and SEM) for determining the particle size distribution of nanomaterials. 3.5.313 These standards are not currently referenced in the existing endpoint specific guidance (ECHA, 2008. R.7a). In relation to the counting procedures of static image analysis, in ISO 13322-1 it is stated in the standard that "it is a prime requirement of the method that measurements shall be made on isolated particles. There should be as few particles as possible touching each other". This may have implications for nanomaterials, which have a high tendency to agglomerate. 3.5.314 ISO/TS 13762:2001 outlines the principles of the small-angle X-ray scattering (SAXS) technique, applicable to particle sizes ranging from 1 - 300 nm, and - 113 - provides guidance for sample preparation (including preparation of dry powder samples and colloidal solutions), the experimental procedure and calculation and expression of results (pointing to ISO 9276-1 and ISO 9276-2 on the representation of the results of particle size analysis). 3.5.315 Given its coverage of the nano-size range, ISO/TS 13762:2001 and the SAXS method are therefore relevant to consider in relation to determination of particle size under REACH. The success of this technique is mainly based on the fact that SAXS effect results from the difference in electron density between particles and their surroundings such that the measurement always indicates the size of a primary particle rather than the internal crystallite or external agglomerate size. Thus, the requirement of particle dispersion of a sample for SAXS analysis is not as strict as that for other methods. However, SAXS does have limitations: o The method cannot distinguish pores from particles and the interference effect between particles will arise as the sample is available only in concentrated form. o In the data analysis for this method, it is assumed that particles isotropic and spherically shaped and it is stated that this technical specification does not apply to powders containing particles whose morphology is far from spherical. o This method would therefore not be relevant to consider for non-spherical nano-objects such as carbon nanotubes. o The method cannot be used for powders consisting of porous particles, which may also limit its applicability to the measurement of certain nanomaterials. 3.5.316 ISO/13320:2009 and the method of laser diffraction is relevant to consider in relation the characterising the particle size distribution, although no specific advice for sub- 100 nm particles is provided. It is important to highlight that this technique assumes a spherical particle shape. It is stated in the standard that test products should have no extreme aspect ratios, with a restriction of 1:3 for non-spherical particles. This method is therefore unlikely to be applicable to the measurement of high-aspect- ratio nanomaterials such as carbon nanotubes. Another major source of error arises - 114 - from incomplete deagglomeration of the particles, due to an improper dispersion procedure. This highlights the importance of establishing suitable dispersion protocols for nanomaterials. 3.5.317 ISO/22412:2008, which specifies a method for the application of Dynamic Light Scattering (DLS) to the estimation of an average particle size and the measurement of the broadness of the size distribution of mainly sub micrometre-sized (approx 1 - 1000 nm) particles or droplets dispersed in liquids, is not currently referenced in the existing endpoint specific guidance (ECHA, 2008. R.7a). As outlined in the literature section of RNC/RIP-oN2/B3/2/FINAL, the method of DLS does have several limitations in relation to the characterisation of nanomaterials (e.g. lack of discrimination between agglomerates of nanoparticles and larger particles) which would need to be acknowledged in the amended guidance. It is also likely that the DLS method would need to be used in combination with other techniques. 3.5.318 Related to this standard is ISO/13321:1996 which provides more detailed information on the procedures to allow the determination of the correct particle size using photon correlation spectroscopy (PCS). It is highlighted that samples should consist of well-dispersed particles in a liquid medium, which may have implications for nanomaterials with a tendency to agglomerate and further highlights the need for reproducible dispersion protocols. Published standard E56 WK8705 from the American Society for Testing and Materials (ASTM) also deals with the measurement of particle size distribution of suspended particles, which are solely or predominantly sub-100 nm, using PCS. It is highlighted within E56 WK8705 that PCS measurement is hydrodynamically based and therefore provides size information in the suspending medium (typically water). Thus the hydrodynamic diameter will almost certainly differ from other size diameters isolated by other techniques and users of the PCS technique need to be aware of the distinction of the various descriptors of particle diameter before making comparisons between techniques. 