ANNEX 3 TO FINAL REPORT...2013/01/09 · IHCP/2011/I/05/27/OC – Annex 3 3 European Commission Annex 3 to Final Report - Impacts on health and the environment in detail Examination

European Commission, Joint Research Centre –

Institute for Health and Consumer Protection – Ispra/Italy

Examination and assessment of consequences for industry, consumers, human health and the environment of possible options for changing the REACH

requirements for nanomaterials

REFERENCE: IHCP/2011/I/05/27/OC

ANNEX 3 TO FINAL REPORT

IMPACTS ON HEALTH AND THE ENVIRONMENT IN DETAIL

09 January 2013

in cooperation with

BiPRO

Beratungsgesellschaft für integrierte Problemlösungen

IHCP/2011/I/05/27/OC – Annex 3 ii

European Commission

Annex 3 to Final Report - Impacts on health and the environment in detail Examination and assessment of consequences for industry, consumers, human health and the environment of possible options for changing the REACH requirements for nanomaterials

Content

1 Introduction ....................................................................................................................... 3

2 Application of option profiles to selected case studies ......................................................... 4

3 Methodology for the qualitative assessment of the exposure and risk associated to

selected nano particles ....................................................................................................... 8

4 Detailed case studies on the impacts of the individual options on selected

nanomaterials .................................................................................................................... 9

4.1 Titanium dioxide nano particles ..................................................................................9

4.2 Impact of the options on risk and exposure scenarios associated to titanium

dioxide nano particles .............................................................................................. 12

4.3 Impact of the option implementation to REACH on risk and exposure (TiO2

nano particles) .......................................................................................................... 13

4.4 Zinc Oxide Nano particles ......................................................................................... 16

4.5 Impact of the options on risk and exposure scenarios associated to zinc oxide

nano particles ........................................................................................................... 19

4.6 Impact of the option implementation to REACH on risk and exposure (ZnO

nano particles) .......................................................................................................... 21

4.7 Nano diamond .......................................................................................................... 24

4.8 Impact of the options on risk and exposure scenarios associated to nano

diamond .................................................................................................................... 26

4.9 Impact of the option implementation to REACH on risk and exposure (nano

diamond) .................................................................................................................. 27

5 Evaluation of knowledge gaps for seven representative nanomaterials due to the lack of

the 21 options discussed above .......................................................................................... 31

5.1 Synthetic amorphous silica (SAS) ............................................................................. 31

5.2 Carbon black ............................................................................................................. 31

5.3 Carbon nano tubes (CNTs) and multiwalled carbon nano tubes (MWCNTs) ........... 32

5.4 Fullerenes ................................................................................................................. 33

5.5 Nano silver ................................................................................................................ 33

5.6 Nano copper ............................................................................................................. 34

5.7 Quantum dots (e.g. Cadmium sulphide) .................................................................. 35

6 References ......................................................................................................................... 36

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1 Introduction

Annex 3 elaborates human health and environmental impacts of 9 of the 21 options as proposed in

the Nano Support Project 20121. The other 12 options are implicitly part of the current REACH

regulation and the guidelines issued by the ECHA (see also baseline definition in the main document).

While this Annex necessarily focuses on the hazards and information needs connected to human

health and the environment it is important to keep in mind, that nanomaterials are exceptionally

useful and hold the promise to be a significant part of the solution to several pressing problems such

the need to lower CO2 emissions, more efficient use of natural resources etc. In connection with a

balanced approach where research and development of new applications are accompanied by

diligent and detailed risk assessment nanotechnology should become widely accepted in the

population.

Three nanomaterials were selected as case studies to highlight the impacts the proposed options

have in dependency of the toxicity already recognized from their bulk form, the tonnage bands and

the pre-existing knowledge. Knowledge gaps resulting from failing to apply individual options arise in

dependence tonnage bands and prior knowledge on the nanomaterial in question.

Additionally seven nanomaterials are discussed in broader terms without addressing individual

options within their context. The application of endpoint specific options is very dependent on the

tonnage band in which the individual materials are produced/imported. This presents a problem not

only with regard to nanosafety (it has been proposed by authorities among them the German BAuA1,

UBA2, BfR3 and the SRU4 to alter tonnage boundaries for nanomaterial risk assessment requirements)

but also because one of the principal options (option 2) highlights an undecided issue concerning the

treatment of nanomaterial surface treatment/modification as either a characteriser or an identifier

(for elaboration see Annex I – Option Profiles). This issue remains without definite answer since the

RIP-oN1 project.2 Depending on which decision is reached the tonnage bands vary greatly due to

inclusion of the nanomaterial within the general dossier of the substance or within individual

dossiers for each nanomaterial surface modification. In light of these legal issues pending resolution

it was decided to apply all options to each case study material regardless of whether or not the

production volume justifies the application under the current legal framework. The discussion of the

knowledge gain and the risk minimization concerning human health and the environment within this

annex is therefore a best case scenario when considered in light of risk minimization. Options that do

not apply to the individual nanomaterial due to the current tonnage bands are currently not relevant

in practice but might become relevant if tonnage bands for nanomaterials are shifted.

1

Bundesanstalt für Arbeitsschutz und Arbeitsmedizin; Federal Institute for Occupational Safety and Health

2

Umweltbundesamt

3

Bundesinstitut für Risikobewertung; Federal Institute for Risk Assessment (BfR)

4

Sachverständigenrat für Umweltfragen; German Advisory Council on the Environment

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2 Application of option profiles to selected case studies

The 9 options out of the 21 options proposed for the modification of REACH to enhance its

applicability to nanomaterials (also referred to as nano particle where appropriate) will be analysed

individually for each selected case study. The consequences of retaining the current legislative status

quo and of implementing a given option will be explored and labelled as A) status quo and B) option

implementation, respectively. To estimate the impact of this choice on human health and the

environment, extrapolations from the current state of knowledge on the specific bulk- and

nanomaterial properties as well as from the general information of nanomaterial behaviour are

made. However, in many cases the knowledge on nanomaterial properties along the entire lifecycle

is severely limited or lacking.

Three general considerations have direct impact on the toxicokinetics, the environmental fate and

toxicity of nanomaterials. They are of particular importance beyond the actual substance the nano

particles are made of and are important for every nanomaterial known up to now.3

All of them hinge on the particularly small dimension of nano particles. Knowing that 0.1 nm (1 Å)

equals the size of one hydrogen atom one can imagine that a nano particle made up of a few dozen

to a few hundred atoms displays these atoms in a very different fashion to the surroundings than

bulk materials do. Due to the dimensions and the high curvature of the nano particle the surface

atoms are highly exposed and have much fewer surrounding atoms of the same type to bind to. As a

consequence the melting point of nanomaterials is severely reduced in contrast to their bulk material

form (provided both have at least the same crystal structure).4 More importantly for the following

considerations however, is the much higher surface area, made up of more exposed atoms compared

to bulk material. To provide an idea of the differences between bulk and nano material surface areas:

A carbon micro particle with a diameter of 60µm has a mass of 0.3 µg and a surface area of

0.01 mm². 0.3 µg of carbon in nano particle form has a surface area of 11.3 mm² if the nano particles

each have a diameter of 60 nm. Considering that 0.3 µg of 60 nm sized carbon nano particles consist

of 1 * 109 nano particles the ratio of surface area to volume/mass for a nano particle is 1000 times

higher than for a micron sized particle.5 This goes along with a roughly 1000 fold increase of reactivity

of nanomaterials versus bulk materials.

The three specific properties are discussed thereunder:

1) The size of the primary nano particles and their agglomerates in the specific application,

upon internalization, contact with the living organism, or an environmental compartment is a

major determinant for the distribution, dissolution, and persistence patterns. As shown for

small mammals, the body, especially the lungs, are less efficient in clearing nano particles

than micrometre sized particles.6 In this context, interspecies variations have to be taken into

account. Larger mammals are frequently more efficient at clearing nano particles from their

lungs than smaller mammals (humans vs. rats).7 Also if nano particles gain access to the

blood stream or are administrated intravenously their accumulation patterns are dependent

on their size (among other determinants). Accumulation e.g. in the liver is frequently

facilitated by the particle being smaller than 50 nm. In the spleen however objects

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(liposomes) of ca. 100 nm are not efficiently accumulated whereas an increase in size also led

to a higher uptake rate3.

The formation of agglomerates is another important factor which is dependent on: size and

material of the original nano particle, the application the nano particle is used in, and the

ubiquitous surface modifications (see p.7).

2) The protein coat that every nano particle acquires when entering a living organism8 due to its

high reactivity is called corona.9–16 Proteins binding to the nanomaterial have a profound

impact on the retention time in an organism as they modulate the uptake rate in organs and

thus the distribution within the organism.

