Why so many uncertainties about the risks associated with nanos?

Why so many uncertainties about the risks associated with nanos?

Although the share funding for studies on health and environmental risks of nanos in the public budgets devoted to research in nanosciences and nanotechnologies stay weak and are much more limited than research on the expected benefits of nanomaterials, research on the potential risks associated with nanomaterials is the subject of several scientific publications. However, many uncertainties remain, for various reasons detailed below.

However, the absence of certainty about the risks should not be equated with the absence of risks – nor lead to inaction, on the contrary: we must not repeat the mistakes of the past (leaded gasoline, asbestos, etc.)!

Limits encountered in nano risk assessment 

Insufficient characterization of the nanomaterials tested 

The physico-chemical characteristicsiwhat nanomaterials tested have long been insufficiently described¹Improvements are already noticeable. Various working groups have defined the parameters which should be systematically specified in all the articles (the detailed description of the experimental conditions is also essential). Following theeditorial from the August 19, 2012 review Nature Nanotechnology, the scientific community has continued to define the information needed to stabilize the characterization base that all nanotoxicology articles should include. See in particular The dialogue continues, Nature Nanotechnology, 8, 69, February 2013: The nanotoxicology community has numerous ideas and initiatives for improving the quality of published papers., or too heterogeneously to be able to reproduce the experiments and/or compare the results between the different studies. Gold these characteristics play a very important role in the toxicity of these materials, undermining a key principle of toxicology according to which “everything is poison, nothing is poison: it is the dose that makes the poison”²Phrase of the doctor and alchemist Paracelsus who founded toxicology and is very often invoked to assess the risks associated with synthetic chemical substances.

On the one hand because the toxicity and ecotoxicity nanoparticles vary according to their physico-chemical characteristics (dimension, shape, structure, state of charge, degree of agglomeration, composition, solubility, etc.) which themselves vary according to the conditions under which the nanoparticles are synthesized, stored, possibly coated, integrated into a product then releaseded in the environment. For example, in humans or animals, fibrous nanoparticles are more likely to generate inflammatory effects than spherical nanoparticles.³See for example Carbon nanotubes, but not spherical nanoparticles, block autophagy by a shape-related targeting of lysosomes in murine macrophages, Autophagy, 14:8, 1323-1334, DOI: 10.1080/15548627.2018.1474993.

On the other hand, it is also necessary take into account what the nanomaterials considered – or their residues – will get in touch : living plants, animals, micro-organisms, and other chemical substances. For example, in the biological environment, nanos can be quickly coated with proteins, forming a protein crown (corona) which can also impact their toxicological effects.⁴See for example:
- in French : Protein crown around nanoparticles: a big deal!, IRAMIS and Joliot Institutes, I2BC, March 2021
- in English : The bio-corona and its impact on nanomaterial toxicity, Westmeier D et al.,  European Journal of Nanomedicine, 7(3), 2015.

Any assessment of the risks associated with nanomaterials is therefore very complex. However, avenues for improvement are proposed by the scientific community.⁵See for example:
- Environmental Risk Assessment of Nanomaterials in the light of new obligations under the REACH regulation ‐ Which challenges remain and how to approach them?, Integrated Environmental Assessment and Management, Schwirn K et al., March 2020 Experts call for updated guidance on nanomaterial risk assessment, ChemicalWatch, March 26 2020
- Harmonizing across environmental nanomaterial testing media for increased comparability of nanomaterial datasets, Geitner NK et al., About. Science: Nano, 7, 13-36, 2020.

Studies often difficult to extrapolate to humans

Nanomaterials are mostly tested vitro, on different cell strains (human, animal, plant), on many micro-organisms (bacteria, viruses, fungi, etc.). These studies mainly give indications in terms of carcinogenesis and cell viability. They cannot completely replace the tests in vivo.

But the studies in vivo They also have limitations: animal models of toxicity pose ethical and financial but also methodological problems: their extrapolation to humans is certainly more reliable than the tests vitro but is not guaranteed⁶See for example How the rat test fails to protect humans, Stephane Foucart, Le Monde, Oct. 22 2012.

There is a real need to have data on the human body. Thanks to the progress of nano-metrology and by respecting obviously essential ethical considerations, it is possible to advance knowledge at the human level⁷Nanotoxicology: the need for a human touch?, Miller M & Poland C, SmallJuly 2020.

Studies sometimes carried out under conditions not representative of actual exposure

Since industrial applications are currently not well known or quantified, they can only be estimated. The “probable” exposure of the population and the environment can therefore only be estimated. On what criteria and with what reliability?

