9: Toxicology - Geosciences
Chapter 9 Forensic Toxicology - PowerPoint PPT Presentation
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Cannabis Dependence and Tolerance
Cannabinoids appear to affect the same reward systems as alcohol, cocaine and opioids (34).Evidence for cannabis dependence is now available from epidemiological studies (6, 8) of long-term users (58, 59), clinical populations (75, 77) and controlled experiments on withdrawal and tolerance (35, 36, 37, 38). Tolerance to cannabis can occur in relation to mood, psychomotor performance, sleep, arterial pressure, body temperature, and antiemetic properties. The critical elements of cannabis dependence include preoccupation with its use, compulsion to use and relapse or recurrent use of the substance (39). Over 50% of cannabis users appear to have ‘impaired control’ over their use (40).Symptoms such as irritability, anxiety, craving and disrupted sleep have been reported in 61-96% of cannabis users during abstinence (36, 41, 42, 43).
Psychiatric Conditions Associated with Cannabis Abuse
In addition to producing dependence, cannabis use is associated with a wide range of psychiatric disorders (44).While there is a clear relationship between the use of cannabis and psychosis, different hypotheses for the same have been propounded. One such, which describes psychosis occurring exclusively with cannabis use has limited evidence. There is strong evidence that cannabis use may precipitate schizophrenia or exacerbate its symptoms. There is also reasonable evidence that cannabis use exacerbates the symptoms of psychosis (37).
Heavy cannabis(30-50mg oral and 8-30 mg smoked) use can specifically cause a mania-like psychosis and more generally act as a precipitant for manic relapse in bipolar patients (37, 44, 45). It is possible that cannabis exposure is a contributing factor that interacts with other known and unknown (genetic and environmental) factors culminating in psychiatric illness (46). It is noticed that in many developed countries, persons with severe mental disorders are more likely to use, abuse, and become dependent on psychoactive substances especially cannabis as compared to the general population (47, 48).The same phenomenon has not been established so far in India.
Toxicology Testing and Evaluation
3.04.7.2 Repeated Dose
As with pharmaceuticals, repeat-dose toxicity studies are expected to be of similar route of administration and duration to the proposed clinical trial ( ICH Harmonised Tripartite 2008 ). In addition to safety pharmacology parameters discussed above, studies should include body weight measurements, food consumption measurements, toxicokinetics, ophthalmologic examinations, measurement of clinical pathology parameters, measurement of urinalysis parameters, PD measurements, and immunogenicity assessments, and may include specific immune parameter measurements based upon the product or product class. Studies should also include organ weights and macroscopic and microscopic evaluations. Other end points may also be included on a case-by-case basis based on the specific product attributes and the specific questions that are being asked (e.g., inclusion of specific proliferation markers to better assess potential preneoplastic changes in chronic toxicity studies).
Additional animals are included in control and high dose or all dose groups to assess recovery/reversibility rather than delayed toxicity. The duration of the treatment-free period can vary based upon duration of PK/PD effects and the extent and measurement of the immune response. For example, there should be sufficient time so that the level of the biopharmaceutical is below the level that would interfere with detection of antibodies to the product.
In general, 6-month duration for chronic dose studies is supported by a retrospective evaluation of approved biopharmaceuticals ( Clarke et al. 2008 ) ( Table 3 ). As noted in ICH S6, specific considerations may require a longer-duration study in some cases and shorter duration may also be acceptable in some cases. Formation of neutralizing antibodies, however, can limit utility of longer-term dosing.