The composition of the corona may vary greatly and is dependent on the properties of the

nano particles, such as surface charge, morphology (curvature/size), and the substance.17 In

addition, nanomaterials may associate with a specific set of proteins when subjected to

different exposure routes (e.g. intravenous administration, inhalation etc.). Within the blood

stream, proteins such as albumins might attach and protect the nano particles from

aggregation, thus regulating retention in the blood stream, organs the nano particles

accumulate in and clearance pathways. In the lung, the bronchial surfactant induces

agglomeration of metal oxide nano particles and thus an increase in particle size which

lowers the dispersion in the lung and enhances the clearance rate from the lung.18

In principal, two major classes of associated proteins were shown to significantly influence

toxicokinetics of nanomaterials, opsonins and dysopsonins. Opsonins are molecules that

target nanomaterials for an immune response and act as a binding enhancer for

phagocytosis. This leads to faster clearance from the organism by the oral route in the lung

or via excretory systems in the body.

Dysopsonins are proteins that lead to the opposite effect and extend the retention time in

the bodily compartment the nanomaterial is currently located in. A hydrophilic, non-charged

(neutral) surface disfavours the binding of opsonins to nano particles.

Additionally the small size and the extreme curvature of the nano particles are the causes for

the proteins that interact with the artificial particle to be subjected to extreme interface

conditions. Under many circumstances this leads to the loss of the delicate protein structure

(i.e. denaturation) and thus inactivation of the protein or severe changes in protein function.

The structure of the proteins, their size relative to the nano particle in question, the nano

particle shape, size, material, and functionalisation (etc.) obviously all play important parts in

such considerations.19–21 In some cases also an extreme stabilization of the proteins attached

to specific nano particles was reported which essentially freezes the protein in a state. Steric

changes of the protein structure are however essential to the function of most proteins.

‘Freezing’ in state is just as harmful to protein function as partial or full denaturation.

In the environment humic acids can fulfil the role of opsonins and increase dispersibility.

3) The surface charge and hydrophobicity of a nanomaterial dictate large parts of its

interactions with other molecules because its surface is so large in comparison to its

volume/mass. Within blood plasma hydrophobic particles are coated with opsonins much

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more quickly than hydrophilic ones.22 Positively charged particles attract different proteins

than nano particles with a negative surface charge. This is due to the protein specific

isoelectric points. In general hydrophobicity and surface charge are key determinants for the

selection of surface bound proteins. To a lesser degree the substance of the nano particles,

the shape and the size also influence the selection process. The coating of the nano particles

heavily influence aggregation, motility, bioavailability and retention time within any

compartment of the living organism that the nano particles are located in.

These three general considerations are applicable to all nanomaterials and, as hinted at above, they

are the reason why nano particles share certain non-substance specific, toxicological characteristics

when living systems are exposed to them. These toxicological properties are, broadly put, size

specific. If the nano particles are soluble then these basic considerations apply for the lifetime of the

nano particle form and are changed and then lost over time together with the physical shape.

Because solubility is heavily dependent on the environment and the substance of the nano particle

total dissolution of the nano particle might happen quickly thus rendering size specific effects at the

dissolution location unimportant. The dispersion to a location where the conditions are favourable

for dissolution is still dependent on the parameters imposed by the small size of the nano particles. If

the dissolution of the nano particle proceeds very slowly then the size specific effects described in

this chapter are in effect for a longer time and become relevant for risk assessments.

The hardest property to quantify in hazard assessments is the ability of nano particles to act as

vectors or shuttles23 for a broad variety of chemicals including bacterial toxins. In conjunction with

toxic chemicals the toxic effects can be enhanced due to better access to the organism when bound

to nano particles much as the targeted deliverance of pharmaceuticals to sites of pathology inside an

organism can be optimized with the aid of nano particles.24 The variety of potential interaction

partners of ‘luggage’ for a given nano particle depends heavily on its lifecycle.

nano particles inhaled by air and/or water breathing organisms are prone to create similar effects in

connection with the sensitive membranes that compromise breathing apparatuses in large parts of

the animal kingdom. Excessive exposure to nano particle-matter can cause lung overload and/or

inflammation due to inflammatory reactions via secondary generation of reactive oxygen species (i.e.

oxidative stress).25–27 nano particles are also able to trigger fibrotic responses and carcinogenic

effects if inhaled chronically at large enough doses. The size distribution of inhalable nano particles

has serious impacts on their inflammatory potency and their persistence time in the respiratory

tract28, because it affects the distribution in the breathing apparatus. Smaller nano particles with less

agglomeration tendency penetrate much deeper into the organ than larger or strongly agglomerated

particles. The individual effects obviously vary in their severity of disease symptoms from cell type to

cell type29, species to species, in a dose-, and exposure dependent manner.

Beyond these general effects of nano particles, individual nano particle substances may have

additional, substance specific toxic effects of varying severity.30 These will be discussed below in the

context of the selected case studies.

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Also the ligands that are accumulated by the nano particles on their surfaces during the course of

their lifecycle will vary from substance to substance and pose specific challenges with regard to the

hazard assessment for each nano particle substance (see below).31 The exposure scenarios can

significantly from substance to substance and from application to application.

The high surface area of nano particles makes them particularly susceptible to surface modifications

(the corona described above is a special, protein related case). In fact every nano particle is surface

modified. This is true from the first contact of atmosphere and nano particle throughout the life cycle

of the product, because of the high surface area and consequently the high reactivity of the nano

particles and their agglomerates.32 In the discourse on safety of nanomaterials the awareness of

intentional vs. unintentional surface modifications is essential. The impacts of intentional surface

modifications are substantial and often well characterized. They are used to maintain granular

distributions, mediate targeting in medical applications, convey photochemical properties etc.

However, because of the high reactivity of nano particles described above, pre-imparted surface

treatments and modifications are easily altered, lost or substituted along the life cycle of any nano

particle. Current studies on nano particles suggest taking into account, that intermediate or final

forms of the nano particle are neither in the same agglomeration / aggregation state, nor are they

likely to carry the same unintended surface modifications. Such alterations of nano particle surface

chemistry have profound impacts on toxicological and exposure related considerations.9,10,33–36

Modifications that provide hydrophilicity or hydrophobicity are among the most straight forward

impacts on the nano particle distribution in the organism and the ecosystems. The shades of gray

between these two extremes and the additional properties that might be imparted by different

ligands such as membrane permittivity, bioreceptor recognition, the accumulation of organochemical

toxins on the nano particle surface, loss of solubility for specific solvents, as well as the potential for

the nano particles to become available for the accumulation in the food chain are examples for some

of the many possible modifications with harmful potentials towards human population and

ecosystems.

A thorough understanding of the physico-chemical and toxicological properties of nano particles with

their intentional surface modifications is the important first step to encompassing and controlling the

inherent hazard. The second step consists of understanding which unintentional modifications are

imparted and the properties they convey to the nano particle during its application life cycle.

It is important to stress that the very same modifications might also prove to be desirable as they

might assist in removing the nano particles from major exposure routes by significantly changing the

agglomeration state or inducing the formation of complexes that are less toxic. Full life cycle analysis

(LCA) would however be the only way to conclusively show positive or negative effects.

The 21 options proposed for the modification of the current REACH regulation provide a basis

towards ensuring a responsible and complete risk assessment. The following section outlines the

potential impacts on human health and the environment, as exemplified by detailed case studies on

nano particles of: zinc oxide (ZnO nano particles), titanium dioxide (TiO2 nano particles), and nano

diamond). Seven additional examples are presented in brief.

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3 Methodology for the qualitative assessment of the exposure and

risk associated to selected nano particles

We decided to follow accepted evaluation patterns by dividing the hazard assessments for the

individual nanomaterials into human and environmental hazards. Human health hazards are grouped

into acute effects, chronic effects and mortality. Environmental hazards were grouped similarly into

acute and chronic effects but as these two groupings frequently include studies on mortality we

added a third category covering accumulation/persistency to address long term effects of the specific

nanomaterial. In this regard accumulation and persistency are of particular concern as the food chain

magnifies the toxicological concerns in dependence of those two aspects.

The considerations above are briefly illustrated in the flow charts below and the considerations

elaborated in the risk and impact assessments below follow these patterns.

Figure 1: Grouping of risk and exposure data for humans and the environment

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4 Detailed case studies on the impacts of the individual options on

selected nanomaterials

4.1 Titanium dioxide nano particles

As with all nano particles there are certain general toxicological characteristics that TiO2 nano

particles (we address specifically TiO2 nano particles and will use this short form henceforth) share

with other nano particles. These have been extensively elucidated in the introduction.

Beyond these general hazards inherent to inhalable nanomaterials TiO2 nano particles have an

additional property to consider. In contrast to ZnO nano particles TiO2 nano particles can be

manufactured in different crystal forms. Rutile and anatase are mainly manufactured where rutile is

the preferred crystal form in products with consumer exposure as it generates less ROS than anatase.