Unrealistic exposure pathways 

For practical reasons, the nanomaterials tested are often introduced directly into certain body parts and organs (e.g. intracerebral, intraperitoneal injections for example), according to modalities which are sometimes very different conditions by which the environment or the population are really exposed, preventing us from properly taking into account what happens during the important mechanisms that come into play "in real life" (processes involved in digestion / fermentation / detoxification, for example).

Turnkey progress is nevertheless being made in terms of the environment, with studies carried out in mesocosms for example – huge aquariums reproducing a mini ecosystem in which the behavior of nanoparticles in contact with plants, fish, soil and water is studied at different dosages⁸See MESONNET: Use of networked terrestrial and aquatic mesocosms to assess the risk associated with the dispersion of manufactured nanoparticles, CEREGE project; and the equipment of theINERIS : Lessons from synthetic ecosystems, Le Monde, 20 Nov 2013.

Studies carried out over too short periods 

The studies are often conducted over periods that are far too short to reflect realistic exposure conditions, largely chronic in this case (cases of accidents are also to be taken into account, but according to very specific configurations).

Epidemiological studies in humans concerning nano risks are almost non-existent. A single epidemiological surveillance program for workers potentially exposed to nanomaterials has been set up in France (EpiNano), but it is encountering such difficulties that its results will not be known or usable for many years.

Synthetic nanomaterials different from those to which ecosystems and human populations are exposed

The nanomaterials considered are often synthesized in the laboratory and therefore different from the nanomaterials (and nanomaterial residues) to which ecosystems and human populations are actually exposed.⁹See for example Yang Y et al., Characterization of Food-Grade Titanium Dioxide: The Presence of Nanosized Particles, About. Science. Technology., 2014, 48 (11), pp 6391-6400, often more complex and mixed with elements from the living. For the time being, scientists indeed have very limited knowledge of the types of nanomaterials that are incorporated into products currently on the market, and a fortiori nanomaterial degradation residues released into the environment throughout the “life cycle” of these products; they also ignore a lot of things about mobility and transformations suffered by them in the environment.

Little consideration of the life cycle 

Nanomaterials can transform during their " life cycle" , whether it be in the environment ou in the human body : many parameters come into play, such as the degree of acidity or salinity for example. The limits listed above apply a fortiori for the degradation residues of nanomaterials released into the environment from their design until their end of life.

Doses tested higher than actual exposure  

The doses of nanomaterials tested are often higher than the concentrations to which ecosystems and human populations are actually exposed (particularly because of limitations of detection and measurement devices used in the laboratory). However, the observed effects (or others) could also occur at lower concentrations; certain nanomaterials (silica in particular) are thus more genotoxic at low doses than at high doses¹⁰See Results of the European program Nanogenotox on the genotoxicity of nanomaterials, presented in French at ANSES, during the Restitution of the national environmental and occupational health research program: Chemical substances and nanoparticles: models for the study of exposure and health effects: Participant's file et Slideshow, November 2013. And "The toxicological assessment of nanomaterials must evolve, according to a European research project", APM International, November 14, 2013. More generally, we are beginning to better understand the effect of low doses and to realize that these effects can be just as harmful as high doses or have antagonistic effects depending on the doses. Dose-effects considerably complicate toxicology research. See for example The health problem of low doses, Elizabeth Grossman, July 2012; The second death of the alchemist Paracelsus, Stéphane Foucart, April 11, 2013. In addition, the high concentrations make it possible to simulate acute and occasional contamination situations (for example an accidental spill on a production site, or even during transport). At the end of 2019, a study also showed that a significant fraction of the nanoparticles tested in nanotoxicity and nanomedicine studies can remain in the plastic syringes used to dose the nanoparticles! This calls into question the reliability and reproducibility of the studies¹¹See Failure to launch: nano toxicity studies may be affected by nanoparticles staying behind in syringes, European Union Observatory for Nanomaterials, 25 November 2019 and Unpredictable Nanoparticle Retention in Commonly Used Plastic Syringes Introduces Dosage Uncertainties That May Compromise the Accuracy of Nanomedicine and Nanotoxicology Studies, Holtzwarth U et al., Frontiers in pharmacology, November 2019..

Hard-to-detect nanos

Due to their small size and the transformations they undergo during their journey in the body or in ecosystems, nanos are difficult to detect, quantify, characterize and track in the human body and in ecosystems.

Gaps in data

In the end, many aspects relating to the toxicity and ecotoxicity of nanomaterials remain to be investigated. In April 2020, an analysis of the literature highlighted, for example, the lack of data concerning the impact of nanomaterials on female fertility and the need for studies on their effects on reproductive capacities.¹²See Female fertility data lacking for nanomaterials, European Observatory of Nanomaterials, April 6, 2020 and A critical review of studies on the reproductive and developmental toxicity of nanomaterials, ECHA / Danish National Research Center for the Working Environment, April 2020.

