Development of International Standard on Nano Aerosol Generation for Inhalation Toxicology Study Toxicology: Open Access

Development ISO TR 19601 : Aerosol generation for NOAA (nano-objects and their aggregates and agglomerates) air exposure studies is completed recently. The technical report (TR) reviews methods for generating aerosols of NOAA for in vivo and in vitro inhalation studies. The goals of this technical report is to aid in selecting appropriates NOAA aerosol generator to perform a planned toxicology design. The TR describes how to approach air exposure study design after considering workplace exposure scenario with providing a flow chart to select a proper NOAA generator for aimed study. The TR presents variety of NOAA generator currently used, and describes the principles of operation, advantage at limitation of the NOAA generators. This TR will assist investigators on NOAA inhalation toxicity testing how to design inhalation exposure study with selection of proper generators. This mini-review summarizes contents of the technical report and provides the current status of science in NOAA aerosol generation.


Introduction
Inhalation is a main route of exposure to aerosolized nanomaterials. New term, nano-objects and their aggregates and agglomerates (NOAA), is frequently used instead of nanomaterials. The NOAA include nano-objects with one, two, are three dimensions in the nanoscale from approximately 1-100 nm which might be spheres, fibers, tubes and others as primary structures. NOAA consists of individual primary structures in the nanoscale and larger than 100 nm with aggregated or agglomerated structure [1]. The toxicity of nanomaterials has been frequently tested based on the OECD test guidelines (TG). Currently acute, subacute and subchronic inhalation test guidelines are under revision. OECD TG 403 [2], is an acute inhalation toxicity test guidelines to obtain LC50. OECD TG 436 [3], is a new acute toxic class method. OECD TG 412 [4], is a repeated dose inhalation toxicity TG for 14-28 day study. OECD TG 413 [5], is a subchronic inhalation toxicity TG. Recently these test guidelines are being revised to accommodate traditional chemical exposure as well as nanomaterial exposure, because existing inhalation TGs are not sufficient to satisfy nanomaterial inhalation toxicity testing. Along with OECD test guideline revision activities, ISO TC 229 (International Organization for standardization technical committee 229-Nanotechnologies) initiated a standard for aerosol generation for NOAA from 2014. This TR complements the activities of the Organization for Economic Cooperation and Development (OECD) working party on manufactured nanomaterials (WPMN) and relevant documents. This TR assists scientists to choose appropriate aerosol generator for their target NOAAs to be tested. This standard ISO TR (technical report) 19601 provides a status of science in producing aerosols of NOAA for inhalation study. Appropriate generation of NOAA aerosols determines a successful inhalation toxicity tests which cost a lot of resources and time.
The TR deals with three critical aspects to consider when designing and conducting nanomaterial inhalation toxicity study : 1) uniform and reproducible nano-objects generation that is relevant to realistic exposures 2) thorough characterization of nanomaterials throughout the duration of testing including starting and generated materials and 3) use of occupational exposure limits (OEL) and reference concentrations (RfC) for dosimetry.

Scope of Standard
The technical report reviews methods for producing aerosols of NOAA for in vivo and in vitro air exposure studies. The purpose of the document is to aid in selecting an appropriate aerosol generator to fulfil a proposed toxicology study design. The document describes characteristics of aerosol generation methods, including their advantages and limitations. This TR does not provide guidance for aerosol generation of specific nano-objects.

Inhalation Study Considerations
In designing an inhalation study for NOAA, an actual workplace exposure scenario should be considered, because the health risk of workers is evaluated by inhalation toxicity study. Appropriate NOAA aerosol generation should reflect actual workplace NOAA exposure and emissions in terms of mass or number concentration, particle size, shape and size distribution, frequency and duration of exposure, and handling and manufacturing conditions. Various methods of NOAA from powder form and suspension in liquid media to solid state materials could be used to generate NOAA aerosols. The NOAA aerosol generation should be in line with existing inhalation testing guidelines such as OECD TG 403, 436, 412, 413 and guidance document (GD) 39 (OECD, 2009) or relevant national or international guidelines. Newly revised OECD TGs for nanomaterials describe that MMAD (mass median aerodynamic diameter) is up to 2 micrometer with a geometric standard deviation (GSD) up to 3. In addition, this TR recommends to consider GHS (Globally harmonized system of classification and labelling of chemicals) categorization when an inhalation study might be used for hazard evaluation, classification and labelling.

