Problems, pitfalls and partial solutions for industry case studies
As one of the EU FP7 flagship programmes, there are high expectations for NanoVALID, which consists of 29 partners from 19 different countries worldwide. The major goal of NanoVALID (http://www.nanovalid.eu/) is to develop a conscientious list of validated methods for use in hazard identification, risk assessment and analysis of engineered nanomaterials (NM). The market place is becoming flooded with NM; the medical industry, the chemical industry, the sporting goods industry, the automotive industry, etc. etc., they are all buying, building, using, removing, aerosolising, collecting, and discarding NM products. This ultimately means our environment is become open to increased levels of exposure to an increasingly diverse collection of materials, and with unsuitable and undetermined testing methods for NM toxicity it is becoming increasingly difficult for effective regulation to be implemented; this is all of great concern to nanotoxicologists. The assortment of methods developed in NanoVALID will provide imperative instruction of how to better operate to keep all these industries, and consumers, safe from the potential hazard of NM.
Within this programme there is a particularly troublesome work-package called 6! It is hard to list work packages in order of importance, but it is safe to say that WP6 will be instrumental in assessing NM exposure and the impact of working within a facility that is actively producing or using NM. During the tasks of WP6, alongside the development of safe handling protocols, the researchers would like to assess real life environments to determine the real life exposure conditions that occur during large-scale synthesis and use of NM; basically to address day to day workplace exposures in real time using real life conditions. This is to include staged accident scenarios, to answer questions such as: what would happen if that truck carrying your CNT is to turn over spilling its cargo? Are the storage containers suitable? Are they safe? What if a drum containing ZnO nanoparticles was to spill in a factory or a warehouse, what would be the exposure zone? Who is affected? Who is safe? What would be the environmental implications? Using real-life exposures systems and mathematical modelling, these are all questions that WP6 would like to find answers to, but there are problems and pitfalls that make this a very difficult task indeed.
For this task to be successful and to overcome the first pitfall considerable input from other work packages, and potentially other project, will be instrumental. To establish reliable readouts for the work place you need to have robust, reproducible, and ultimately easy! assays. Why easy? Because one outcome of this case study exploration is to produce apparatus that will be used by non-scientists with very little training and with limited access to the common, routine biological assessment laboratories. To determine which assays are suitable is a long process; a panel of particles were chosen and an assortment of assays are currently being tested. In this round robin style investigation different readout parameters, such as fluorescence, luminescence, or absorbance, are used to test many endpoints, such as cell death, inflammatory responses, cellular stress, genotoxicity etc., with the ultimate goal of finding robust reproducible assays that can be read via simple mechanisms. One prominent component to be used in these assays is likely to be stable cell lines transfected with promoter induced fluorescence that will emit a fluorescence signal when any one of a number of genes is expressed. This allows an easy, fast, and simple measurement of almost any outcome of cellular stress. So it certainly seems possible that through the work of those members involved in WP2 and WP3, in developing these techniques, the first pitfall can be overcome.
When a suitable selection of assays is made the next dilemma is what will be tested? Which brings us to the second pitfall, can there be actual real-time exposures, or will assessment have to be on collected material? Even if both are possible, each one has its potential complications. To use real-time exposures a functioning exposure chamber is essential, as is a suitable level of exposure material. As for collected material, an advantage would be method selection is irrelevant, all material would be transported back to a biological lab, and therefore a full complement of toxicological assessment would be available. However, with transport there would be particle aging, therefore responses would no longer be relevant to a work place exposure situation. Furthermore, what is taken from the collected material? Is it best to filter out aggregates and agglomerates, should attempts be made to eradicate biological and inorganic contaminants? The primary goal is to investigate the health implications of nanomaterials, but should it be only nanomaterials? Once in any environment a NM is unlikely to be found as a lone entity, it would couple with biological components such as allergens, or form aggregates with other NM or other inorganic contaminants from the production process, so what should be tested? Surely the most relevant would be to investigate the whole material? This would give NM in their true form, couples and all. However, without filtration the sample cannot be confirmed as a respirable material! To handle the question of bystander product contamination the impact of contaminant on a NM has to be assessed, but also the impact of a NM on a contaminant. For example, the binding of an allergen to a NP may aid its transport through a barrier through allergen protease activity, or adversely, the binding of a NP to an allergen may subdue the allergen activity by hiding the allergens active sites, these interactions all need to be considered before the relationship of NM within an environment can be fully understood.
The final pitfall is who is going to allow these measurements to take place?! It is important to say that it is expected that most facilities producing NM are likely to be safe, and exposure levels to be low. If a company is confident in their safety precautions they may grant access, but then how much exposure is really going to be found? Enough to assess the stringently developed test methods? Probably not. If a company is not confident in the safety of their working environment, yes maybe there would be suitable levels of particle exposure to test, but why would they grant assess to a group of scientists whose primary goal is to show how unsafe their working environment is! It is clear that these are hurdles that must be overcome, even if access is granted to cooperative facilities. One option to be considered is the staging of controlled accidents to highlight workplace safety, but also that of the general public with controlled transport accidents. The relevance and importance of such situations should be on the conscience of all produces and users of nanomaterials, even if their factory is safe, as accidents do happen. Data from these controlled accidents, in conjunction with mathematical models, can be used to answer the earlier questions regarding transport vehicles spilling their cargo, the safety of storage containers, and who and what is found within the exposure zone; ultimately this would help to determine safe procedures of NM transport, storage and utilisation.
There clearly are many obstacles to be found within the industrial case study section of the NanoVALID project, but work is on-going to overcome these problems, to provide solutions for this exciting and important work, which may provide vital answers for many groups working within nanotechnology.