Improvements in progress and to come

Ongoing research is leading to significant improvements, particularly at the methodological level¹³See for example: Scientific meeting on microplastics and nanomaterials: environmental and health research, ANSES / ANR, 20 May 2021 and Cahier de la recherche n°17: "Microplastics and nanomaterials" - Understanding where research is at, ANSES, May 2021.

An example, for illustrative purposes: the Nanomique project developed at the CEA in partnership with the Lavoisier Institute (CNRS) of the University of Versailles, is a systematic screening approach to define the toxicity of about fifteen nanoparticles (already used in industry) on human lung cancer cell lines and on three-dimensionally cultured lung tissue. It is based on a high-throughput screening platform: a device allowing a large number of tests to be carried out in parallel on cell cultures. It thus makes it possible to quickly test different concentrations of nanoparticles and different types of cells.¹⁴See “Measurements of the toxicological effects of metallic nano-oxides on human cells vitro“, Chevillard S, in Nanomaterials and health – Understanding where research stands, HANDLES, The research notebooks, October 2015. .

Progress is also being made in environmental matters, with studies carried out in mesocosms for example – huge aquariums reproducing a mini ecosystem in which the behavior of nanoparticles in contact with plants, fish, soil and water¹⁵See MESONNET: Use of networked terrestrial and aquatic mesocosms to assess the risk associated with the dispersion of manufactured nanoparticles, CEREGE project; and the equipment of theINERIS : Lessons from synthetic ecosystems, Le Monde, 20 Nov 2013.

Eventually, the advances in nano-metrology as well as improvements to R-Nano register and registration of nanomaterials in REACH should advance knowledge, by making it possible to work more precisely on nanomaterials produced or imported in France.

Adapting standardized tests used for toxicological testing of conventional chemicals (such as OECD guidelines) to nanomaterials takes time¹⁶Work is underway for this adaptation. See in particular:
- Adapting OECD Aquatic Toxicity Tests for Use with Manufactured Nanomaterials: Key Issues and Consensus Recommendations, Petersen EJ et al., About. Science. Technology., 49 (16): 9532-9547, 2015
- Nanotechnology Regulation and the OECD, CIEL, ECOS, Öko-Institute, January 2015
- Ecotoxicology and Environmental Fate of Manufactured Nanomaterials: Test Guidelines, the Working Party on Chemicals, Pesticides and Biotechnology, OECD, March 2014..

In the meantime, we must not "throw the baby out with the bathwater": the many studies showing toxic effects and published before the development of these standardized tests¹⁷See for example our sheets Risks associated with carbon nanotubes ; Risks associated with nanosilver ; Risks associated with nano titanium dioxide ; Risks associated with nanosilica should not be dismissed out of hand on the pretext that they are not perfect from the point of view of characterization or other limits listed above.

Better financing of risk studies is essential 

In 2009, researchers had estimated that fifty years of work and several hundred million dollars the amount of studies needed to study the risks of nanomaterials already on the market; methods of financing risk studies associated with nanomaterials must therefore be invented.

