Food safety considerations and research priorities for the cultured meat and seafood industry.

Cell-cultured meat and seafood offer a sustainable opportunity to meet the world's increasing demand for protein in a climate-changed world. A responsible, data-driven approach to assess and demonstrate safety of cell-cultured meat and seafood can support consumer acceptance and help fully realize the potential of these products. As an initial step toward a thorough demonstration of safety, this review identifies hazards that could be introduced during manufacturing, evaluates applicability of existing safety assessment approaches, and highlights research priorities that could support safe commercialization. Input was gathered from members of the cultured meat and seafood industry, researchers, regulators, and food safety experts. A series of workshops were held with 87 industry representatives and researchers to create a modular manufacturing process diagram, which served as a framework to identify potential chemical and biological hazards along the steps of the manufacturing process that could affect the safety of a final food product. Interviews and feedback on draft documents validated the process diagram and supported hazard identification and evaluation of applicable safety methods. Most hazards are not expected to be novel; therefore, safety assessment methods from a range of fields, such as conventional and novel foods, foods produced from biotechnology, pharmaceuticals, and so forth, are likely to be applicable. However, additional assessment of novel inputs or products with significant differences from existing foods may be necessary. Further research on the safety of the inputs and associated residues, potential for contamination, and development of standardized safety assessment approaches (particularly animal-free methods) is recommended.

Current characterization methods for cellulose nanomaterials.

A new family of materials comprised of cellulose, cellulose nanomaterials (CNMs), having properties and functionalities distinct from molecular cellulose and wood pulp, is being developed for applications that were once thought impossible for cellulosic materials. Commercialization, paralleled by research in this field, is fueled by the unique combination of characteristics, such as high on-axis stiffness, sustainability, scalability, and mechanical reinforcement of a wide variety of materials, leading to their utility across a broad spectrum of high-performance material applications. However, with this exponential growth in interest/activity, the development of measurement protocols necessary for consistent, reliable and accurate materials characterization has been outpaced. These protocols, developed in the broader research community, are critical for the advancement in understanding, process optimization, and utilization of CNMs in materials development. This review establishes detailed best practices, methods and techniques for characterizing CNM particle morphology, surface chemistry, surface charge, purity, crystallinity, rheological properties, mechanical properties, and toxicity for two distinct forms of CNMs: cellulose nanocrystals and cellulose nanofibrils.

Advancing Risk Analysis for Nanoscale Materials: Report from an International Workshop on the Role of Alternative Testing Strategies for Advancement.

The Society for Risk Analysis (SRA) has a history of bringing thought leadership to topics of emerging risk. In September 2014, the SRA Emerging Nanoscale Materials Specialty Group convened an international workshop to examine the use of alternative testing strategies (ATS) for manufactured nanomaterials (NM) from a risk analysis perspective. Experts in NM environmental health and safety, human health, ecotoxicology, regulatory compliance, risk analysis, and ATS evaluated and discussed the state of the science for in vitro and other alternatives to traditional toxicology testing for NM. Based on this review, experts recommended immediate and near-term actions that would advance ATS use in NM risk assessment. Three focal areas-human health, ecological health, and exposure considerations-shaped deliberations about information needs, priorities, and the next steps required to increase confidence in and use of ATS in NM risk assessment. The deliberations revealed that ATS are now being used for screening, and that, in the near term, ATS could be developed for use in read-across or categorization decision making within certain regulatory frameworks. Participants recognized that leadership is required from within the scientific community to address basic challenges, including standardizing materials, protocols, techniques and reporting, and designing experiments relevant to real-world conditions, as well as coordination and sharing of large-scale collaborations and data. Experts agreed that it will be critical to include experimental parameters that can support the development of adverse outcome pathways. Numerous other insightful ideas for investment in ATS emerged throughout the discussions and are further highlighted in this article.

Evaluating the toxicity of hydroxyapatite nanoparticles in catfish cells and zebrafish embryos.