Anatase is however the commercially most used isoform.37 Both however are able to generate ROS

directly with the aid of UV and visible light.38 Especially with unicellular, light dependant organisms

such as microalgae or soil bacteria this property magnifies the hazard posed by TiO2 nano particles.

Applications

TiO2 nano particles are used in a large amount of products covering all states from tight integration

into complex materials to nearly free (agglomerated) nano particles. It was produced at tonnage

bands of more than 50,000 t/y in 2010. By 2015 the production is projected to increase to 201,500

t.39

TiO2 nano particles find their applications in products where they provide UV-protection, UV-

absorption for catalytic processes, or serve as pigments in paints and varnishes. The ability to absorb

UV light and redirect the collected energy is used in sunscreens (main consumer exposure).40 Here

the collected energy is redirected towards forming other substances than ROS if possible.41,42

The high opacity of the white created by TiO2 particles is used in paints and varnishes among other

applications. Here TiO2 nano particles also serve as one of the main constituents for the ‘self-

cleaning’ properties newer coatings and materials have. In that case the ROS production is desirable

in order to destroy adherent dirt. It should be noted however that applications listing TiO2 as a

pigment are estimated to contain less than 0,1 % (w/w) TiO2-nano particles by the IARC.43,44 The same

function is used in antibiotic coatings of textile fibres and to cleanse water in waste water

applications.

Physico-chemical properties

TiO2 nano particles occur in three crystalline structures: Anatase (tetragonal crystals), brookit (ortho-

rombic crystals, not manufactured commercially) and rutile (tetragonal crystals). As mentioned

above TiO2TiO2O2 is able to absorb UV light. In nano particle form TiO2O2TiO2 is transparent in the

visible spectrum which makes it desirable for applications such as sunscreen lotions. In these

applications the rutile form is preferred. The anatase form of TiO2 nano particles has specific electric,

photocatalytic and thus also antimicrobial properties. Coupled to the capacity to form ROS when

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exposed to UV and visible light is also the ability to form other – potentially undesirable – side

products such as formaldehyde, acetic aldehyde and nitrates. These pose additional risks for human

health and the environment.

Exposure

The exposure of humans and the environment to TiO2 nano particles can occur at all stages of the

TiO2 nano particle life cycle. The likelihood of exposure during the production is comparatively low, if

work places are equipped with appropriate measures. In 2011 Kuhlbusch et al. were unable to detect

significant exposure at workplaces processing TiO2 nano particles.45

Hansen et al. estimate a daily exposure of 57 µg/kg*day for a two year old from TiO2 nano particles

contained in sunscreen lotions (dermal exposure).46 The TiO2 nano particles are not able to

transverse the barrier the skin poses. Even inflamed or otherwise damaged skin poses an effective

barrier to these nano particles.

Some sunscreen products are also offered as a spray application. Inhalative exposure to TiO2 nano

particles and larger particles was calculated to be 3,5 g/m³ in 2011.47 Additionally Powell et al.

(2010)48 estimate a daily oral intake of micro and nanosized titanium in Great Britain at around

5 mg/person due to additives in food added for colouring effects (here nanosized refers to a size

range of 100-200 nm).

The main sources of TiO2 nano particles in the environment are currently judged to be the use of

sunscreens, and the washing-out of nano particles from house paint and the wear imposed on coated

surfaces of construction materials and coated windows. A significant percentage of the nano

particles in house paint are lost in the course of 2 years merely due to environmental effects.49

Surface waters in Europe were estimated to carry between 0.7 µ/L and 24.5 µg/L whereas the

exposure of soil and sediments was estimated to be between 1 and 1.03 g/kg. 50,51

TiO2 in any size range is not water soluble and tends to agglomerate in water. This does not

significantly affect its reactivity33 but the distribution in the environmental compartments might be

affected because of the sedimentation processes, that gain impact with increasing particle size. Once

the TiO2 nano particles have sedimented it is unlikely that they become mobile again.52 Kiser et al.

(2009) have shown that part of the TiO2 nano particles brought into a wastewater treatment plant

are not held back in the sludge but are released to the environment.53

Toxicology

Beyond the general dangers (acute as well as chronic) listed above that inhalable nano particles

potentially pose TiO2 nano particles are suspected to interact with the DNA and thus have direct

genotoxic potential as they have been found inside cellular nuclei in lungs and in cultured cells.5,54–56

These results are interesting, but it is important to note that the studies yielding these results have

been carried out with high concentrations of TiO2 nano particles. Based on inhalation studies in rats

TiO2 nano particles are rated as potentially carcinogenic (group 2B) by the IARC.43,44 Dermal

absorption of TiO2 nano particles could not be shown experimentally, thus making toxicological

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effects due to sunscreens etc. unlikely.40,57 Oral intake of TiO2 nano particles results in exposure of

the gastro intestinal tract and may result in accumulation in internal organs.47 In the presence of UV

light TiO2 nano particles are significantly more toxic due to their photochemical reactivity.

Ecotoxicology

There is little reliable data available concerning the effect of TiO2 nano particles on terrestrial

organisms. Studies have been made with a focus on the effects on aquatic organisms. The lack of

clear characterization of the test material and consistent experimental conditions however make it

difficult to draw any conclusions as the results are contradictory. The German Federal Environment

Agency and the Danish Ministry of the Environment both published studies correlating data of

different experiments on the model organism Daphnia magna, the results of which range from non-

toxic to toxic for TiO2 nano particles.47,52

Experiments with rainbow trout show little acute toxicity but also reveal morphological changes in

gills, the intestinal tract. Oxidative stress was detected in these organs as well as in the brain (see

also studies cited above). Furthermore there is experimental evidence of Arsenic and Cadmium

accumulation in context with TiO2 nano particle presence in fish.58,59 This might be due to the

association of the heavy metals with the nano particles thereby transforming the nano particles into

effective carriers. There are studies that indicate bioaccumulation processes for TiO2 nano particles in

aquatic organisms but further tests are necessary to verify the existing data.47

Crustaceans have been exposed to TiO2 nano particles using the sample organism Daphnia magna

and acute as well as chronic effects were detected. Due to the widespread results it is difficult to

exactly determine the toxicological profile in crustaceans.60–63

Single cellular organisms such as bacteria, certain algae and yeast have also been exposed to bulk

TiO2 and TiO2 nano particles. The toxicity varies considerably and is also enhanced in the presence of

UV light due to the additional production of ROS. Gram-positive bacteria (B. subtilis) are more

susceptible to the antimicrobial activity of TiO2 nano particles than gram-negative bacteria (E. coli).63

Aruoja et al. (2008) showed that the microalgae Pseudokirchneriella subcapitata is significantly more

susceptible for toxic effects originating form TiO2 nano particles (EC50=5.83 mg/l) than to effects

from bulk TiO2 (EC50=35.9 mg/l).64 To yeast neither bulk TiO2 nor TiO2 nano particles were toxic even

at concentrations of up to 2,000 mg/l.65 These results show that the effects of nano particles can vary

strongly from one species to another and very specific requirements and questions have to be

applied to each nanomaterial.

To our knowledge three studies have been published thus far on bioaccumulation of TiO2 nano

particles. In Daphnia magna relatively slow elimination of TiO2 nano particles with an average size of

21 nm diameter led to accumulation. Rainbow trout were exposed to TiO2 nano particles by Ramden

et al. (2010) and they found TiO2 accumulation in gut, liver, spleen, gills, and brain after 8 weeks of

exposure.66 Scown et al. (2010) found bioaccumulation of TiO2 in the kidneys where the nano

particles remained for up to 21 days after intravenous exposure. Clearance was noted after 90 days

from the kidneys but the TiO2 nano particle concentrations found in the liver did not change.

Retention of TiO2 nano particles was longer than the runtime of the experiment.67

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Grading in the hazard table (see Final Report)

TiO2 nano particles are graded with a medium hazard albeit being listed as potentially carcinogenic

(group 2B) by the IARC as they consistently exhibit the toxicity that can be attributed to all inhalable

nanomaterials. Their ability to generate ROS varies in its intensity with the crystal structure (anatase,

brookit, rutile) but is highly dependent on the availability of visible or UV light. Environmental

experiments yield contradictory results with regard to the toxicity in crustaceans. They show a

tendency for the TiO2 nano particles to bio-accumulate, but the acute toxicity is again coupled to the

availability of visible or UV light which is only given in single cellular organisms living in exposed

habitats. The accumulation in higher organisms led to oxidative stress and morphological changes

being detected in rainbow trout for example. TiO2 nano particles lack the ionic effects that pose

additional danger from other nanomaterials.

4.2 Impact of the options on risk and exposure scenarios associated to titanium dioxide

nano particles

The following tables illustrate where option implementation or lack thereof will affect the fields

listed below without implying positive or negative nature of the impact. The tables serve as an

orientation for the reader to illustrate those aspects that will be of particular interest in the following

discussion and the ensuing upscaling of the impacts of the proposed modifications to REACH.