Table 3 . Case-by-case approach to assessment of chronic toxicity
|Target/activity||Biopharmaceutical (generic name)||Molecule type||Indication(s) (approved in the United States)||Duration of chronic toxicity study(ies)|
|G-CSF||Filgrastim||Growth factor||Neutropenia||12 months (rat)|
|EPO||Epoetin alpha||Growth factor||Anemia||12 months (rat)|
|DNA||Dornase-α||Enzyme||Cystic fibrosis||6 months (rat and NHP)|
|PDGF||rhPDGF||Growth factor||Lower extremity diabetic neuropathic ulcers||3 months|
|Laronidase||Recombinant alpha- l -Iduronidase||Enzyme||Mucopolysaccharidosis I (MPS I)||6 months|
|Agalsidase||Agalsidase beta||Enzyme||Fabry disease||6 months (rat and NHP)|
|Alglucosidase||Alglucosidase alpha||Enzyme||Pompe disease||6 months (NHP)|
|Interferon||rhIFN-β1a||Interferon||Relapsing/remitting MS||Not done|
|IL-1 receptor||Anakinra||IL-1 receptor antagonist||RA||6 months (rats)|
|CTLA-4||Abatacept||Fusion protein (hinge CH2 and CH3 domains of IgG1)||RA||12 months (NHP)|
|CD2||Alefacept||Fc fusion||Psoriasis||12 months (NHP)|
|TNF-α||Entanerept||FC fusion (soluble receptor)||RA, AS, psoriatic arthritis, psoriasis||6 months (NHP)|
|PEGylated filgrastim||Pegfilgrastim||PEGylated growth factor||Neutropenia||6 months (rat)|
|PEGylated rIFN-α2b||Interferon||PEGylated interferon||Chronic hepatitis C||Not done due to immunogenicity|
|PEGylated rIFN-α2α||Interferon||PEGylated interferon||Chronic hepatitis C||Not done due to immunogenicity|
|rhEPO||Darbepoetin||Long-acting EPO||Anemia||6 months (NHP)|
|TNF-α||Entanerept||FC fusion (soluble receptor)||RA, AS, psoriatic arthritis, psoriasis||6 months (NHP)|
|IL-1||Rilonacept||IL-1 trap||Cryopyrin-associated periodic syndromes and Muckle–Wells syndrome||6 months (NHP)|
|α 4 Integrin||Natalizumab||Humanized mAb||MS, Crohn’s disease||6 months (NHP)|
|C5||Eculizumab||Humanized mAb||Paroxysomal nocturnal hemoglobinuria||6 months (mouse) (surrogate)|
|CD11a||Efalizumab||Humanized mAb||Psoriasis||6 months (NHP)|
|CD20||Rituximab||Chimeric mAb||NHL, RA|
|EGFR||Cetuximab||Chimeric mAb||Cancer||9 months (NHP)|
|Panitumumab||Fully human mAb||Cancer|
|HER-2||Trastuzumab||Humanized mAb||Cancer||6 months (NHP)|
|IgE||Omalizumab||Humanized mAb||Asthma||6 months (NHP)|
|TNF-α||Adalimumab||Fully human mAb||RA, AS, psoriatic arthritis, psoriasis, Crohn’s disease||9 months (NHP)|
|Infliximab||Chimeric mAb||RA, Crohn’s disease||6 months (rat) (surrogate)|
|Certolizumab pegol||Fab PEGylated||Crohn’s disease||6 months (NHP)|
|VEGF||Bevacizumab||Humanized mAb||Cancer||6 months (NHP)|
If two relevant species exist, then short-term repeat-dose toxicity studies are recommended. However, if the target organ profile is similar across species and/or similar class effects are observed and the dose selected in the clinical trials appears acceptable, then chronic toxicity studies in a single species may be justifiable to reduce animal use.
Toxicology Lab Owner and Marketer Sentenced for Payment of Kickbacks Doctor Pleads Guilty to Receipt of Kickbacks
LEXINGTON, Ky. – Several defendants were recently convicted or sentenced for their roles in a conspiracy to violate the federal Anti-Kickback Statute. On December 4 and December 7, 2020, Uday Shah, 66, of Houston, Texas, and Timothy Andrews, 57, of Deer Park, Texas, were sentenced by Chief United States District Judge Danny C. Reeves to 24 and 15 months’ imprisonment, respectively, for their roles in a conspiracy to pay kickbacks to a physician, Dr. Ghyasuddin Syed, in exchange for Dr. Syed’s referral of urine drug testing to laboratories operated by Shah. On Wednesday, December 2, 2020, Dr. Syed pleaded guilty to soliciting and accepting kickbacks as part of the same scheme.