3.5.319 ISO/22412 2008, ASTM E2490-09 and ISO/13321 1996 are thus also relevant to consider with regards characterising the particle size distribution of nanoparticles in suspensions, potentially in relation to the REACH Granulometry Information Requirement as well as investigating the preparation of stable dispersions for (eco)toxicological testing. - 115 - 3.5.320 ISO/20998-1:2006, which describes ultrasonic methods for determining the size distributions of one or more material phases dispersed in a liquid, is also relevant to consider in relation to determining the particle size distribution of nanoparticles in suspension, potentially in relation to the REACH Information Requirement on Granulometry. Ultrasonic methods can be used to monitor dynamic changes in the size distribution, including agglomeration or flocculation in concentrated systems and may therefore be of use in the preparation of stable dispersions of nanomaterials for (eco)toxicological testing. However, there is limited evidence of the application of this method in relation to nanomaterials in the scientific literature at present, with methods such as DLS preferred. 3.5.321 ISO/21501-2:2007, describes a calibration and verification method for a light scattering liquid-borne particle counter (LSLPC), which is used to measure the size and particle number concentration of particles suspended in liquid. The typical size range of particles measured by this method is between 0.1 μm and 10 μm in particle size. This standard and the method of LSLPC may be relevant to the characterisation of the particle size distribution of nanomaterials in suspension, potentially in relation to the REACH Granulometry Information Requirement. However, as with acoustic methods, there is limited evidence of the application of LSLPC in the scientific literature at present. 3.5.322 BS EN 13925-1:2003 describes the general principles of X-ray diffraction (XRD) for polycrystalline and amorphorphous materials, but does not go as far as to define a specific or detailed standard for each field or type of analysis. XRD allows estimation of the average particle size by mathematical adaption of a simulated diffractogram, with sensitivity down to 1 nm. BS EN 13925-1:2003 outlines the general principles of the method, characteristics of powder diffraction line profiles, types of analysis and experimental conditions. This standard is further supported by BS EN 13925-2:2003, which specifies the basic procedures of the XRD method, and BS EN 13925-3:2005, which sets of the characteristics of instruments used for XRD as a basis for their control and hence quality assurance of the measurements made by this technique. BS EN 13925-1 2003, BS EN 13925-2 2003 and BS EN 13925-3:2005 and the method of XRD are relevant to consider in the context of the REACH Granulometry Information requirement for nanomaterials. - 116 - 3.5.323 ISO/AWI TS 10797 (which describes the characterisation of SWCNT using TEM, ISO/AWI TS 10798 (which describes the characterisation of SWCNT using SEM and energy dispersive X-ray spectrometry analysis) are currently at Committee Draft stage, while work on ISO/NP TS 10812 (which outlines the use of Raman Spectroscopy for the characterisation of SWCNT) has just recently commenced. It is recommended that these documents are reviewed in detail when they reach FDIS stage in relation to their potential to inform the determination of particle size distribution in the REACH context. 3.5.324 ISO/NP TS 10868 (currently at FDIS stage) provides guidelines for the characterisation of compounds containing SWCNTs by using optical absorption spectroscopy. Although this standard does not address particle size/size distribution per se, the information provided in relation to diameter determination may be relevant to consider under the REACH Granulometry Information Requirement, depending on the coverage of this term. 3.5.325 ISO/AWI TR 13014 (which aims to provide guidance on physico-chemical characterisation for manufactured nano-objects for toxicological testing) and ISO/DTR 10929 (which outlines a collection of measurement methods for the characterisation of multi-walled carbon nanotubes (MWCNT)), currently at Committee Draft stage, may offer information of relevance to determining the particle size/geometrical properties (e.g. length/diameter) of nanomaterials. It therefore recommended that these documents are reviewed for their relevance in the REACH context when they reach FDIS stage. 