Table 4-1: Human health impacts of TiO2 nano particles

options with impacts pathology

acute effects

workers 6,11,12,21 local inflammation of the lung

consumers 21

via the environment 21

chronic effects

workers

6,12, 21 fibrotic changes in the lung tissue, systemic accumulation in organs such as kidney, liver, brain, etc.

consumers accumulation of TiO2 nano particles via the food chain

via the environment 6,13,16,17,18,19,21

mortality

workers 6,12,21 cancer

via the environment 6,13,16,17,18,19,21

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Table 4-2: Environmental impacts of TiO2 nanoparticles

options with impacts effects

acute effects

soil and sediment organisms 6,13,18,21

bactericide

aquatic organisms

6,13,17,21

bactericide, algaecide, pathogenic to higher organisms (fish, crustaceans), increased mortality rates

chronic effects


bactericide

aquatic organisms 6,13,17,21

pathogenic to higher organisms (fish, crustaceans), increased mortality rates

accumulation/ persistency

soil and sediment 6,13,21

persistent

aquatic sphere 13,21

biota 6,13,17,18,21 bioaccumulation (see effects above)

4.3 Impact of the option implementation to REACH on risk and exposure (TiO2 nano

particles)

Option 6 – Include information on dustiness

A) status quo

The need for inhalation studies in the endpoint section is not apparent which has the potential to

lead to incomplete exposure assessments. The implications for workers, consumers and ecological

habitats are considerable as damage has to become apparent before further research is initialized.

Furthermore this option specifies the need for these studies independent from the production or

import quantities. Inhalation studies are otherwise required at a production/import tonnage of 10 t

or more.

Without the data gained from inhalation study the need for specialized air filters and breathing

masks at exposed workplaces might not become apparent.

B) option implementation

TiO2 nano particles are a dusty substance and risk assessments as well as necessary endpoint related

studies are determined by this property. Inhalation studies with various size distributions and in

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different environments are necessary and the obtained data will be applied in security considerations

along the lifecycle of products containing TiO2 nano particles in inhalable form.

Option 11 – Require acute toxicity data for the most relevant route of exposure.

The toxicity data provided by the registrant should be coupled to the most relevant route of

exposure of humans to a given substance and nanoform.

The amount of TiO2 nano particles produced requires the full range of toxicity tests and inhalation is

clearly a relevant exposure route. This option has no impact on TiO2 nano particles.

Option 12 – Change particles to ‘(nano)particles’ for repeated dose toxicity studies

(inhalation)

This option relates to the phrasing in the Annexes VIII, 8.6.1, IX, 8.6.2, and IUCLID section 8.5.2. The

boundaries of the definitions here should be extended and specified by exchanging all instances of

the word ‘particles’ for ‘(nano)particles’. Furthermore the following addition should be considered:

‘Especially for (nano)particles it should be justified when inhalation is not considered a relevant route

of exposure.’

Either the test methods (for sub-acute, sub-chronic or combined repeated dose toxicity/reproductive

screening study) or the guidance should refer to extended pathology/histology determinations and

examination of relevant parameters in BAL (bronchoalveolar lavage) fluid.

A) status quo

Without the imposed fine resolution of the inhalation studies with regard to the employed size

ranges of the nano particles and their modifications, little information is gained beyond the particular

experimental setup. Specific applications and the alterations to the surface properties of the nano

particles during the usage are the first ‘experiments’ indicating hazards to humans


As stated for the option 11 here also the option has little impact on the practice as TiO2 nano

particles are already tested for inhalation toxicity due to the volume of production. In relation with

previous options however a finer detail is achieved here by testing explicitly the nanoforms of TiO2

for health risks connected to inhalation in dependence from their size distribution. Hazards

connected to TiO2 nano particle applications in which inhalation is imaginable are well characterized

thus further minimizing the risk to humans. Especially work place related hazards are detected early.

Option 13 – Require non-bacterial, in vitro gene mutation study

Genotoxicity of TiO2 nano particles has been shown in various studies (see introduction TiO2 nano

particles) although primary-, secondary-ROS generation, and direct particle DNA interaction have so

far not been sufficiently distinguished to determine the cause of genotoxicity.

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Option 16 – Consider water solubility in relation to test waving

Nano particles are often not soluble in water. The lack of water solubility is not a reliable indicator for

the lack of bioavailability. Nano particles have other options available to them to interact with their

environment and biological cells.

A) status quo

TiO2 nano particles are not water soluble and thus testing of bioavailability, -accumulation, and

persistence can be waived. In all three aspects solid data shows TiO2 nano particle activity/presence

in spite of the lack of solubility per se.


TiO2 nano particles are tested for all relevant endpoints and accurately described for hazard and risk

assessment within humans, other organisms, and the environment. As numerous results show the

lack of water solubility does not correlate with unavailability in the biosphere. This is especially true

for nano particles.

Option 17 – Specify that long term testing should not be waived based on lack of short

term toxicity.

Some nanoforms have a tendency to accumulate in biological systems and should thus not be

discounted when considering toxicity just because they do not exhibit short term toxicity.

TiO2 nano particles exhibit short and long term toxicity in all organisms exposed to them with the

exception of yeast which one study shows to be impervious. This option has no effect on TiO2 nano

particles

Option 18 – Require that testing on soil and sediment organisms is prioritised

A) status quo

As TiO2 nano particles are not soluble the deposition in soil and sediments is relevant but potentially

overlooked. Bioaccumulation in the soil and in sediments might play an important role in creating a

hazardous situation over time.


The exposure characteristics of TiO2 nano particles are easily altered by changing surface

modifications in the course of the TiO2 nano particle lifecycle. The testing of soil and sediment

organisms will quantify the amount of hazard in relation to surface modifications and in dependence

of applications of the TiO2 nano particles. These data are interlinked with the assessment of

bioaccumulation processes in this habitat.

Option 19 – Specify that algae testing should not be waived based on insolubility

See Option 16

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Option 21 – Require considerations of most appropriate/relevant metrics

The appropriate metrics should be chosen also to develop and present a realistic exposure

assessment and risk characterization. Mass metrics are not necessarily appropriate as a point of

reference for the evaluation of nanomaterials.

4.4 Zinc Oxide Nano particles

The specific properties of ZnO nano particles will be briefly highlighted below. As described above,

some toxicological properties are specific to the substance the nano particle is made of and are

cumulative with the general considerations described above. The impact of the individual options for

the modification of REACH will become more apparent with the information given here in mind.

Applications

33.400 t/y of ZnO nano particles were produced worldwide in 2011.68 It is used in micro and

nanoform as a UV protection agent. As such, it is a functional part of sunscreen lotions, cosmetic

products, paints and varnishes. It is used in plastics and rubber where it is embedded in the product

matrix and finds applications in scientific research and the medical sector as bactericide and in the

treatment of cancer. Furthermore, ZnO nano particles are used in the electronics industry for the

production of e.g. semiconductors or flexible TFT-screens.52,69

Physico-chemical properties

ZnO in every form dissociates in aqueous environments releasing Zn2+ ions. Zincoxide is able to

absorb UV-A and UV-B irradiation. The harvested energy can be employed to produce reactive

oxygen species (ROS).70,71 ZnO nano particles in comparison to coarser particles do not scatter light in

the visible spectrum and thus appear translucent.72

Exposure

The main exposure routes for to humans and the environment are determined by the extensive

usage of ZnO nano particles as UV protection agent. Humans can be exposed to ZnO nano particles

either via direct, topical application on the skin in the form of cosmetics and sunscreens or

unintentionally via the use of paints and varnishes (workers and amateurs alike). The latter are likely

to release nano particles to the environment as was also shown for nano silver and titanium dioxide

nano particles.49,73,74 It is also possible that ZnO from coatings is lost via ion leaching. Further research

is necessary to determine the exposure arising from such applications. Open applications such as

cosmetics easily release ZnO nano particles with or without specific surface modifications to the

environment. In all likelihood, nano particles from the applications mentioned above end up in

agglomerated or actual nano particle form in the wastewater system from where a fraction is

distributed into the ecosystem.36,51,75,76

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The complexed forms of the ZnO nano particles found in e.g. plastics and rubber pose no particular

risk to consumer health as they are usually not released from their matrix and consequently are not

bioavailable.