According to their plea agreements, Shah owned and operated several toxicology laboratories, including Pinnacle Laboratory in Lexington. Andrews worked as a marketer on behalf of Shah’s labs. Shah and Andrews admitted that between November 2014 and August 2017, they paid $475,992 in kickbacks to Dr. Syed, a Houston-area physician, and Dr. Syed’s wife, Shazana Begum. The kickbacks were often disguised as lease payments for office space owned by Dr. Syed and Begum. In his plea agreement, Dr. Syed disputed the exact amount of kickbacks, but acknowledged receiving them from Shah and Andrews, and referring urine drug testing for his patients to Shah’s labs in exchange. All of the defendants agreed that Pinnacle and Shah’s other labs billed the Medicare program for the urine drug testing tainted by these kickbacks, and that Medicare paid the labs $325,739 to which they were not entitled.
Andrews pleaded guilty in June 2019, and Shah pleaded guilty in October 2019. In addition to their respective terms of incarceration, Shah and Andrews were ordered to pay $325,739 to the Medicare program in restitution, jointly and severally. Under federal law, Giles and Wallace must serve 85 percent of their prison sentences. Upon their release, they will be under the supervision of the U.S. Probation Office for three years.
Dr. Syed is scheduled to be sentenced on March 12, 2021, in Lexington. He faces up to five years in prison for the conspiracy to violate the Anti-Kickback Statute, and a maximum fine of $250,000. However, any sentence will be imposed by the Court, after its consideration of the U.S. Sentencing Guidelines and the applicable federal sentencing statutes.
Dr. Syed’s wife, Shazana Begum, has entered into a pretrial diversion agreement wherein she admitted her role in the offense, and agreed to be under the supervision of the United States Probation Office for 12 months, to pay restitution of $325,739 along with Shah and Andrews, and to perform community service.
Preclinical Development of Ipilimumab and Nivolumab Combination Immunotherapy: Mouse Tumor Models, In Vitro Functional Studies, and Cynomolgus Macaque Toxicology
The monoclonal antibodies ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) have shown remarkable antitumor activity in an increasing number of cancers. When combined, ipilimumab and nivolumab have demonstrated superior activity in patients with metastatic melanoma (CHECKMATE-067). Here we describe the preclinical development strategy that predicted these clinical results. Synergistic antitumor activity in mouse MC38 and CT26 colorectal tumor models was observed with concurrent, but not sequential CTLA-4 and PD-1 blockade. Significant antitumor activity was maintained using a fixed dose of anti-CTLA-4 antibody with decreasing doses of anti-PD-1 antibody in the MC38 model. Immunohistochemical and flow cytometric analyses confirmed that CD3+ T cells accumulated at the tumor margin and infiltrated the tumor mass in response to the combination therapy, resulting in favorable effector and regulatory T-cell ratios, increased pro-inflammatory cytokine secretion, and activation of tumor-specific T cells. Similarly, in vitro studies with combined ipilimumab and nivolumab showed enhanced cytokine secretion in superantigen stimulation of human peripheral blood lymphocytes and in mixed lymphocyte response assays. In a cynomolgus macaque toxicology study, dose-dependent immune-related gastrointestinal inflammation was observed with the combination therapy this response had not been observed in previous single agent cynomolgus studies. Together, these in vitro assays and in vivo models comprise a preclinical strategy for the identification and development of highly effective antitumor combination immunotherapies.
Conflict of interest statement
The studies described in this manuscript were sponsored by Bristol-Myers Squibb. The funder provided support in the form of salaries for all authors (MJS JJE RJJ LL MH KT DY MQ JV CW BC PMC DB AJK) and was involved in study design, data collection and analysis, decision to publish, and preparation of the manuscript. The specific roles of these authors are articulated in the Authors’ Contributions section. In addition, the funder provided support in the form of salaries to individuals listed in the Acknowledgements section (LW MS IC CB).