3.5.326 In addition, ISO/CD 12025 (which aims to provide a general framework for determining nano-object release from powdered engineered nanoparticles into the gaseous surroundings by means of analysis of the generated aerosols particles) and the methods it advocates, is relevant to consider in relation to REACH Granulometry Information Requirement, in terms of measurement related to dustiness as well as methods for the generation of nanoaerosols for studying parameters such as particle size distribution. The release of nano-objects from nanomaterials into the surrounding air is an important consideration in relation to the hazard potential of nanomaterials. However, this document is currently at Committee Draft stage with the content still under development. It is recommended that it should be reviewed in further detail when the document reaches FDIS stage. - 117 - 3.5.327 Agglomeration/ aggregation state 3.5.328 A number of the methods and standards described above for determining the particle size distribution of particles will also apply to determining the level of agglomeration/aggregation of particles in powder and suspension form, of relevance to consider in the context of REACH. Of key importance are ISO/20998-1:2006 (which describes ultrasonic methods for determining the size distributions of one or more material phases dispersed in a liquid), ISO/13322-1:2004 (which describes static image analysis for determining the size distribution of particles in both powder and suspension form) and ISO/TS 13762:2001 (which describes the small angle X- ray scattering method, also applicable to both powders and suspensions). 3.5.329 A number of the standards are currently at Committee Draft level may also offer information of value to consider in relation to characterising the agglomeration/aggregation state of nanomaterials, specifically ISO/AWI TS 10797 (which describes the characterisation of SWCNT using transmission electron microscopy), ISO/AWI TS 10798 (which describes the characterisation of SWCNT using scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry analysis) and ISO/AWI TR 13014 (which aims to provide guidance on physico-chemical characterisation for manufactured nano-objects for toxicological testing). It is recommended that these standards should be reviewed with regards their relevance for informing characterisation under REACH they reach FDIS stage. 3.5.330 Crystallite or grain size 3.5.331 A number of the methods and standards described above for determining the particle size distribution of particles will also apply to determining the crystallite or grain size of nanomaterials, of possible relevance to consider in the context of REACH. Of particular relevance is BS EN 13925-1:2003 which describes the general principles of X-ray diffraction (XRD) for polycrystalline and amorphorphous materials. XRD can be used to study single crystal or polycrystalline materials in powder or dispersion form, with sensitivity down to 1 nm, providing a wealth of information, including crystallite size, as well as crystalline phase composition, lattice strain and defects, crystallographic orientation and charge distribution. BS EN 13925-1:2003 outlines the general principles of the method, characteristics of powder diffraction line profiles, types of analysis (including analysis of crystallite size) and experimental conditions. This standard is further supported by BS EN - 118 - 13925-2:2003, which specifies the basic procedures of the XRD method, and BS EN 13925-3:2005, which sets of the characteristics of instruments used for XRD as a basis for their control and hence quality assurance of the measurements made by this technique. 3.5.332 In addition, ISO/AWI TS 10797 on the use of TEM for charactering SWCNT, currently under development, will also be relevant to consider in this context. It is recommended that this document be fully reviewed when it reaches FDIS status. 3.5.333 Aspect ratio/ shape 3.5.334 The static image analysis methods described in ISO/13322-1:2004 and the FDIS standard ISO/NP TS 10868 which provides guidelines for the characterisation of compounds containing SWCNTs by using optical absorption spectroscopy, covering determination of the mean diameter may be particularly useful for studying the shape of nanomaterials. 3.5.335 It is recommended that the following draft standards under development should be reviewed when they reach FDIS status in regards to their relevance to inform the measurement of the aspect ratio/shape of nanoparticles in the context of REACH: ISO/AWI TS 10797 (which describes the characterisation of SWCNT using TEM), ISO/AWI TS 10798 (which describes the characterisation of SWCNT using SEM and energy dispersive X-ray spectrometry analysis), ISO/AWI TR 13014 (which aims to provide guidance on physico-chemical characterisation for manufactured nano-objects for toxicological testing), ISO/DTR 10929 (which outlines a collection of measurement methods for the characterisation of multi-walled carbon nanotubes (MWCNT)) and ISO/DTS 11888 (which addresses the determination of mesoscopic shape factors of MWCNT). 