At the workplace the classical exposure route is possible during welding processes. However, in

modern industrial workplaces protective measures are set in place. In Germany a maximum

workplace concentration of airborne ZnO nano particles is set at 1 mg/m³.77

Model calculations of ZnO nano particles in the surface water bodies of Europe span a wide range

between 0,03 µg/l and 76 µg/l.51

Toxicology

The ion release into the aqueous surroundings (including tissues and cells) is the main toxicological

effect to be considered in humans and the environment.64,65,70,78 In general, ion leaching is dependent

on the particle stability which is heavily influenced by the coating of the nano particles among other

factors. The ZnO nano particles themselves are able to generate ROS much like other nano

particles.79

Beyond the basic set of toxicological concerns common to all inhalable nano particles (see

introduction), ZnO nano particles are not perceived as particular risk to humans yet. This is due to the

fact that Zinc is an essential trace element. A daily dose of around 70 mg/day and individual human

does not lead to any adverse health effects. Up to this dose and with the oral uptake route the ions

do not represent a problem for humans.36

However, if humans are exposed to airborne ZnO nano particles in relevant doses they are affected

by metal fever. The symptoms appear as influenza like pathology resulting from the inflammation of

the lung that is reversible within 48 h if no further particles are inhaled. Consumers are rarely

exposed to fumes or inhalable dust containing ZnO nano particles. Only workers in the particular field

of industry or researchers have an increased chance of being exposed to large enough doses of

inhalable dust. To our knowledge, the first scientific documentation of this pathology was presented

in 1927.80,81 Furthermore, the chronic inhalation or the overload of the lung with dusty nano particles

can lead to fibrotic changes in the lung and consequently to cancer (most chronic effects are likely

due to be particle related effects with added impact due to nano particle distribution patterns in

living systems). Due to the high dissolution rate of ZnO nano particles sufficient concentrations for

particle effects to present themselves would only be achieved via coatings that decrease the rate, via

chronic exposure, or via a large initial dose. In case of a large dose the dissolution equilibrium would

have to be reached before the nano particles dissolve entirely. As Xia et al. (2008) have shown ZnO

nano particles triggered all three tiers of oxidative stress and are thus significantly more effective in

this regard than TiO2 nano particles.28,36,70,82,83

In the case of uncoated (= pristine; relative term – see discussion on unintentional surface

modifications p. 7) ZnO nano particles, current research attributes the pathologic effects to two

sources. Firstly, the nano particle-form of zinc makes it inhalable for humans when the nano particles

are airborne.84,85 Secondly, ZnO nano particles release Zn2+ ions in aqueous environments. These

conditions are found inside living organisms and cells as well as in aquatic habitats. The dissolution

processes are also responsible for the toxicity rating of bulk ZnO78,86 and are responsible for altered

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exposure patterns87. The high surface area of ZnO nano particles can benefit the release of Zn2+ ions

in comparison to bulk sized ZnO. If ZnO nano particles are inhaled or are absorbed in the gastro-

intestinal tract the effects of the leeched ions become more pronounced. Zn2+ ions are able to

interact with cellular structures and proteins by either inactivating or denaturating them. This is a

stochastic process with many potential cellular targets. The same is true for the generation of ROS

and the effects of the radicals on the surrounding biomolecules.

The acquisition of a corona upon ingestion or inhalation may profoundly change the distribution

patterns of ZnO nano particles in the human body, further complicating the assessment of associated

risks. There is some evidence, however, that inhalation and interaction with the BAL

(bronchoalveolar lavage; lung fluids) leads to nano particle agglomeration and thus clearance from

the lung is facilitated.

The most prominent exposure of consumers to ZnO nano particles is via their skin through the

application of cosmetics and sunscreens. No negative effects have been documented to the present

time. ZnO ions and nano particles are able to penetrate the outer skin barrier to a limited degree that

is not considered to be a cause for concern.57,88–91

Ecotoxicology

Because of the increasing industrial and cosmetic applications ZnO nano particles are used in, it is

likely that their concentration in the environment will rise in the future. Zn2+ ions are the main

concern in the environment alongside with the potential of Zn2+ ions to accumulate in the food

chain.92,93 Zinc (ZnO) in bulk form is listed as hazardous substance, with particular danger to aquatic

organisms and the potential to inflict long term damage to aquatic habitats (Category 1 H400, H410

GHS94; WGK 2, Germany95; CLP Index No.: 030-013-00-75). ZnO nano particles tend to agglomerate in

the aquatic compartment. This effect is further enhanced by the presence of organic interaction

partners. Within this compartment they are able to release Zn2+ ions to the surrounding water. The

ZnO nano particles are persistent in soil and sediments.52 Ion release is higher from nano particles

than from bulk material due to the increased surface area of the ZnO nano particles. Additionally the

distribution of ZnO nano particles is altered compared to the distribution of bulk ZnO because of the

higher dispersion rate inherent to nanomaterials.75 The potential for the acquisition of unintentional

surface modifications of the ZnO nano particles is hard to determine into hazard considerations

because there is little research along the life cycle of ZnO nano particle containing products available

at this point. Bacteria and higher organisms such as crustaceans and fish can be heavily affected by

rising Zn2+ ion concentrations resulting in increased adult and embryonal mortality and embryonal

deformations.61,96 The NOEC for crustaceans was set to 30 µg/l. In soil and sediments ZnO nano

particles are persistent for up to 14 days97 and here also displayed their negative effects on the

reproduction rate of worms98 and the survival of soil bacteria.99,100

5

Classification, Labelling and Packaging; http://clp-inventory.echa.europa.eu/SummaryOfClassAndLabelling. aspx? SubstanceID=93&HarmOnly=no?fc=true&lang=en

http://clp-inventory.echa.europa.eu/SummaryOfClassAndLabelling

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ZN nano particles were graded as highly hazardous because even without the unintended, potentially

harmful surface modifications that the nano particles can pick up during their life cycle, ZN nano particles

already exhibit high toxicity mainly via the effects of their ions in the environment. Especially in the aquatic

environment their nano-morphology just heightens the ion related effects. In addition zinc oxide acquires

pathological properties upon inhalation, ingestion and the potential to disperse even wider systemically as

well as environmentally when in nano-morphology. Counting in unintended modifications that impart an

ubiquitous hazard to all nano particles some known modifications can lead to retardation in the ion leaching

process and higher dispersibility in the aquatic habitat or to increased retention times within organisms for

example.

4.5 Impact of the options on risk and exposure scenarios associated to zinc oxide nano

particles

The following tables illustrate where the implementation of the additional options or lack thereof will

affect the fields listed below without implying a positive or negative nature of the impact. The tables

serve as an orientation for the reader to illustrate those aspects that will be of particular interest in

the following discussion and the ensuing upscaling of the impacts of the proposed modifications to

REACH.

Table 4-3: Human health impacts of zinc oxide nano particles


acute effects

workers 6,11,12,21

influenza like symptoms; 48 h, local inflammation of the lung

consumers 21 see above


chronic effects

workers 6,12,13,21 fibrotic changes in the lung tissue

consumers 6,21 accumulation of ZnO nano particles via the food chain


mortality

workers

6,12,13,21

fibrotic changes in the lung tissue, cancer, unknown effects of distribution and accumulation in the body due to surface modifications

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Table 4-4: Environmental impacts of zinc oxide nano particles

options with impacts effects

acute effects


bactericide, increase in mortality rates for key indicator organisms

aquatic organisms 6,13,17,18,21

bactericide, algicide, toxic to higher organisms (fish, crustaceans), increased mortality rates

chronic effects

soil and sediment organisms 6,13,21

lowering of animal reproductive rates, bactericide

aquatic organisms 6,13,17,18,21

lowering of reproductive rates, deformations


soil and sediment 6,21

persistent

aquatic sphere 6,21 increase of ZnO2+ ion concentration

biota 6,17,18,21 bioaccumulation (see effects above)

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4.6 Impact of the option implementation to REACH on risk and exposure (ZnO nano

particles)

Option 6 – Include information on dustiness (Annex VI)

A) status quo

The need for inhalation studies in the endpoint section is not apparent. The implications for workers,

consumers and ecological habitats are considerable as damage has to become apparent before

further research is initialized. Furthermore this option specifies the need for these studies

independent from the production or import quantities. Inhalation studies are otherwise required at a

production/import tonnage of 10 t or more.

Without the data gained from inhalation study the potential need for air filters and breathing masks

at exposed workplaces might not become apparent.


ZnO nano particle is a dusty substance and risk assessments as well as necessary endpoint related



along the lifecycle of products containing ZnO nano particles in inhalable form.

Option 11 – Require acute toxicity data for the most relevant route of exposure (Annex VII)



The amount of ZnO nano particles produced requires the full range of toxicity tests and inhalation is

clearly a relevant exposure route. This option has no impact on ZnO nano particles.


(inhalation)





of exposure.’




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A) status quo






As stated for the option 11 here also the option has little impact on the practice as ZnO in powder

form is already tested for inhalation toxicity. In relation with previous options however a finer detail

is achieved here by testing explicitly the nanoforms of ZnO for health risks connected to inhalation in

dependence from their size distribution. Hazards connected to ZnO nano particle applications in

which inhalation is imaginable are well characterized thus further minimizing the risk to humans.

Especially work place related hazards are detected early.