Fig 1. Antitumor Responses of Anti-CTLA-4 and…
Fig 1. Antitumor Responses of Anti-CTLA-4 and Anti-PD-1 Antibodies in Staged MC38 and CT26 Tumor…
Fig 2. Immunohistochemistry of MC38 Tumors: Detection…
Fig 2. Immunohistochemistry of MC38 Tumors: Detection of CD3 + T Cells and PD-L1 +…
Fig 3. FACS Analysis of Tumor and…
Fig 3. FACS Analysis of Tumor and Spleen T Cell Populations from MC38 Tumor-Bearing Mice.
Fig 4. FACS Analysis of Tumor and…
Fig 4. FACS Analysis of Tumor and Spleen T Cell Populations from CT26 Tumor-Bearing Mice.
Fig 5. CD8 + :Treg TIL Ratios…
Fig 5. CD8 + :Treg TIL Ratios in Treated MC38 and CT26 Tumor-Bearing Mice.
Fig 6. Ipilimumab (Anti-CTLA-4) and Nivolumab (Anti-PD-1)…
Fig 6. Ipilimumab (Anti-CTLA-4) and Nivolumab (Anti-PD-1) Antibodies Potentiate IL-2 Secretion in SEB-Stimulated Human PBMC.
Fig 7. Ipilimumab (Anti-CTLA-4) and Nivolumab (Anti-PD-1)…
Fig 7. Ipilimumab (Anti-CTLA-4) and Nivolumab (Anti-PD-1) Antibodies Potentiate IL-2 Release in an Allogeneic MLR…
How Is It Given?
Most of the time, a sample of your blood or urine will be tested. Blood is drawn from a vein in your arm or you’ll be asked to pee into a cup. The sample will then be tested at a lab.
Sometimes, sweat, a strand of hair, or saliva from your mouth is used instead of blood or urine. In extreme cases, other body fluids are checked. If your stomach is pumped at the hospital, a sample of your stomach contents may be tested.
Before your test, let your doctor know what drugs you’ve taken in the past few days. Make sure to include over-the-counter medicines and supplements. Some of them can show up in your system like other drugs and cause a “false positive” on your test.
Depending on the type of test you have, you should get your results within 24 to 48 hours.
Nanomaterials have at least one primary dimension of less than 100 nanometers, and often have properties different from those of their bulk components that are technologically useful. Because nanotechnology is a recent development, the health and safety effects of exposures to nanomaterials, and what levels of exposure may be acceptable, is not yet fully understood.  Nanoparticles can be divided into combustion-derived nanoparticles (like diesel soot), manufactured nanoparticles like carbon nanotubes and naturally occurring nanoparticles from volcanic eruptions, atmospheric chemistry etc. Typical nanoparticles that have been studied are titanium dioxide, alumina, zinc oxide, carbon black, carbon nanotubes, and buckminsterfullerene.
Nanotoxicology is a sub-specialty of particle toxicology. Nanomaterials appear to have toxicity effects that are unusual and not seen with larger particles, and these smaller particles can pose more of a threat to the human body due to their ability to move with a much higher level of freedom while the body is designed to attack larger particles rather than those of the nanoscale.  For example, even inert elements like gold become highly active at nanometer dimensions. Nanotoxicological studies are intended to determine whether and to what extent these properties may pose a threat to the environment and to human beings.  Nanoparticles have much larger surface area to unit mass ratios which in some cases may lead to greater pro-inflammatory effects in, for example, lung tissue. In addition, some nanoparticles seem to be able to translocate from their site of deposition to distant sites such as the blood and the brain.
Nanoparticles can be inhaled, swallowed, absorbed through skin and deliberately or accidentally injected during medical procedures. They might be accidentally or inadvertently released from materials implanted into living tissue.    One study considers release of airborne engineered nanoparticles at workplaces, and associated worker exposure from various production and handling activities, to be very probable. 
