Final report May 2008
Defra CB403
Comparison of risk assessment approaches for manufactured nanomaterials
Comparison of risk assessment approaches for manufactured nanomaterials
Report compiled as part of Defra project (CB403)
Final report
30thMay 2008
Report compiled by Dr S Rocks, Prof S Pollard, Dr R Dorey, Prof L Levy, Dr P Harrison (Cranfield University) and Dr R Handy (University of Plymouth)
Final report May 2008
Defra CB403
Comparison of risk assessment approaches for manufactured nanomaterials
Table of Contents
1. Introduction3
1.1. Aims and statements3
1.1.1. Aims and objectives3
1.1.2. First report summary4
1.2. Toxic effects of manufactured nanomaterials6
1.3. Summary7
2. Assessment of hazard9
2.1. Assessment methods9
2.1.1. General overview9
2.2. Physicochemical testing21
2.2.1. Overview22
2.2.2. Testing methods29
2.2.3. General knowledge gaps30
2.3. Toxicity testing30
2.3.1. Overview31
2.3.2. Testing methods36
2.3.3. General knowledge gaps38
2.4. Ecotoxicological testing39
2.5. Addressing areas of concern39
2.5.1. Physicochemical tests40
2.5.2. Toxicity tests40
2.6. Summary41
3. Risk Assessment Framework42
3.1. General overview42
3.2. Risk assessment frameworks47
3.2.1. Pharmaceutical risk assessment framework47
3.2.2. Occupational risk assessment framework52
3.2.3. Chemical risk assessment framework56
3.3. Risk assessment tools58
3.3.1. Human health risk assessment tools60
3.3.2. Environmental risk assessment tools61
3.4. Reported opinion on the appropriateness of current
risk assessment frame works for application to manufactured nanomaterials 61
3.5. Summary75
4. Workshop outcomes79
4.1. Overview79
4.2. Issues covered79
4.2.1. Inventory of evidence79
4.2.2. Strength of evidence83
4.2.3. Weight of evidence88
4.4. Significant knowledge gaps and recommendations90
5. Summary92
6. Bibliography94
7. Abbreviations99
Section 1
Introduction
1.1 Aims and statements
1.1.1. Aims and objectives
The overall aim of this project is to evaluate and make recommendations on risk assessment approaches for manufactured nanomaterials through information exchange (across the Organisation for Economic Co-operation and Development; OECD) and, through an understanding of any unique challenges nanomaterials present, to identify opportunities to strengthen and enhance risk assessment capacity.
This overarching aim will be achieved through the following objectives:
1.exchanging, collating and synthesising information on current risk assessment approaches for industrial chemicals that may apply to manufactured nanomaterials;
2.undertaking a gap analysis of current risk assessment approaches as these apply to manufactured nanomaterials;
3.making recommendations to the OECD Steering Group for addressing and filling identified gaps; and
4.recognising that there will be limitation to the applicability of risk assessment to engineered nanomaterials given the current evidence base on dose-response assessment and exposures beyond occupational settings.
The first report (Rocks et al., 2008) addressed Objective 1. The purpose of this report is to build on the conclusions of the first report and convey the findings of a gap analysis on the risk assessment approaches (with particular emphasis on those that apply to industrial chemicals) as they apply to manufactured nanomaterials, and to make recommendations for addressing and filling the identified gaps. This report addresses the final three objectives and reports the results from a workshop held at CranfieldUniversity on 15thMay 2008.
1.1.2. First report summary
The first report considered the current regulatory frameworks available across the world (concentrating on those from US, UK, EU and Australia) and identified areas for further discussion as to whether manufactured nanomaterials would be sufficiently covered by these guidelines. Data have been collected by extensive literature searches and responses to the OECD’s questionnaire to the Working Party on Manufactured Nanomaterials.
The regulatory guidelines were considered in terms of the manufacture or importation of a chemical species. The described guidelines give the general principles applied to the risk assessment of chemicals and pointed to more detailed information and resources where available.
A summary of a general regulatory framework was produced (Figure 1.1), and it was determined that international risk assessment frameworks mainly followed the same overall procedure, with the notable exception of the regulation of chemical species in New Zealand where the act of manufacture or importation is enough to require the manufacturer to start the risk assessment process. The industrial chemical risk assessment in the European Union (EU) is covered under the Registration, Evaluation, Authorisation and Restriction of Chemical substances (REACH; Regulation (EC) No 1907/2006) which applies to existing and new chemicals being manufactured or imported in amounts greater than 1 tonne/year, which are collated by the European Chemicals Agency (ECHA). Regulation of chemical substances under REACH is based on the principle “that industry should manufacture, import or use substances or place them on the market in a way that, under reasonably foreseeable conditions, human health and the environment are not adversely affected” (ECHA, 2000).