Option 13 – Require non-bacterial, in vitro gene mutation study (Annex VII)

Bacterial mutagenicity tests are widely used to assess basic mutagenic properties of a given

compound but are not always appropriate to do so for nanomaterials. As elucidated in RIP-oN2 and

ECHA-12-G-03-EN bacterial tests might lead to false negatives because inorganic (nano)particles

cannot permeate the bacterial wall and membrane, thereby creating false results. Instead an in vitro

test for the mutagenicity of nanomaterials is suggested.

A) status quo

If genotoxicity is dependent on the size distribution of the ZnO nano particles genotoxicity would

only show up under very specific circumstances which are dissimilar for bacteria and eukaryotes.

Tests with living organisms are also often confounded by the fact that the secondary ROS production

triggered by the inflammatory effects of nano particles in general can lead to gene deregulation. This

is then however due to the presence of the nano particle as an irritant and not a danger that is

related in particular to ZnO nano particles. Without non-bacterial tests, it is impossible to assess

potential genotoxicity inherent to the ZnO nano particles in particular.


ZnO nano particles are not known to be genotoxic up to this point. It is unclear however if this is

entirely true until non-bacterial tests are documented. The results dictate the appropriate risk

prevention measures.


Dissolution of ZnO-Ions is well known also for bulk ZnO and constitutes one of the two identified

routes of toxicity. The consequences of this option do not apply to ZnO nano particles.

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term toxicity.



ZnO nano particles and bulk-ZnO both exhibit aquatic short and long term toxicity. This option has no

effect on ZnO nano particles.

Option 18 - Specify that algae testing should not be waived based on insolubility

As described above the lack of water solubility should not be a valid reason for the waiving of aquatic

tests in the case of nanomaterials. Many are able to persist in aqueous media and influence their

surroundings via agglomeration or surface modification with molecules found in the environment.

This issue is also taken up by the ECHA in their new guidance document ECHA-12-G-05-EN.

This option has no effect on the ZnO nano particle testing.


A) status quo

The exposure characteristics of ZnO nano particles are easily influenced by changing surface

modifications in the course of the ZnO nano particle lifecycle. While the nano particles will also end

up in soil and sediments (if their modifications slow down the dissolution) the main toxicity arises

from the dissolution in aqueous environments. The testing of soil and sediment organisms will

quantify the amount of hazard in relation to surface modifications and in dependence of applications

of the ZnO nano particles. The impact on soil and sediment organisms is assessed. This data is

interlinked with the assessment of bioaccumulation processes in this habitat.


Without the prioritisation it is probable that comparatively weaker effects in soil and sediment

remain undocumented next to the more obvious hazards to aqueous habitats. Bioaccumulation in

the soil and in sediments might play an important role in creating a hazardous situation for

organisms dwelling or growing in these habitats an over time.



assessment and risk characterization. Mass metrics are not necessarily appropriate as a point of

reference for the evaluation of nanomaterials.

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4.7 Nano diamond

Applications

Nano diamonds are registered with REACH for a tonnage band of 1-10 t/y. According to

communication with the FEPA the production range within Europe lies slightly above 1 t/y.6 After the

initial discovery in the 1960s and the subsequent evolution of high throughput production processes

in the 1990s the nano diamonds have found wide ranging applications in research and medicine as:

substitutes for some quantum dots, nanoscale magnetic sensors, drug delivery platforms, surgical

implant technology and medical instrument construction. In all of these fields the research and the

subsequent medical implication is ongoing and picking up speed as more nano diamond becomes

available. Similarly in industry nano diamonds find increasing application in tribology and lubrication,

energy storage composites and catalysis.101 and citations therein

Physico chemical properties

Nano diamonds are also made of pure carbon but are polycrystalline in contrast to the

monocrystalline conventional diamonds. They are translucent in appearance but consist of many

granular crystals with a size range between 2-8 nm102 or 10-20 nm103 depending on the production

process. The assortment of randomly oriented crystals conveys the exceptional mechanical

properties that surpass the monocrystalline structure commonly associated with the term ‘diamond’.

They are uniformly, exceptionally hard within a Knoop hardness range of 110-140 whereas the range

of monocrystalline diamond is 60-120 on the same scale, due to the dependence on directionality of

the applied pressure on a given crystal.103 The nano diamond is temperature stable up to 1200 °C in

an inert atmosphere. The initial production process via chemical-vapour deposition (months) has

been replaced since, first by high-pressure synthesis at 2300-2500 °C for a few minutes103 and later

by an oxygen depleted TNT/hexogen (RDX) detonation.104

Exposure

The majority of the nano diamonds are used in industrial applications where they are either used in

closed systems (lubrication) with little to no chance for inhalation or as complex materials where a

release of the nano diamonds is unlikely. The production of the nano diamonds requires complex

technological setups that are also closed systems. The inhalation of the nano diamonds is overall

unlikely outside of the scenario of an accident.

In research and in medical context only low quantities are used and especially in the latter the nano

diamonds are again either complexed or administered intravenously. Implants are set into place

surgically. None of these applications show an obvious exposure hazard in light of the high

biocompatibility nano diamonds are attributed with so far.

Environmental exposure is unlikely due to the highly specialized applications and the usage of nano

diamonds within complexed materials in many cases. It has been estimated that nano diamonds

6

Federation of European Producers of Abrasives: http://www.fepa-abrasives.org/DesktopDefault.aspx?

portalname=&language=&folderindex=0&folderid=0&headingindex=0&headingid=0&tabindex=0&tabid=0

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decrease the consumption of fossil fuels by ca. 5 % if they are added to lubricants used in motors.

Here nano particles might be released into the environment via the exhaust fumes or during repair

works or recycling.

As there are numerous possible modifications of the nano diamond surface the environmental

distribution could affect soil and water compartments but there are no studies on the environmental

distribution of nano diamonds up to now.

Environmental behaviour

Currently data on bioaccumulation and environmental distribution of nano diamonds is lacking.

Toxicology

Overall toxicity of nano diamonds is exceptionally low according to current research. Although lung

overload still leads to inflammatory reactions of lung tissue the effects are reported to be muted in

contrast to other nanomaterials in general and to carbon based materials in particular.96,105–108

Some reports indicate low toxicity in dependence of the nano diamond source (i.e. surface

modification or contamination from the manufacturer side) and of the acquired corona (see initial

nanomaterial hazard statements).109

Upon inhalation or instillation nano diamonds show no toxicity in the low dosage ranges 0,1-1 mg/kg

and following that (0,8-20 mg/kg) a dosage dependent accumulation and pathology in lung, liver,

kidney and blood.110,111 Neither long-term oral exposure of mice to nano diamonds nor subcutaneous

exposure led to detectable pathologies or exhibited effects on the reproduction rate and the

offspring.112,113 The genotoxicity results are contradictory and require further research.114,115

There is no information available concerning environmental toxicity at this point but the test results

for vertebrates indicate low toxicity. Further studies in invertebrates and bacteria are necessary to

determine the environmental risk of nano diamonds.


Nano diamonds are graded ‘low hazard’ in relation to other nanomaterials in this study because of

the toxicology described above. Nano diamond is used as an example for nanomaterials of very low

toxicity, even if in the case of nano diamond not all data is available to prove that. Although they

show toxicity upon inhalation – as all nanomaterials do – the exhibited toxicity is comparably low.

They are not water soluble and their distribution in the aquatic habitat, while possible from the

physico-chemical characteristics, is not sufficiently studied yet. Judging from the physico-chemical

properties little toxicity is expected.

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4.8 Impact of the options on risk and exposure scenarios associated to nano diamond

The following tables illustrate where option implementation or lack thereof will affect the fields

listed below without implying positive or negative nature of the impact. The tables serve as an

orientation for the reader to illustrate those aspects that will be of particular interest in the following

discussion and the ensuing upscaling of the impacts of the proposed modifications to REACH.

Table 4-5: Human health impacts of nano diamond


acute effects

workers 6,11,12,21 local inflammation of the lung

chronic effects

workers 6,12,21 fibrotic changes in the lung tissue

Table 4-6: Environmental impacts of nano diamond

options with impacts environmental impacts


biota 6,13,17,18,19,21 bioaccumulation

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4.9 Impact of the option implementation to REACH on risk and exposure (nano

diamond)

Option 6 – Include information on dustiness

A) status quo

The need for inhalation studies in the endpoint section is not apparent which has the potential to

lead to incomplete exposure assessments. The implications for workers, consumers and ecological

habitats are considerable as damage has to become apparent before further research is initialized.

Furthermore this option specifies the need for these studies independent from the production or

import quantities. Inhalation studies are otherwise required at a production/import tonnage of 10 t

or more.

Without the data gained from inhalation study the need for specialized air filters and breathing

masks at exposed workplaces might not become apparent.


Nano diamonds are a dusty substance and risk assessments as well as necessary endpoint related



along the lifecycle of products containing nano diamonds in inhalable form.

Option 11 – Require acute toxicity data for the most relevant route of exposure.