Considerations in Selection of Proper Generators
When conducting guideline based study, the standardized testing guidelines such as OECD, EPA OPPTS or EU. The study should be conducted following guidelines including number of animals, duration of exposure, observation period, and test material characterization. Studies driven by research hypothesis are more flexible than test guideline based studies. The basic scheme of study consideration is described in the Table 1. The physicochemical characterization of the pristine or manufactured nanomaterial is important before generation of a NOAA aerosol. Because nanomateial are manufactured by various synthetic procedures that impart those unique properties designed for specific applications, the nanomaterial could have a complex structure including impurities and different surface properties. The physicochemical properties nanomaterial influence toxicity of nanomaterials. Useful physicochemical properties nanomaterial include, but not limited to particle size, size distribution, shape, aggregation/agglomeration, surface characteristics, crystalline structure, dustiness, composition and purity. NOAA exposure information on use or handling and manufacturing in terms of particle mass, concentration, number, size, dispersion or shape is very important in designing the inhalation study. Particle shape and concentration similar to workplace exposure should be determined for NOAA inhalation study. Exposure characteristics including duration and frequency of exposure, worker activities, NOAA manufacturing handling and release or emission scenarios would be very useful in designing an inhalation study. Two types of inhalation exposure chambers, whole-body and nose-only are widely used. Nose-only exposure is a principle method of exposure recommended in the OECD TGs, reduces skin and oral exposure potential and consumes less quantity of test nanomaterials, while whole-body is more relevant to human exposure and causes less pain. NOAA particle should be characterized by real-time and off-line monitoring devices. Real-time monitoring of particle size and number including DMAS (differential mobility analyzing system) and ELPI (electrical low pressure impactor) will give particle size distribution and particle number concentration in real-time. Off-line filter sampling can be used to determine mass concentration of NOAA. In addition, off-line filter or EM grid sampling can be prepared for transmission electron microscope (TEM) observation for size and shape of NOAA and analyzed for composition by EDX (energy dispersive X-ray analyzer). The filter sampling can be further analyzed for chemical composition. The stability of NOAA aerosol concentration in inhalation chamber during exposure period should be monitored regularly according to test guidelines or test protocols. OECD TG recommends to concentration deviates within 20% during exposure period.
Step Considerations

NOAA Aerosol Generators
NOAA aerosol generators have several modes of generation: dry dissemination, wet dissemination, phase change, chemical reaction and liquid phase filtration/dispersion. The generation techniques, principle of operation, advantage and disadvantages are summarized in the Table 2.   Table 4).

Experimental Integration
The NOAA aerosol generator needs to be integrated exposure method with NOAA aerosol concentration, particle properties, electrostatic charge, flow rate, gas concentrations, temperature and relative humidity. The gas stream from the aerosol generator needs to be conditioned before and monitored before introduction to the exposure system. The objective of an NOAA air exposure study is to establish a quantitative relationship between toxicological result and NOAA exposure in relation to nanomaterial characteristics, precise characterization of the NOAA is essential for an inhalation exposure study. Nanoparticle and nano-object composition number and mass concentrations, median and mean size and size distribution, surface area, electrical charge, surface properties, hygroscopicity and shape are important parameters for dosimetry [6].

Systems
During preparation of the NOAA aerosol generation system and exposure chamber, aerosol particle composition, size distribution, and purity should be measured. Stability of NOAA concentration in inhalation chamber should be ensured over exposure time period planned. Inhalation chamber and supporting equipment should be prepared in accordance with relevant test guidelines. NOAA aerosol can be deposited on chamber walls by Brownian diffusion and particle size can change due to aggregation/agglomeration. This deposition process depends on the particle size, electrostatic charge, particle number concentration and residence time. To reduce deposition losses, conductive tubing of the minimum length practical to use with the tubing diameter is selected to interface with instrumentation. All the measurement equipment should be calibrated. Recently in vitro air exposure study has been developed to reduce substantive time, cost, and animal numbers to substitute traditional in vivo study. To be predictive of human effects, in vitro air exposure study should include certain parameters in the assay design, 1) the choice of relevant cell types in a physiologically relevant configuration, 2) characterization of the test-material throughout the assay, including life cycle transformations, 3) the choice of realistic test-material concentration and form relevant to real exposures, 4) the use of context-specific dispersants and 5) the use of appropriate exposure route and duration. To compare and assess inhalation toxicity of NOAA, an ALI (air-liquid interface) cell exposure system (rather than submerged cell exposure systems) is preferred as it is more closely resembles in vivo conditions in the lungs and allows for physiologically relevant delivery of aerosolized nanoparticles to the cells [7].

Conclusions
Nanotechnology is developing rapidly and expected to affect every aspect of global industry and society. International standardization on nanotechnologies will contribute to improving quality of life, public health all environment, most of all, improving economic development. Currently, many new manufactured nanomaterials coming to market and workplace raise concerns on occupational safety and health. Inhalation is considered to be the primary route by NOAA entering the bodies of workers. Inhalation toxicity testing is a primary test in evaluating hazards of NOAA. To conduct appropriate inhalation toxicity testing, it is important to design or choose appropriate NOAA aerosol generator. This review presents NOAA aerosol generators described in the ISO TR 19601 [8]. The standard providing the status of NOAA aerosol generators, and further discuss the principles of generation the advantages and limitations of the respective NOAA generators.