3.5.336 Specific surface area 3.5.337 ISO/9277:2010 (which outlines a method for determination of the total specific external and internal surface area of disperse or porous solids by measuring the amount of physically absorbed gas according to the BET method) and ISO/18757:2005 (which provides guidelines for the determination of the total specific external and internal surface area of disperse or porous (pore diameter > 2 nm) fine - 119 - ceramic materials by the BET method) are relevant to consider in the context of characterising the specific surface area of nanomaterials. 3.5.338 Although the BET method assumes a mono-dispersed spherical system, it is commonly applied to determine both the surface area and average particle size (not size distribution) of nanomaterials. ISO/9277:2010 highlights that, in order to ensure proper working conditions and correct data evaluation, the apparatus performance should be monitored periodically using a surface-area reference material, with certified reference materials listed in an annex. This highlights the importance of defining suitable reference nanomaterials. 3.5.339 ISO/TR 27628:2007 (which contains guidelines on characterising occupational nanoaerosol exposures against a range of metrics) provides general information of relevance to consider in relation to characterising the surface area of aerosolised nanoparticles. However, no specific methodological guidance or protocols are detailed. It is important to note that within this standard it has highlighted that the BET method has been used with some success for measuring aerosol surface area, but that it suffers the disadvantages of requiring the collection of relatively large amounts of material, and measurements are influenced by particle porosity and collection/support substrate. 3.5.340 ISO/TS 13762:2001 (which specifies a method for determining particle size distribution of ultra-fine powders by the small-angle X-ray scattering (SAXS) technique, applicable to particle sizes ranging from 1 - 300 nm) can also be used to derive the specific surface area of a powder sample from the measured particle size distribution and the mass density of the powder particle. 3.5.341 Similarly, the static image analysis techniques described in ISO/13322-1:2004 enable determination of the specific surface area of particles from the particle size measurements. 3.5.342 The DMAS system described within ISO 10808:2010 can be used to calculate the surface area based on the determined particle diameter. However, it is noted that for non-spherical particles (e.g. carbon nanotubes), estimation of diameter and mass concentration by DMAS can result in significant error. - 120 - 3.5.343 Three published ISO standards which address determination of the pore size distribution of solid materials by mercury porosimetry and gas adsorption using related methods (ISO 15901-1 2005; ISO 15901-2 2006; ISO 15901-3 2007) may enable indirect determination of the specific surface area of the characterised material. Indeed, ISO 15901-1:2005 provides an equation to calculate a specific surface area of the intruded pores. However, these methods are quite specific and unlikely to obtain widespread use for the determination of specific surface area alone. 3.5.344 It is recommended that ISO/AWI TR 13014 (which aims to provide guidance on physico-chemical characterisation for manufactured nano-objects for toxicological testing) should be reviewed in further detail when the document reaches FDIS stage in the context of identifying methods for the determination of surface area under REACH. 3.5.345 Surface charge/zeta potential 3.5.346 No published standards of relevance for determining the surface charge of nanomaterials have been identified. 3.5.347 It is recommended that draft standards under development ISO/CD 13099-1 and ISO/CD 13099-2 (which both relate to the measurement of zeta potential) as well as ISO/AWI TR 13014 (which aims to provide guidance on physicochemical characterisation for manufactured nano-objects for toxicological testing), should be reviewed when the reach FDIS stage in relation to their relevance in providing methods for the characterisation of surface charge of nanomaterials 3.5.348 Surface chemistry 3.5.349 ISO has published a wide-range of standards related to surface chemical analysis using three principle techniques: X-ray photoelectron spectroscopy (XPS), Auger electron spectroscopy (AES), and secondary-ion mass spectrometry. However, these documents are highly specific and they most relevant to consider in the context of determining the surface chemical composition of materials. 