According to current knowledge nano diamond can be classified as non-hazardous and exposure

assessments are thus not required.


(inhalation)





of exposure.’




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A) status quo






This option will require the generation of inhalation specific toxicity data. The specific test methods

dictated here will ensure good comparability to other materials.

Option 13 – Require non-bacterial, in vitro gene mutation study

A) status quo

If nano diamonds are genotoxic the circumstances of genotoxic activity might be dissimilar for

bacteria and eukaryotes. Tests with living organisms are also often confounded by the fact that the

secondary ROS production triggered by the inflammatory effects of nano particles in general can lead

to gene deregulation. Without non-bacterial tests, it is impossible to assess potential genotoxicity

inherent to nano diamonds in particular.


Nano diamonds are not known to be genotoxic up to this point. It is unclear however if this is entirely

valid until non-bacterial tests are documented. The results dictate the appropriate risk prevention

measures.


Nano particles are often not soluble in water. The lack of water solubility is not a reliable indicator for

the lack of bioavailability. Nano particles have other options available to them to interact with their

environment and biological cells.

A) status quo

Nano diamonds are not water soluble and thus testing of bioavailability, -accumulation, and

persistence can be waived. But as it is also the case with other nano particles the lack of solubility is

no deterrent for one of the three processes to occur. Without the necessary research done the risk

assessment is likely to be imprecise.


Nano diamonds are tested for all relevant endpoints and accurately classified for hazard and risk

assessment within humans, other organisms, and the environment. As numerous results show the

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lack of water solubility does not correlate with unavailability in the biosphere. This is especially true

for nano particles.


term toxicity.



A) status quo

Nano diamonds exhibit remarkably low toxicity and mild, acute effects show only when

comparatively large doses are administered. Using this as an indicator, further experiments for long

term toxicity can be waived justifiably. Long term effects in humans or the environment might be

overlooked depending on the manufacturer of the dossier.


Nano diamonds are tested for long term pathological effects in humans and the environment. The

information from such experiments is crucial for application specific risk assessment.


As many nanomaterials are basically hydrophobic the current praxis of assessing the fate of

nanomaterials in the environment on the base of equilibrium partitioning, could be reviewed.

Especially sediments and soils are expected to be sinks for nanomaterials and the organisms living

there should be given appropriate consideration, as the concentrations and modifications of the

nanomaterials might be significantly higher and different than in the aqueous phase.

A) status quo

As nano diamonds are not soluble the deposition in soil and sediments is relevant but potentially

overlooked. Bioaccumulation in the soil and in sediments might play an important role in creating a

hazardous situation over time.


The exposure characteristics of nano diamonds are easily altered by changing surface modifications

in the course of the nano diamond lifecycle. The testing of soil and sediment organisms will quantify

the amount of hazard in relation to surface modifications and in dependence of applications of the

nano diamonds. The impact on soil and sediment organisms is assessed. This data is interlinked with

the assessment of bioaccumulation processes in this habitat and adds to the complete profile of the

nano diamonds for later risk assessment.

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Option 19 – Specify that algae testing should not be waived based on insolubility

In accordance with the options 14-17 in this document algae testing should not be waived based on

lack of water solubility.

As described above the lack of water solubility should not be a valid reason for the waiving of aquatic

tests in the case of nanomaterials. Many are able to persist in aqueous media and influence their

surroundings via agglomeration or surface modification with molecules found in the environment.

This issue is also taken up by the ECHA in their new guidance document ECHA-12-G-05-EN.

See Option 16



assessment and risk characterization.

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5 Evaluation of knowledge gaps for seven representative

nanomaterials due to the lack of the 21 options discussed

above

5.1 Synthetic amorphous silica (SAS)

In 2009 the yearly production rate of nanoparticular SiO2 was estimated at 1.590.000 t (METI

2009116). It is used mainly as high precision polishing agent, in plastics and rubber, paint, pigments,

cosmetics, PET-bottles, drying agents and waterproofing spray. The surface area of the nano particles

is highly dependent on the production technique. Exposure routes include oral ingestion of food or

additives, dermal exposure via usage of cosmetics and toothpaste, and via inhalation of the

nanoscale fraction of SiO2 in concrete, rubber tires (abrasion), and paints.117 As only very low

amounts of most SiO2 nano particles are water soluble the nano particles are mainly present in

agglomerated form and tend to sediment quickly. If the agglomeration is hampered however the

nano particles are difficult to separate from wastewater.47

Inhalation of nano particles in high doses showed some pathological effects and the toxicity is

inversely proportional to the particle size (basic risk of inhalable nanomaterial). High concentrations

of nano particles in the aquatic compartment cause increased mortality rates and deformations

within the local fauna.47,63 The International Agency for Cancer Research (IARC) defines amorphous

SiO2 nano particle as not classifiable with regard to their carcinogenicity towards humans.118

Currently SAS would be grouped somewhere between nano diamonds and TiO2 nano particles

because it is not classified as carcinogenic and does not by itself produce ROS. Nano diamonds

however are not yet fully assessed with regard to their environmental impact. Thus SAS and nano

diamonds might still find themselves grouped in the same hazard category. Due to different uses and

surface chemistry exposures might vary considerably. In relation to the other nanomaterials in this

report we group SAS nano particles in the low hazard category.

5.2 Carbon black

Carbon black is produced at an estimated tonnage of 8.100.000 t/y (ICBA7). It is used mainly in the

production of rubber (e.g. tires), as black pigment in paints, finishes and toner cartridges, in paper,

plastics and fibres. Due to its electrical conductivity it is also used in electrodes and carbon brushes.

In is water insoluble and is transferred into the environment mainly via tire abrasion, combustion

processes and the usage of printers. In high concentrations it has been shown to be mutagenic,

genotoxic, and potentially carcinogenic in humans. The presence of polycyclic aromatic hydrocarbons

(PAHs) is unavoidable due to the production process but the PAHs do not contribute to the toxicity

evaluations.119,120 The IARC groups carbon black as possibly carcinogenic to humans (Group 2B).43

There are further indications of negative effects on the blood circulatory system and it is suspected

of aggravating allergic reactions. Aquatic organisms show very little effects upon exposure to high

doses of carbon black. Because of the high doses necessary to observe the pathological effects and

7 International Carbon Black Association (http://www.carbon-black.org/what_is.html)

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the impurities in form of PAHs (attributed with the more toxic effects observed) present in previous

experiments the danger to humanity and the environment is deemed low.52

Due to the carcinogenic rating, the high production volumes, and the emphasis on toxicological

effects on humans and not so much on the environment we group carbon black roughly with TiO2

nano particles concerning the pathologic potential. As always the hazards detailed above have to be

factored in with the exposures (heavily dependent on application, intentional and unintentional

surface modifications).

5.3 Carbon nano tubes (CNTs) and multiwalled carbon nano tubes (MWCNTs)

An estimated amount of maximum 900 t of CNTs were produced per year in 2011.121 They are used in

composite materials to mechanically strengthen them while adding little weight or/and to convey

electrical conductivity. They are also used as antistatic coatings and are being investigated for the use

as carriers for pharmacologically interesting molecules and substances. CNTs are mechanically

resilient, can be single or multi-walled and display a high temperature conductivity while being water

insoluble. In their pristine state they tend to agglomerate and accumulate at hydrophilic /

hydrophobic interfaces. They are, however, easily modified by a multitude of organic molecules

which convey new characteristics to them. Most commonly water solubility increases with

modifications. Additional toxicological effects depend strongly on the type of modification. In the

course of the production process they are also typically contaminated with trace metals from the

catalysts which imbue them with toxic properties.52 Gustavsson et al. (2011)121 come to the

conclusion that no direct evidence is available for carcinogenic effects of CNTs on humans but that

this might be due to large information gaps. They find that “toxicological data is inadequate but

indicates that there is a risk of imflammatory reaction and pulmonary fibrosis when inhaled at

relatively low doses; there is also a risk of producing a DNA-damaging effect. They advise utmost

precaution in production and handling facilities and the need to reduce the airborne exposure to a

minimum. In addition to surface modifications and the composition of the nano tubes the aspect

ratio is of critical importance when considering acute and chronic toxicity as well as the carcinogenic

potential of CNTs. The higher the aspect ratio gets the higher the potential for frustrated

phagocytosis and effects similar to those of carcinogenic asbestos fibres becomes. Besides length and

aspect ratio rigidity plays another important role. Also here risk assessment is marred by the lack of

knowledge and comparability between the many studies.122,123 The reason for the decision to include

CNTs in this study is that production capacities about ten times higher than the current production

volume are already available. Furthermore, due to the various fields of application that are currently

investigated by the developers and producers, the production volume is very likely to increase

significantly until 2022. If on-going research can be used as an indicator CNTs have the potential to

find widespread application in many fields.