Size is a key factor in determining the potential toxicity of a particle.  However it is not the only important factor. Other properties of nanomaterials that influence toxicity include: chemical composition, shape, surface structure, surface charge, aggregation and solubility,  and the presence or absence of functional groups of other chemicals. The large number of variables influencing toxicity means that it is difficult to generalise about health risks associated with exposure to nanomaterials – each new nanomaterial must be assessed individually and all material properties must be taken into account.
Metal based nanoparticles (NPs) are a prominent class of NPs synthesized for their functions as semiconductors, electroluminescents, and thermoelectric materials.  Biomedically, these antibacterial NPs have been utilized in drug delivery systems to access areas previously inaccessible to conventional medicine. With the recent increase in interest and development of nanotechnology, many studies have been performed to assess whether the unique characteristics of these NPs, namely their large surface area to volume ratio, might negatively impact the environment upon which they were introduced.  Researchers have found that some metal and metal oxide NPs may affect cells inducing DNA breakage and oxidation, mutations, reduced cell viability, warped morphology, induced apoptosis and necrosis, and decreased proliferation.  Moreover, metal nanoparticles may persist in the organisms after administration if not carefully engineered. 
The latest toxicology studies on mice as of 2013 involving exposure to carbon nanotubes (CNT) showed a limited pulmonary inflammatory potential of MWCNT at levels corresponding to the average inhalable elemental carbon concentrations observed in U.S.-based CNT facilities. The study estimated that considerable years of exposure are necessary for significant pathology to occur. 
One review concludes that the evidence gathered since the discovery of fullerenes overwhelmingly points to C60 being non-toxic. As is the case for toxicity profile with any chemical modification of a structural moiety, the authors suggest that individual molecules be assessed individually. 
Other classes of nanomaterials include polymers such as nanocellulose, and dendrimers.
There are many ways that size can affect the toxicity of a nanoparticle. For example, particles of different sizes can deposit in different places in the lungs, and are cleared from the lungs at different rates. Size can also affect the particles' reactivity and the specific mechanism by which they are toxic. 
Dispersion state Edit
Many nanoparticles agglomerate or aggregate when they are placed in environmental or biological fluids. The terms agglomeration and aggregation have distinct definitions according to the standards organizations ISO and ASTM, where agglomeration signifies more loosely bound particles and aggregation signifies very tightly bound or fused particles (typically occurring during synthesis or drying). Nanoparticles frequently agglomerate due to the high ionic strength of environmental and biological fluids, which shields the repulsion due to charges on the nanoparticles. Unfortunately, agglomeration has frequently been ignored in nanotoxicity studies, even though agglomeration would be expected to affect nanotoxicity since it changes the size, surface area, and sedimentation properties of the nanoparticles. In addition, many nanoparticles will agglomerate to some extent in the environment or in the body before they reach their target, so it is desirable to study how toxicity is affected by agglomeration.
The agglomeration/deagglomeration (mechanical stability) potentials of airborne engineered nanoparticle clusters also have significant influences on their size distribution profiles at the end-point of their environmental transport routes. Different aerosolization and deagglomeration systems have been established to test stability of nanoparticle agglomerates.
Surface chemistry and charge Edit
NPs, in their implementation, are covered with coatings and sometimes given positive or negative charges depending upon the intended function. Studies have found that these external factors affect the degree of toxicity of NPs.
Inhalation exposure is the most common route of exposure to airborne particles in the workplace. The deposition of nanoparticles in the respiratory tract is determined by the shape and size of particles or their agglomerates, and they are deposited in the lungs to a greater extent than larger respirable particles. Based on animal studies, nanoparticles may enter the bloodstream from the lungs and translocate to other organs, including the brain.  The inhalation risk is affected by the dustiness of the material, the tendency of particles to become airborne in response to a stimulus. Dust generation is affected by the particle shape, size, bulk density, and inherent electrostatic forces, and whether the nanomaterial is a dry powder or incorporated into a slurry or liquid suspension. 