3.5.350 It is highly recommended that ISO/WD TR 14187 (which looks specifically at the surface chemical analysis of nanostructured materials), as well as ISO/NP TS 10812 (which outlines the use of Raman Spectroscopy for the characterisation of SWCNT) - 121 - and ISO/AWI TR 13014 (which aims to provide guidance on physico-chemical characterisation for manufactured nano-objects for toxicological testing), should be reviewed when they reach FDIS stage to assess their relevance for informing the characterisation of the surface chemistry of nanomaterials in the REACH context. 3.5.351 Dustiness 3.5.352 The standard current referenced in the REACH Guidance on Granulometry for determining the dustiness of bulk materials is EN 15051:2006. The current limitation of these methods in relation to nanomaterials is that they measure the mass, rather than e.g. the particle number, of the resultant dust and provide no size- based information. In addition, rotating drum dustiness tests are usually performed as three replicate tests and need quite large amounts of test material, typically 300– 600 g. The EN 15051 continuous single-drop method requires a total amount of 500 g for the required five single-test runs. Modified methods for measuring the dustiness of nanomaterials have been described in the FP6/7 section and the literature section of RNC/RIP-oN2/B3/2/FINAL. 3.5.353 It is highly recommended that draft standard under development ISO/CD 12025 (which aims to provide a general framework for determining nano-object release from powdered engineered nanoparticles into the gaseous surroundings by means of analysis of the generated aerosols particles, using adapted procedures for determining dustiness) should be reviewed in further detail when the document reaches FDIS stage as it will likely contain information of importance to consider in relation to REACH guidance updates relating to dustiness measurements for nanomaterials, 3.5.354 Porosity 3.5.355 The three published ISO standards which describe methods for determination of the pore size distribution of solid materials by mercury porosimetry and gas adsorption ISO 15901-1 2005; ISO 15901-2 2006; ISO 15901-3 2007) may be applicable to determining the porosity of nanomaterials. 3.5.356 Photocatalytic activity 3.5.357 ISO 22197-1:2007 (which specifies a test method for determination of the a test method for the determination of the air-purification performance of materials that - 122 - contain a photocatalyst or have photocatalytic films on the surface) may be applicable for the determination of the photocatalytic activity of nanomaterials, However it has not been established whether the interpretation of data gathered by this method, and used for hazard assessment purposes, will be meaningful. Whilst it is being investigated under the OECD Sponsorship Programme, it is stated that the method does not apply to powder or granular photocatalytic materials, thus restricting its use for some forms of nanomaterials. 3.5.358 Sample preparation 3.5.359 ISO/14887:2000 (which provides general guidance to assist in the preparation of good dispersions from various powder/liquid combinations) covers procedures for wetting a powder into a liquid; deagglomerating the wetted clumps; selecting dispersing agents to prevent reagglomeration; evaluating the stability of the dispersion against reagglomeration. This standard is applicable to particles ranging in size from approximately 0.05 to 100 µm, and is therefore relevant to consider for informing the preparation of nanomaterials dispersions in the REACH context. For examining the resultant dispersion, ISO 14887:2000 states that, for particles smaller than 1 µm, the standard method of optical microscopy should be replaced with some other form of evaluation. 3.5.360 The characterisation of particle properties like size, form and specific surface area requires very careful sampling and sample splitting practices to be followed. It is considered vital that the test aliquot used for measurement is representative of the sample of particulate material. While this standard does not specifically address procedures for nano-sized materials, ISO 14488:2007 (which specifies methods for obtaining a test alliquot from a defined sample of particulate material that can be considered to be representative of the entire sample with a defined confidence level) offers general information and good practice of relevance to powder sampling of a range of materials of value for further consideration in relation to the updated REACH Guidance. 