Here it is necessary to distinguish between workers exposed to CNTs or MWCNTs in the course of the

production cycle and consumers which are usually exposed only to nano tubes tightly bound in the

matrix of complex materials. Because some CNTs exhibit asbestos like characteristics upon inhalation

they are graded with ‘medium hazard’. Nonetheless many CNT variations do not exhibit such

properties and show only basic toxicology attributable to inhalable nanomaterials if they are not

further modified. MWCNTs as they are mostly produced in Europe do not fulfil the fibre criterion of

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the WHO by not being long or rigid enough to do so. The family of CNTs is highly diverse and a

detailed analysis of all possible structural variations and aspect ratios would exceed the boundaries

of the report.

5.4 Fullerenes

Fullerenes were produced at approximately 3 t/y worldwide in the year 2009.116 They have industrial

and possibly medical applications. Industrially they are used as catalysts, in semi-conductors, as

superconductors, base material for artificial diamonds, lubricants for high temperature applications,

batteries, solar cells, fuel cells and sports devices. They are not dispersible in water in their pristine

form just like the CNTs and the main exposure routes would be either via unskilled handling of the

raw product or via their presence within cosmetic products (very few and not legally available on the

European market). Though they are not toxic by themselves they show the same ability that CNTs

have to accumulate other organic molecules on their surface. These convey additional characteristics

to the Fullerene which can also include toxicity and the ability to penetrate the blood-brain barrier to

unknown effect.52,124 For this reason Kahru et al. 2010 rated Fullerenes more hazardous than CNTs

and TiO2 nano particles.23 Although we agree with the inclusion of unintentional modifications into

the risk assessment for nano particles we chose not to rate Fullerenes as toxic as Kahru et al. did as a

consequence of an extensive literature search. The stochastic nature of the unintended nano particle

surface modifications needs to impact on this assessment instead of focussing exclusively on toxic

modifications. Regarding future production volumes, growth rates similar to CNT can be assumed.

Hence, in analogy to CNT, Fullerenes have been selected as a case study despite of their relatively

low current production volume. Large scale studies will be necessary to fully evaluate the risk posed

by Fullerenes to human health as well as to the environment with an eye towards the exposure

characteristics and the modifications imparted along the life cycle of specific applications.125

Due to the possible variations of surface modifications within specific applications and along the life

cycle Fullerenes are grouped with TiO2 nano particles concerning the risk they pose. It is important

however to keep in mind that the surface modifications are the crucial elements determining the

hazard. In pristine state they show no enhanced toxicological properties and the potentially harmful

surface modifications would hopefully be acquired unintentionally by the Fullerenes and thus be

stochastic in nature. Lacking stronger, intrinsic toxicological properties and considering the other

nanomaterials in this report we assign the relative grade ‘low hazard’ to Fullerenes.

5.5 Nano silver

In 2012, up to 550 t/y were produced.126 Nano silver is used as anti-microbial agent in textiles and

packaging, medical products, cosmetic products, washing agents, paints and anti-biofouling coatings

for marine vessels. Silver nano particles tend to aggregate as much as ZnO nano particles and the

main toxicological effects stem from the ability to produce reactive oxygen species and the leaching

of silver ions. The main exposure route is the loss of nano particles out of paints, finishes and

textiles.74,127 Once in the waste water pathway, silver is oxidized to silver salts which have very low

water solubility and therefore tend to accumulate in sediments and soil. Nano silver is rated as highly

hazardous to water (water hazard class 3) but displays very little toxicity towards humans.52 On the

one hand large knowledge gaps are still hindering conclusive risk assessment as also discussed by

Christensen et al. and Johnston et al. (2010)128,129, on the other hand many assessments point

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towards the toxic properties of silver nano particles and still remain unheard due to non-scientific

issues.130

Due to the high toxicity towards aquatic habitats and the lack of data concerning many human health

aspects we group nano silver with ZnO nano particles. The toxic effects mediated via ions similarly

add to the toxic potential of both metal nano particles.

5.6 Nano copper

The yearly production of nano copper is between 1-10 t worldwide. It is widely used due to its cheap

base material and its interesting industrial and end-user oriented applications. It is used in inks,

lubricants, heat conductor fluids, coatings/catalysts, semiconductors and machine tools. It is also

reported to be beneficial towards wound-healing131 and is found in some cosmetic products132. The

exposure to Cu nano particles can thus be via inhalation or via oral uptake. Inhalation of Cu nano

particles leads to an increased probability for the occurrence of lung infections most probably due to

the fact, that the neutrophil cells of the immune system are busy removing the nano particles and

are thus less available for the immune defence against bacteria that cause the lung inflammation.133

In this regard it is not clear whether Cu nano particles are more prone to facilitate lung infections

than other nano particles. After all the involvement of the neutrophils in nano particle removal is

universal so the added stress of invading bacteria would apply in any case of inhalatory exposure to

nano particles. The inhalation of Cu nano particles does also trigger the mucociliary escalator which –

also similar to any other inhaled nanomaterial – can lead to ingestion of the nanomaterial

transported out of the lung. In comparison with many other metal oxides, Cu nano particles are very

toxic upon ingestion. In spite of the fact that a daily uptake of Cu in the range of 70 and 100 µg/kg

per adult human is normal ingestion of higher amounts of Cu quickly leads to heavy metal poisoning.

This is mainly due to the Cu ions generated in the interaction between the gastric acid and the

copper. As decreasing volume of a particle emphasizes the surface area disproportionally the smaller

the Cu nano particles ingested are the larger the toxicological effects become.134 In case of Cu nano

particles this increase in toxicity from bulk to nano form was quantified with a factor of 51.23 Besides

the ion derived effects which affect mainly the lipid profile, pH values of blood and tissues, and tend

to accumulate in renal tissue and the liver leading to dysfunction and cell death135, the nano particles

themselves are able to generate ROS. In spite of the fact that increasing surface oxidation and

intentional modifications strongly diminish the amount of ROS produced the remaining ROS

production is still sufficient to induce DNA damage and thus be carcinogenic.136

Due to the increased toxicity of Cu nano particles vs. Cu bulk material on both pathways (ions and

ROS generation) the Cu nano particles have been rated class 3 (moderately toxic) on the Hodge and

Sterner Scale, while the bulk sized particles only warrant class 5 (practically non-toxic).135 Karlsson et

al. (2008)137 classify Cu nano particles as the most toxic nano particles in comparison to TiO2-, ZnO-,

Fe2- and Fe3-nano particles in comparative inhalation studies. The same toxic effects described here

can also be found in organisms of the aquatic habitat and Cu nano particles are known for their

bactericidal and fungicidal properties.138,139

Concerning their impact on human health when ingested orally the Cu nano particles should be

grouped above the TiO2 nano particles. There is little information on large scale environmental

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effects but it can be expected that the negative ionic effects of Cu on the aquatic environment are

within the same order of magnitude as ZnO ions. Cu salts are also grouped in the same category WGK

28 in Germany as ZnO is.140 Therefore we group Cu nano particles with ZnO and Ag nano particles in

the category ‘high hazard’.

5.7 Quantum dots (e.g. Cadmium sulphide)

Remark: Quantum dots represent a diverse group of nanosized heavy metals by themselves and are

listed here as representatives of a host of nanomaterials that are manufactured at less than 1 ton

per year. Toxicological and exposure considerations will be exemplified using cadmium sulfide based

particles.

In 2010 we estimate that less than 100 kg of quantum dots (QDs) were produced based on an

average price range between 2500 and 6000 $ per gram and a market volume of ~67 mio $ in that

year.141 Within this group several different types of nanomaterials are used. Due to their cost in

production and the heavy metals contained their application range was limited to research and some

LED applications. With the development of new QDs without heavy metals and the sinking prices QDs

are investigated for the use in Electronics, Optoelectronics, Optics, and solar energy and a large

growth of the market is predicted (BCC Research 2008142). In their original and still widely applied

incarnation QDs contain cadmium or selenium for example. Both metals display highly toxic

properties in bacteria and mammals but in the context of quantum dots show highly diverging results

concerning their toxicity. This is most probably due to extensive variations in their shell and outer

coating compositions.143–145 The newer generation of QDs is designed without the need for a heavy

metal core which might alleviate the toxicity significantly. With the redesign of the QDs their surface

has also become hydrophilic thus opening new routes of exposure and ecological compartments that

might be affected by their widespread usage.

Due to the usage of heavy metals in the original incarnation of QDs we grade these as highly

hazardous.

8 WGK: Wassergefährdungsklasse 2; water hazard class 2 denominates a substance that is considered

hazardous to water dwelling organisms. In the German classification system there are three water hazard classes with class 3 being highly hazardous and class 1 weakly hazardous

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ANNEX 3 TO FINAL REPORT...2013/01/09 · IHCP/2011/I/05/27/OC – Annex 3 3 European Commission Annex 3 to Final Report - Impacts on health and the environment in detail Examination