The grouping of nanomaterials with similar toxicity potential (read-across) is a strategy recommended by certain industrial players, but it is also disputed as there are many methodological pitfalls.¹⁸See about the grouping of nanomaterials (“grouping” and “read-across”):
- 1st Innovative and complex materials: Towards grouping to support hazard and risk assessment, Stakeholder Workshop, BMBF-project InnoMat.Life, June 15 2021
- NanoApp, an ECETOC project, launched in December 2020 (“this tool is used to establish and justify sets of nanoforms and identify poorly soluble – low toxicity (PSLT) nanoforms”).
- A framework for grouping and read-across of nanomaterials- supporting innovation and risk assessment, Stone V et al., NanoToday, 35, December 2020
- Grouping all carbon nanotubes into a single substance category is scientifically unjustified, Bengt Fadeel & Kostas Kostarelos, Nature NanotechnologyMarch 2020
- Categorize nanomaterials for effective hazard and risk assessment, Cordis, NanoREG II, December 17, 2019
- Material-specific properties applied to an environmental risk assessment of engineered nanomaterials – implications on grouping and read-across concepts, Wigger H and Nowack B, Nanotoxicology, 13(5): 623-643, February 2019
- Understanding the legal term Nanoform in REACH (and 'set of similar nanoforms') – A discussion Workshop between ECHA and Industry Experts, CEFIC & NIA, October 16, 2018
- Nanotechnology experts from across the globe join forces to advance nanomaterials safety testing through Grouping and Read Across, NanoReg2 and Gracious, September 2018
- Criteria for grouping of manufactured nanomaterials to facilitate hazard and risk assessment, a systematic review of expert opinions, Landvik NE et al., Regulatory Toxicology and Pharmacology, 95: 270-279, Jun 2018
- GRACIOUS: Grouping, Read-Across, CharacterIsation and classificatiOn framework for regUlatory risk assessment of manufactured nanomaterials and Safer design of nano-enabled products, research project H2020, 2018-2021
- Grouping and read-across for nanoforms, ECHA, 30 November 2017
- Conference on Categorization of Next Generation Nanomaterials, FutureNanoNeeds, November 30 and December 1, 2017
- Considerations about the relationship of nanomaterial's physicalchemical properties and aquatic toxicity for the purpose of grouping, UBA, November 2017
- Effects of nanoparticles on human immune cells, Denis Girard, IRSST, November 2017: “All of the results clearly show that it is difficult to classify NPs strictly according to their potential to modify one or other of the functions studied. It is preferable to present a more nuanced picture in which the effects caused by a given NP on the biology of human EOs in vitro must be taken into consideration to clarify its mode of action. The effects of NPs are therefore extremely varied and the present study aims to demonstrate that they do not all act in the same way.
- Approaches on nano-grouping/equivalence/read-across concepts based on physical-chemical properties (Gera-PC) for regulatory regimes, OECD, January 2016
- NanoToxClass – Assessment of the health effects of industrially used nanomaterials to be made more efficient, BfR, 18 January 2016
- ECETOC concept allows assessing the safety of nanomaterials undertaking animal testing only as a very last resort, ECETOC, 16 December 2015
– the work of the European research project NanoSolutions (2013-2017), which seeks to identify the characteristics of engineered nanomaterials that determine their biohazard potential. It will allow the development of a safety classification model for these nanomaterials, based on an understanding of their interactions with living organisms
- Grouping of nanomaterials: a tool for assessing risks, FOPH, August 2015: The grouping concept proposed by the FOPH first provides for classifying nanomaterials designed in a very similar way in the form of entities based on a set of criteria. In a second phase, the entities are attached to “clouds”. Within the same “cloud”, the entities can be evaluated with the same test strategy.Cf. Walser & Studer, Sameness: The regulatory crux with nanomaterial identity and grouping schemes for hazard assessment, Regulatory Toxicology and Pharmacology, 72(3): 569-571, Aug 2015
- Grouping nanomaterials – A strategy towards grouping and read-across, RIVM, June 2015
- EU toxicology body publishes grouping framework for nanomaterials – Risk assessment tool contributes to sustainable development of nano products, ChemicalWatch, April 2, 20115 and A decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping), Arts JHE et al., Regulatory Toxicology and Pharmacology, 71(2): S1-S27, March 2015
- A critical appraisal of existing concepts for the grouping of nanomaterials, Regulatory Toxicology and Pharmacology, 70(2): 492-506, November 2014
- Grouping of nanomaterials for risk assessment, Bolt HM, Archives of ToxicologyNovember 2014
- A strategy for grouping of nanomaterials based on key physico-chemical descriptors as a basis for safer-by-design NMs, NanoToday, 9(3): 266-270, Jun 2014.

Some scientists recommend working on “model nanoparticles”¹⁹See in particular:
- "Imogolite nanotubes: a new model material in nanotoxicology?" by Rose J et al., in Participant's file prepared for the Restitution of the National Environmental and Occupational Health Research Program (PNREST)October 2015
-“Towards a model material in nanotoxicology? », Rose J and « Measurements of the toxicological effects of metallic nano-oxides on human cells vitro“, Chevillard S in Nanomaterials and health – Understanding where research stands, HANDLES, The research notebooksOctober 2015; we are therefore still a long way from obtaining knowledge on the toxicity and eco-toxicity of the nanoparticles used by manufacturers...

The OECD Working Party on Manufactured Nanomaterials is looking at hazard and exposure assessment and guidance for different types of manufactured nanomaterials. He suggested at the end of 2015 to give priority to manufactured nanomaterials contained in gases or liquids, for which the risk of exposure is higher than for solids, since gases and liquids spread more quickly and enter the human body more easily by inhalation or ingestion²⁰See OECD, Nanomaterials in waste streams (Chapter 1, overview), November 2015. Further publications are expected.

For the moment, the uncertainties give rise to divergent interpretations

These uncertainties and difficulties explain why the results cannot be generalized and should be considered with caution.

When some minimize the risks by arguing that the experiments were carried out on the basis of a "worst case scenario" (for "worst case scenario" in English, involving for example nanoparticles used in dispersed form and in very high doses ), others underline a contrario that the conclusions lead to ringing the alarm bell.