Figure 1.1. Schematic showing a general summary for the risk assessment of chemical substances. The schematic indicates the areas in which testing is normally required with the type of tests involved. The normal trigger for the risk assessment of a chemical substance is for the production to exceed 1tonne/year.
The initial requirement for the risk assessment of a chemical substance occurred generally after the amount produced exceeded 1 tonne/year (as seen in Table 1.1).
Table 1.1. International weight limits for the request for more information (i.e. triggers) in the risk assessment of chemicals
The amount of substance manufactured or imported is considered to be the initial trigger for the risk assessment process. Further weight triggers require more information to be generated about a chemical; however toxicological findings could also trigger this request.
1.2.Toxicological effects of manufactured nanomaterials
Chemicals, and manufactured nanomaterials, can enter the body via the lungs, skin and gastro-intestinal tract (Klaassen, 2001). The extent of initial entry into the body is likely to depend on the size and surface properties of the nanomaterial (Nemmar et al, 2002a;Gieser et al., 2005;). There has been some indication that the surface properties of nanomaterials are less important than the size and shape of the material (Poland et al., 2008; Ferin et al., 1990; Oberdoerster et al, 1990; Brown et al., 2001; Dankovic et al., 2008), however it is likely that a combination of a number of physicochemical characteristics and the chemical properties will cause the overall toxic effect of nanomaterials (Nemmar et al, 2002b; Figure 1.2), which is supported by similar observations in ultrafine particles (Kreyling et al, 2004).
Figure 1.2. Schematic of the physicochemical properties of nanomaterials and their likely effect on biological interactions (after Stone et al., 2008).
The size of the nanomaterial has been shown to affect the surface area and therefore the chemical reactivity (Duffin et al, 2002; Duffin et al, 2007) as well as the translocation potential of nanomaterials, which will in turn increase the toxicological effects of nanomaterials (Stone et al., 2007). Therefore, it is likely that a combination of many different characteristics and properties determine the toxicological effect of nanomaterials. However, the likelihood of exposure must also be assessed before the associated risk of manufactured nanomaterials can be determined.
1.3. Summary
The manufacture and importation of manufactured nanomaterials will be covered under REACH in the EU and, apart from the initial requirement for a weight trigger, will generally be appropriate for the risk assessment of nanomaterials depending on the quality of data used.
This report firstly considers the physicochemical and toxicity methods (as adopted by OECD; Section 2), the knowledge gaps presented by these methods,the risk assessment framework and determination of quality of data (Section 3), potential risk assessment tools and the reported opinions on whether the risk assessment frameworks are appropriate for use with nanomaterials. We then present the findings from the workshop identifying further knowledge gaps with associated recommendations (Section 4).
Section 2
Assessment of hazard
2.1 Assessment methods
2.1.1. General overview
The assessment of the effects of chemical exposure on human health and organisms in any environment involves the consideration of a range of properties, principles and characteristics. The starting point is normally an assessment of the physicochemical and toxicological properties of a substance (Klaassen, 2001; Barille, 2004). The latter requires an understanding of how the substance behaves in different environments, including consideration of its persistence, bioavailability, internal distribution and bioaccumulation, which can be indicated by its physicochemical properties (van Leeuwen and Vermeire, 2007). The evaluation of the relative significance of the possible exposure pathways is essential as this determines not only the extent to which various tissues might be exposed, and therefore which toxicity data are most relevant, but also whether significant exposure is likely to occur at all (Harrison and Holmes, 2006).
In chemical risk assessment, a range of critical toxicity endpoints and associated test guidelines have been established by regulatory bodies worldwide (including OECD, Environmental Protection Agency (EPA), EU). These are described in more detail below (along with their suitability to determine the toxicity of nanomaterials), but typically involve the assessment of acute toxicity (e.g. lethal dose for 50% of test animals; LD50), repeat dose toxicity, irritancy, sensitization potential, mutagenicity, clastogenicity, carcinogenicity and reproductive toxicity. The specific tests conducted and the routes of exposure used in the testing regime are governed by the physicochemical properties of the substance, as well as its likely use and human exposure scenarios. Exposure routes include oral (delivered in the feed or by gavage), dermal, and inhalation, as well as other, less common, routes such as sub-dermal, intravascular and intraperitoneal,which may be used if appropriate in the health risk assessment of pharmaceuticals and in special circumstances (e.g.determination of toxicological mechanisms of action; van Leeuwen and Vermeire, 2007). The use of toxicological endpoints in risk assessment frameworks is discussed in more detail in Section 3.