Animal studies indicate that carbon nanotubes and carbon nanofibers can cause pulmonary effects including inflammation, granulomas, and pulmonary fibrosis, which were of similar or greater potency when compared with other known fibrogenic materials such as silica, asbestos, and ultrafine carbon black. Some studies in cells or animals have shown genotoxic or carcinogenic effects, or systemic cardiovascular effects from pulmonary exposure. Although the extent to which animal data may predict clinically significant lung effects in workers is not known, the toxicity seen in the short-term animal studies indicate a need for protective action for workers exposed to these nanomaterials. As of 2013, further research was needed in long-term animal studies and epidemiologic studies in workers. No reports of actual adverse health effects in workers using or producing these nanomaterials were known as of 2013.  Titanium dioxide (TiO2) dust is considered a lung tumor risk, with ultrafine (nanoscale) particles having an increased mass-based potency relative to fine TiO2, through a secondary genotoxicity mechanism that is not specific to TiO2 but primarily related to particle size and surface area. 
Some studies suggest that nanomaterials could potentially enter the body through intact skin during occupational exposure. Studies have shown that particles smaller than 1 μm in diameter may penetrate into mechanically flexed skin samples, and that nanoparticles with varying physicochemical properties were able to penetrate the intact skin of pigs. Factors such as size, shape, water solubility, and surface coating directly affect a nanoparticle's potential to penetrate the skin. At this time, it is not fully known whether skin penetration of nanoparticles would result in adverse effects in animal models, although topical application of raw SWCNT to nude mice has been shown to cause dermal irritation, and in vitro studies using primary or cultured human skin cells have shown that carbon nanotubes can enter cells and cause release of pro-inflammatory cytokines, oxidative stress, and decreased viability. It remains unclear, however, how these findings may be extrapolated to a potential occupational risk.   In addition, nanoparticles may enter the body through wounds, with particles migrating into the blood and lymph nodes. 
Ingestion can occur from unintentional hand-to-mouth transfer of materials this has been found to happen with traditional materials, and it is scientifically reasonable to assume that it also could happen during handling of nanomaterials. Ingestion may also accompany inhalation exposure because particles that are cleared from the respiratory tract via the mucociliary escalator may be swallowed. 
The extremely small size of nanomaterials also means that they much more readily gain entry into the human body than larger sized particles. How these nanoparticles behave inside the body is still a major question that needs to be resolved. The behavior of nanoparticles is a function of their size, shape and surface reactivity with the surrounding tissue. In principle, a large number of particles could overload the body's phagocytes, cells that ingest and destroy foreign matter, thereby triggering stress reactions that lead to inflammation and weaken the body's defense against other pathogens. In addition to questions about what happens if non-degradable or slowly degradable nanoparticles accumulate in bodily organs, another concern is their potential interaction or interference with biological processes inside the body. Because of their large surface area, nanoparticles will, on exposure to tissue and fluids, immediately adsorb onto their surface some of the macromolecules they encounter. This may, for instance, affect the regulatory mechanisms of enzymes and other proteins.
Nanomaterials are able to cross biological membranes and access cells, tissues and organs that larger-sized particles normally cannot.  Nanomaterials can gain access to the blood stream via inhalation  or ingestion.  Broken skin is an ineffective particle barrier, suggesting that acne, eczema, shaving wounds or severe sunburn may accelerate skin uptake of nanomaterials. Then, once in the blood stream, nanomaterials can be transported around the body and be taken up by organs and tissues, including the brain, heart, liver, kidneys, spleen, bone marrow and nervous system.  Nanomaterials can be toxic to human tissue and cell cultures (resulting in increased oxidative stress, inflammatory cytokine production and cell death) depending on their composition and concentration. 
