Complete Data Reporting in Nanotoxicological Studies
Issues pertaining to detailed documentation and complete reporting of experimental findings have been frequently raised in the past NanoTOES project meetings. It has been recognised that reports on “no-effect” or negative results are important in the field of nanotechnology, in particular nanotoxicology, and are relevant in the context of NanoTOES. Toxicological studies of nanomaterials on humans and environment have not been straightforward and existing literature can be confusing at times. Due to lack of standardisation and incomplete reporting that selectively focused on adverse effects, the quality and appropriateness of these data remain questionable. Since studies on nanotoxicology are likely to continue for the foreseeable future, effort needs to be taken to encourage accurate and proper dissemination of these findings.
Nanoparticles on their own display a number of characteristics of shape, size, composition, and surface chemistry. Different nanoparticles also exhibit varying degrees of aggregation and agglomeration when suspended in solution. These multiple physico-chemical properties of nanoparticles will determine their biological and environmental effects, which very often are difficult to predict. To add to the complexity and (sometimes) confusion, these properties will be modified as the nanoparticles are exposed to and/or interact with biological molecules in the body and environment. When it comes to the actual process of nanotoxicological testings, more variables are then introduced as the choice of assays, the endpoints, dosing, and route of exposure will result in different outcomes. Therefore, it is important that methodological and toxicological data on nanomaterials be properly documented, and subsequently reported in full detail, to avoid confusion and to ease the process of interpreting results. If done systematically, this will allow better understanding of the impact that nanomaterials have on health and the environment.
Nanotechnology is undeniably a promising and fast progressing branch of science, affecting many aspects of human lives and the environment. The availability of toxicological data on nanomaterials forms the basis for gazetting the regulatory guidelines for their use. At the same time, however, lack of information as well as misinformation from existing data have added to societal concerns relating to the safety of nanotechnology. Bias reporting in science is not uncommon. Positive or adverse results are preferentially reported and published, whilst negative or no-effect results very often remain hidden. This practice can be worryingly misleading, because if safety guidelines and regulations are to be based upon published data, then all results no matter good or bad should be considered. Only when comprehensive findings are disseminated will true evaluation of the benefits and risks of nanomaterials be properly assessed. Moreover, only when these toxicological data are of acceptable quality and rigour will public doubts on nano-object safety be addressed.
Most of today’s policy-making is facilitated by research-based evidence and systematic reviews of published literature which provide a means to address specific questions, identify gaps, as well as to guide implementation strategies related to new policy. The reliability of a systematic review depends on the appropriateness of the methods used to gather relevant information, whilst also wherever possible recognising possible biases and limitations. Therefore, in the context of nanotoxicology, the availability of complete, high-quality data on both positive and negative effects of nanomaterials is of extreme importance. To ensure data of high quality, an experiment has to be well-designed and adequately controlled. Experimental conditions must be meticulously documented, while choices of assays and endpoints must be relevant and defensible.
As mentioned earlier, detailed documentation of experimental details is particularly important in nanotoxicological studies, given the multiple characteristics of nanoparticles and their unpredictable interaction with biological molecules and environment. Therefore, assay design and methodology, especially the presentation of nano-objects into the assay system, need to be specific, reproducible and fit for purpose, to allow better correlation of nanoparticles properties to the observed effects. This will fundamentally improve the ability to interpret results. Whilst this principle applies throughout the analytical process, from the start materials right to the assay endpoints, some particular points of variance are worth highlighting.
- The start material, which may be a nanoparticle being investigated, needs to be adequately characterised, used within its shelf life, and checked to be devoid of secondary contaminants which could greatly affect the result outcomes.
In immunotoxicology for instance, presence of endotoxin in test materials, even in minute amount could lead to the activation of the immune response. This will prompt unwary scientists to draw false conclusions, as the true effects of the nanoparticles would have been masked by the effects contributed by the contaminating endotoxin.
- Assays selected for nanotoxicological studies should be appropriate for the task. The assays should preferably provide mechanistic understanding of the nanoparticle effects and the assay readouts should not be interfered by the test materials. Additionally, solvent effects also need to be investigated to avoid false interpretation of the results.
- Assays also need to be properly validated. Validation is of great importance if assays are to be demonstrably fit for their intended purpose. The validation process will vary with the assay target and objective; however, the common feature is validation will demonstrate the accuracy, precision and sensitivity of the measurements being made. By bench-marking assays in this way, the availability of validated and robust assays for nanotoxicological testing will greatly enhance end-user assurance in and use of the information generated, through confidence in the predictive value of the analytical system used to obtain it.
- Use of reference materials and appropriate experimental controls must be included to ensure the reliability of the assay.
The lists of issues for consideration in nanotoxicological studies do not stop here. For instance, dosing regimens need to be properly considered and documented. Acute versus chronic treatment, exposure route, and the measure of dose will affect the outcomes of the assay, and ought to bear some relevance to a real-life situation. For example, the assay platform should include cells representative of the point of exposure to the nanoparticle, or to the most likely site of its major effect(s).
Quantification of dosage is similarly not a straightforward matter: since there is not yet any standard measure of dose or concentration applicable across nanotoxicology, different dosing systems, such as expression in particle numbers, mass, and/or surface area are used in different laboratories. This practice has added to the confusion and complicates data interpretation as direct comparison of results between laboratories could not be easily made, or are sometimes impossible because key information was not recorded. Then, there will be questions regarding the actual dose given. Are these doses physiological and realistic? During in vivo studies for example, are the exposure methods and doses of nanoparticles administered appropriate to represent actual exposures (intentional versus unintentional/accidental)? Conversely, during in vitro testing – and assuming that the in vitro model bears meaningful resemblance to the physiological reality, is the experimental exposure reflective of a meaningful exposure scenario? The former concern can be addressed by the use of physiological-relevant human primary cells in place of animal cell lines which have existed in culture for a long time, thereby losing the characteristics of their tissue of origin. The latter must be determined by robust assessment of actual exposure, for example under acute (high-level accidental) or chronic (low-level environmental) challenge.
These considerations culminate in a need to promote standard methodology and practice in nanotoxicological studies. A standardised approach to understanding the benefits and risks of nanomaterials could be a great tool not only for scientists, but also to other stakeholders and policy-makers. Coupled with the dissemination of complete and high-quality data, comparison of results with the literature could be done with ease and without bias. With this clear and precise understanding on the impact of nanomaterials to health and environment, better decision could then be made by policy-makers to address safety, ethical and public concerns regarding nanotechnology.
As the development of regulatory guidelines for safe nanomaterial use is still very much a work in progress, efforts should be started to aid this process. This can be done by ensuring full reporting of toxicological data, thereby allowing access to accurate information on the pros and cons of nanomaterial use. In order to promote reporting of negative or no-effect results, awareness on the consequences of biased publication should be raised and this ought to be targeted to the scientific community, journal editors and publishers, as well as policy-makers. A change in the current editorial policy which favours publication on positive and significant results is needed. An alternative is to set up electronic databases that hold such data, which could be made available to interested parties. This is an attractive, but perhaps less accessible option, unless accompanied by some objective collation of findings into end-user friendly formats. The challenges ahead may be tough, but by ensuring that the bits and pieces are being done right from the beginning, it will pave the way for better dissemination, communication, and understanding in this advancing field of nanotechnology.
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