The European Chemicals Bureau (ECB) controls the implementation and harmonisation of test methods on chemical substances in the EU, in close collaboration with the OECD and other International Organisations. The legally binding EU standardised Testing Methods to determine the hazardous properties of chemicals are contained in Annex V of Dir 67/548/EEC on the Classification, Packaging and Labelling of Dangerous Substances (accessed at The tests enable the determination of the intrinsic properties of chemicals, but further testing requirements have been determined for particulate materials and man-made fibres (EUR 20268 EN, 2002) which are currently being developed. The knowledge of these properties allows the identification and assessment of the hazards that the chemicals pose and provide the information needed for exposure assessment as well as fate and pathways of chemicals in the environment. These tests aim to identify any adverse effects that the chemicals have an inherent capacity to cause and, where appropriate, estimation of the relationship between dose or level of exposure to a substance and the incidence and severity of an effect. The Testing Methods are split into three parts: Part A, physicochemical properties; Part B, human health effects; and Part C, environmental effects. The Testing Methods are summarised in Table 2.1, where the corresponding OECD Technical Guidance Document number (OECD TG) is also quoted as well as the general concerns over the use of the methods for the risk assessment of manufactured nanomaterials. The discussion of those points is continued below.
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Final report May 2008
Defra CB403
Comparison of risk assessment approaches for manufactured nanomaterials
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterialsA. / Physicochemical properties
Substance state
Melting freezing temperature (OECD TG102) / Capillary method in liquid bath or in metal block (visual identification)
Kofler hot bar (visual identification)
Melt microscope (using microscope hot stages)
Method to determine the freezing temperature (temperature measured)
Apparatus with photocell detection
Differential Thermal Analysis (DTA)
Differential Scanning Calorimetry (DSC)
Boiling point (OECD TG103) / Ebulliometer
Dynamic method
Distillation method
Method according to Siwoloboff
Photocell detection
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Boiling point (OECD TG103) (continued) / Differential Thermal Analysis (DTA)
Differential Scanning Calorimetry (DSC)
Relative Density*(OECD TG109) / Hydrostatic balance2
* nanomaterials assumed to be solid and not liquid / Pycnometer2
Air comparison pycnometer2
Vapour Pressure (OECD TG104) / Dynamic method (Cottrell’s method, only if low melting point)
Static method
Isoteniscope
Effusion method: vapour pressure balance2
Effusion method: loss of weight2
Gas saturation method
Spinning rotor
Surface tension (OECD TG115) / Plate method4
Stirrup method4
Ring method4
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Water Solubility (OECD TG105)5 / Preliminary test (visual determination of dissolved amount)
Column elution method
Flask method
Partition Coefficient (n-octanol/ water) / Shake-flask method (OECD 107)3,5,8,
HPLC method 3,5,8
Flash point / Liquids only
Flammability (solids) / Preliminary screening test2
Burning rate test (NF T20-042)2
Flammability (contact with water) / Step-by-step testing (not suitable for substances that spontaneously combust with air)
Self-ignition Temperature (Pyrophoric) / Powdery solid poured from height and observed (NF T20-039)2,6
Relative Self-ignition Temperature (solids; NF T-20-036)
Explosive Properties (NF T20-038) / Thermal sensitivity (DIN 1623)
Mechanical sensitivity (shock)
Mechanical sensitivity (friction)
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Oxidising Properties (NF T20-035) / Preliminary test7 mixture of solid with cellulose by weight
Train test7 mixture of solid with cellulose by weight
Grannulometry
Stability in organic solvents and identity of relevant degradation products
Dissociation constant
Viscosity
Particle size distribution / Microscopy examination (OECD TG110) using light or electron microscopy
Sieving (OECD TG110)6,12
Sedimentation (gravitational settling; OECD TG110)
Electrical sensing zone (OECD TG110)
Phase Doppler anemometry (PDA) – assumes particles are spherical and have a known refractive index
Determination of fibre length and diameter distributions (OECD TG110)
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Particle size distribution (contd) / Cascade impaction
Laser scattering/diffraction
Rotation drum method
Elutriation (OECD TG110)
Air jet sieve
Cycone
A. / Toxicity testing / Histopathological examination in all studies should include electron microscopy
Eye Irritation/Corrosion / Acute eye irritation (OECD TG405)2,7,9
Skin Sensitisation / Guinea pig maximisation test (OECD TG406)2,7,9
Buehler test (OECD TG406)2,7,9
Acute Oral Toxicity / Fixed dose procedure (OECD TG420)2,7,9 – administration of substance by tube (volume required for standard doses)