3.5.361 It is highly recommended that ASTM draft standard E56 WK10417 (which outlined standard practice for the preparation of nanomaterial samples for characterisation) is reviewed when published. - 123 - 3.5.362 Other information of relevance 3.5.363 Although ISO/DTR 13121 (which describes a process for evaluating, addressing, making decisions about, and communicating the potential risks of developing and using engineered nanoscale material) does not address specific methods for characterising the physico-chemical properties of nanomaterials, it highlights a number of key issues of importance to consider further in development of recommendations for guidance updates, specifically: o The importance of characterising the physico-chemical properties of a nanomaterial over its entire life cycle is highlighted; o It is recommended that any anticipated changes in relevant physical and chemical properties across the lifecycle of the material should be noted. It may be necessary to characterize the material at multiple points unless there is good reason to expect that the material will remain unchanged; o It is recommended that the properties of the nanomaterial should be compared to those of the corresponding bulk (non-nanoscale) materials, where appropriate, to determine the nature and extent to which the properties are different. 3.5.364 It is highly recommended that the following draft standards ISO/AWI TS 11931-1 and ISO/AWI TS 11937-1 which describe characteristics and measurement methods for nano-calcium carbonate and nano-titanium dioxide, respectively currently under development are reviewed when published to assess their relevance in the context of the updated REACH guidance. - 124 - 3.5.365 TOXICOLOGICAL INFORMATION 3.5.366 Those ISO/CEN documents published or classified as being at Final Draft International Standard (FDIS), Draft International Standard (DIS) stage or at an early stage of development (i.e. Committee Draft stage or lower) were reviewed and commented upon. It is clear that whilst there a number of standards at ballot level or under development, there is to date only one published document pertaining to the toxicity of nanomaterials (ISO 10801:2010). It is expected that this standard will provide a useful contribution to supporting good experimental design in the mammalian toxicity testing under hazard identification within the context of REACH. In summary, at the current time there exists only one fully published method from ISO which may be considered directly relevant for fulfilling the REACH Information Requirements relating to toxicity. 3.5.367 ECOTOXICOLOGICAL INFORMATION 3.5.368 On the basis of the review carried out, it is considered that no work has so far been published by ISO and CEN that is considered directly relevant for fulfilling the REACH Information Requirements in regard to ecotoxicity. - 125 - 3.6 GAP ANALYSIS OF RELEVANT INTRINSIC PROPERTIES FOR NANOMATERIALS POSSIBLY NOT ADDRESSED BY STANDARD TEST GUIDELINE METHODS AND REQUIRING FURTHER DEVELOPMENT OF IN VITRO, IN VIVO OR OTHER METHODOLOGIES (TASK B4) 3.6.1 The gap analysis of relevant intrinsic properties for nanomaterials, which may not be addressed by standard test guideline methods and for which further development of in vitro, in vivo or other methodologies is required, has assembled and further developed the findings from the examination of existing REACH Guidance related to information requirements and testing (information generation) strategies (Task B1), the identification of additional relevant specific intrinsic properties for nanomaterials (Task B2), the assessment of relevance and applicability of testing, endpoints and methods described in the scientific literature and on-going international work relevant to the fulfilment of the data requirements under REACH (Task B3). 3.6.2 The structural framework used for the gap analysis considers physico-chemical properties and toxicological and ecotoxicological endpoints, and integrates the existing and additional properties/endpoints to meet the objective of identifying those which may and may not be addressed by standard test guideline methods and where further development of in vitro, in vivo or other methodologies is required. 3.6.3 The outcomes of the gap analysis, specifically addressing the aforementioned objective, are summarised below. (The information developed for the gap analysis has been used subsequently in the development of specific guidance updates and recommendations for research & development (two of the objectives of Task B5)). 3.6.4 The following intrinsic properties, identified from the gap analysis, are relevant to nanomaterials, with suspected important differences between nano and non- nanomaterials, which may not be addressed by standard test guideline methods and require further development of in vitro, in vivo or other methodologies. Those considered to be of low priority for further research and development are indicated using an asterisk. Those for which standards, or other important documents, are known to be in preparation are indicated with a . 3.6.5 Physico-chemical properties Existing Information Requirements  R.7.1.7 Water Solubility  R.7.1.10 Flammability - 126 -  R.7.1.11 Explosive properties Additional Relevant Specific Intrinsic Properties  Porosity  Surface energy  Surface acidity  Surface charge (zeta potential)  Redox potential 3.6.6 Toxicological endpoints  None identified. 3.6.1 Ecotoxicological endpoints 