Oxidative stress Edit
For some types of particles, the smaller they are, the greater their surface area to volume ratio and the higher their chemical reactivity and biological activity. The greater chemical reactivity of nanomaterials can result in increased production of reactive oxygen species (ROS), including free radicals. ROS production has been found in a diverse range of nanomaterials including carbon fullerenes, carbon nanotubes and nanoparticle metal oxides. ROS and free radical production is one of the primary mechanisms of nanoparticle toxicity it may result in oxidative stress, inflammation, and consequent damage to proteins, membranes and DNA. 
A primary marker for the damaging effects of NPs has been cell viability as determined by state and exposed surface area of the cell membrane. Cells exposed to metallic NPs have, in the case of copper oxide, had up to 60% of their cells rendered unviable. When diluted, the positively charged metal ions often experience an electrostatic attraction to the cell membrane of nearby cells, covering the membrane and preventing it from permeating the necessary fuels and wastes.  With less exposed membrane for transportation and communication, the cells are often rendered inactive.
NPs have been found to induce apoptosis in certain cells primarily due to the mitochondrial damage and oxidative stress brought on by the foreign NPs electrostatic reactions. 
Metal and metal oxide NPs such as silver, zinc, copper oxide, uraninite, and cobalt oxide have also been found to cause DNA damage.  The damage done to the DNA will often result in mutated cells and colonies as found with the HPRT gene test.
Characterization of a nanomaterial's physical and chemical properties is important for ensuring the reproducibility of toxicology studies, and is also vital for studying how the properties of nanomaterials determine their biological effects.  The properties of a nanomaterial such as size distribution and agglomeration state can change as a material is prepared and used in toxicology studies, making it important to measure them at different points in the experiment. 
With comparison to more conventional toxicology studies, in nanotoxicology, characterisation of the potential contaminants is challenging. The biological systems are themselves still not completely known at this scale. Visualisation methods such as electron microscopy (SEM and TEM) and atomic force microscopy (AFM) analysis allow visualisation of the nano world. Further nanotoxicology studies will require precise characterisation of the specificities of a given nano-element: size, chemical composition, detailed shape, level of aggregation, combination with other vectors, etc. Above all, these properties would have to be determined not only on the nanocomponent before its introduction in the living environment but also in the (mostly aqueous) biological environment.
There is a need for new methodologies to quickly assess the presence and reactivity of nanoparticles in commercial, environmental, and biological samples since current detection techniques require expensive and complex analytical instrumentation.
Toxicology studies of nanomaterials are a key input into determining occupational exposure limits.
The Royal Society identifies the potential for nanoparticles to penetrate the skin, and recommends that the use of nanoparticles in cosmetics be conditional upon a favorable assessment by the relevant European Commission safety advisory committee.
The Woodrow Wilson Centre's Project on Emerging Technologies conclude that there is insufficient funding for human health and safety research, and as a result there is currently limited understanding of the human health and safety risks associated with nanotechnology. While the US National Nanotechnology Initiative reports that around four percent (about $40 million) is dedicated to risk related research and development, the Woodrow Wilson Centre estimate that only around $11 million is actually directed towards risk related research. They argued in 2007 that it would be necessary to increase funding to a minimum of $50 million in the following two years so as to fill the gaps in knowledge in these areas. 
The potential for workplace exposure was highlighted by the 2004 Royal Society report which recommended a review of existing regulations to assess and control workplace exposure to nanoparticles and nanotubes. The report expressed particular concern for the inhalation of large quantities of nanoparticles by workers involved in the manufacturing process. 
Stakeholders concerned by the lack of a regulatory framework to assess and control risks associated with the release of nanoparticles and nanotubes have drawn parallels with bovine spongiform encephalopathy (‘mad cow's disease'), thalidomide, genetically modified food, nuclear energy, reproductive technologies, biotechnology, and asbestosis. In light of such concerns, the Canadian-based ETC Group have called for a moratorium on nano-related research until comprehensive regulatory frameworks are developed that will ensure workplace safety.