Acute toxic class method (OECD TG423) 2,7,9 – administration of substance by tube (volume required for standard doses)
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Acute Inhalation Toxicity / Acute inhalation toxicity (OECD TG403)2,6,7,9 - range of doses not exceeding 5% volume of test chamber
Acute Dermal Toxicity / Acute dermal toxicity (OECD TG402)2,7,9
Acute Dermal Irritation/Corrosion / Acute dermal irritation/corrosion (OECD TG404)2,7,9,10
Repeated Dose (28 days) Toxicity / Oral administration (OECD TG407)2,7,9
Inhalation administration (OECD TG412)2,7,9
Dermal administration (OECD TG412)2,7,9
Sub-Chronic Oral / Repeated dose 90-day in rodents (OECD TG408)2,5,6(in diet dried form),7 – administration by gavage/diet/drinking water (dependant on material) should ensure that dose is constant
Repeated dose 90-day in non-rodents (OECD TG409)2,5,7 – administration dependant on material and species
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Sub-chronic Dermal / Repeated dose 90-day in rodents (OECD TG411)2,5,6,7 – solution applied to uncovered skin daily
Sub-chronic inhalation / Repeated dose 90-day in rodents (OECD TG413)2,5,7 – six hours exposure daily
Chronic / Chronic Toxicity Test (OECD TG452)2,5,6,7 daily administration for major proportion of life span by an appropriate route, see sub-chronic studies
Prenantal Developmental Toxicity Study (Tetratogenicity, OECD TG414) 2,5,7 – oral administration may not be the most appropriate route, route determined by material properties
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Chronic Tests (continued) / Carcinogenicity Test (OECD TG451)2,4,5,6,7 – if substance is made available continuously (e.g. in water or diet) then it should be monitored to ensure a constant exposure level
Combined Chronic Toxicity/Carcinogencity Test (OECD TG453)
One-Generation Reproduction Toxicity Test (OECD TG415)2,4,5,7,11 – normally administered in diet or drinking water
Two-Generation Reproduction Toxicity Test (OECD TG416)2,4,5,7,11 – normally administered in diet or drinking water
Toxicokinetics (OECD TG417)2,7,9,11 – single or repeated doses by appropriate route, human exposure may be by more than one route.
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Chronic Tests (continued) / Neurotoxicity study in rodents (OECD TG 424)2,4,5,7,11 – oral administration over 28, 90 or 360+ days, inhalation may be more appropriate.
Mutagenicity / In vitro mammalian chromosome aberration test (OECD TG473) – test substance dissolved or suspended, range of concentrations administered (up to 5mg/mL or 0.01M)7
Reverse mutation test using bacteria (OECD TG471) - test substance dissolved or suspended, range of concentrations administered (up to 5mg/plate)7
In vivo mammalian chromosome aberration test (OECD TG475) – test substance dissolved or suspended, limit test (2000mg/kg), length of exposure 1 – 14 days2,7,11
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Mutagenicity (continued) / In vivo mammalian erythrocyte micronucleus test (OECD TG474) – test substance dissolved or suspended, limit test (2000mg/kg), length of exposure 1 – 14 days2,7,11
In vitro gene mutation assay Saccharomyces cerevisiae (OECD TG480) – test substance dissolved or suspended, relatively insoluble substances tested up to limit of solubility 2,7
In vitro mitotic recombination assay Saccharomyces cerevisiae (OECD TG481) – test substance dissolved or suspended, relatively insoluble substances tested up to limit of solubility 2,7
In vitro mammalian cell mutation assay (OECD TG476) – test substance dissolved or suspended, maximum concentration 5mg/mL or 0.01M2,7
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Mutagenicity (continued) / DNA Damage and Repair - Unscheduled DNA synthesis mammalian cells in vitro (OECD TG482) – test substance dissolved or suspended, range of concentrations (maximum with some cytotoxic effect)2,7,9
In vitro sister chromatid exchange assay in mammalian cells (OECD TG479) – test substance dissolved or suspended, range of concentrations (maximum with significant toxic effect, non-soluble tested up to limit of solubility)2,7,9
Sex linked recessive lethal test in Drosophila melanogaster – range of exposures (one either maximum tolerated concentration or indications of toxicity)2,7,9
Suitable for use with nanomaterials / Concerns over use with nanomaterials / Unsuitable for use with nanomaterials
Mutagenicity (continued / In vitro mammalian cell transformation tests –– range of exposures (yielding a concentration-related toxic effect), varying duration2,7,9
Rodent dominant lethal test (OECD TG478) – three dose levels, high dose causing some toxicity, generally single administration2,4,7,11
Mammalian spermatogonial chromosome aberration test (in vivo, OECD TG483) – range of doses (maximum to no toxicity), limit test of 2000mg/kg body weight/day, one administration2,7,11
Mouse spot test (in vivo, OECD TG484) – two dose levels (one showing toxicity)
Mouse heritable translocation (in vivo, OECD TG485) – appropriate dose and exposure routes used
1visual identification is not possible without microscope