The toxicity of needle-(nHA-ND) and rod-shaped (nHA-RD) hydroxyapatite (HA) nanoparticles is evaluated in vitro on catfish B-cells (3B11) and catfish T-cells (28s.3) and in vivo on zebrafish embryos to determine if biological effects are similar to the effects seen in mammalian in vitro systems. Neither nHA-ND nor nHA-RD affect cell viability at concentrations of 10 to 300 µg mL(-1) . However, 30 µg mL(-1) needle-shaped nHA lower metabolic activity of the cells. Axial deformations are seen in zebrafish exposed to 300 µg mL(-1) needle shaped nHA after 120 h. For the first time, nHA is reported to cause zebrafish hatching delay. The lowest concentration (3 µg mL(-1) ) of both types of nHA cause the highest hatching inhibition and needle-shaped nHA exposed zebrafish exhibit the lowest hatch at 72 h post fertilization.

Cultured Meat Safety Research Priorities: Regulatory and Governmental Perspectives.

As with every new technology, safety demonstration is a critical component of bringing products to market and gaining public acceptance for cultured meat and seafood. This manuscript develops research priorities from the findings of a series of interviews and workshops with governmental scientists and regulators from food safety agencies in fifteen jurisdictions globally. The interviews and workshops aimed to identify the key safety questions and priority areas of research. Participants raised questions about which aspects of cultured meat and seafood production are novel, and the implications of the paucity of public information on the topic. Novel parameters and targets may require the development of new analytical methods or adaptation and validation of existing ones, including for a diversity of product types and processes. Participants emphasized that data sharing of these efforts would be valuable, similar to those already developed and used in the food and pharmaceutical fields. Contributions to such databases from the private and public sectors would speed general understanding as well as efforts to make evaluations more efficient. In turn, these resources, combined with transparent risk assessment, will be critical elements of building consumer trust in cultured meat and seafood products.

Silver nanoparticles inhibit sodium uptake in juvenile rainbow trout (Oncorhynchus mykiss).

The silver ion (Ag(+)) is well documented to be a potent inhibitor of sodium (Na(+)) transport in fish. However, it has not been determined whether silver nanoparticles (Ag NPs) elicit this same effect and, if so, if the NP itself and/or the dissociation of ionic Ag(+) causes this effect. Citrate-capped Ag NPs were dialyzed in water to determine the dissolution rate of ionic Ag(+) from the NPs and the maximum concentration of free Ag(+) released from the NPs was used as a paired Ag(+) control to distinguish NP effects from ionic metal effects. The maximum concentration of ionic Ag(+) released from these NPs over 48 h was 0.02 µg l(-1). Juvenile rainbow trout were exposed to 1.0 mg l(-1) citrate-capped Ag NPs and dialyzed citrate-capped Ag NPs or 10 µg l(-1) and 0.02 µg l(-1) ionic Ag(+) (as AgNO(3)) as controls. Both nondialyzed and dialyzed Ag NPs and 10 µg l(-1) ionic Ag(+) significantly inhibited unidirectional Na(+) influx by over 50% but had no effect on unidirectional Na(+) efflux. Na(+),K(+)-ATPase was significantly inhibited by the Ag NPs with no discernible effect on carbonic anhydrase activity. This study is the first to show that sodium regulation is disrupted by the presence of citrate-capped Ag NPs, and the results suggest that there are nanospecific effects.

Fluorescently Labeled Cellulose Nanofibers for Environmental Health and Safety Studies.

An optimal methodology for locating and tracking cellulose nanofibers (CNFs) in vitro and in vivo is crucial to evaluate the environmental health and safety properties of these nanomaterials. Here, we report the use of a new boron-dipyrromethene (BODIPY) reactive fluorescent probe, meso-DichlorotriazineEthyl BODIPY (mDTEB), tailor-made for labeling CNFs used in simulated or in vivo ingestion exposure studies. Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) was used to confirm covalent attachment and purity of mDTEB-labeled CNFs. The photoluminescence properties of mDTEB-labeled CNFs, characterized using fluorescence spectroscopy, include excellent stability over a wide pH range (pH2 to pH10) and high quantum yield, which provides detection at low (µM) concentrations. FLIM analysis also showed that lignin-like impurities present on the CNF reduce the fluorescence of the mDTEB-labeled CNF, via quenching. Therefore, the chemical composition and the methods of CNF production affect subsequent studies. An in vitro triculture, small intestinal, epithelial model was used to assess the toxicity of ingested mDTEB-labeled CNFs. Zebrafish (Danio rerio) were used to assess in vivo environmental toxicity studies. No cytotoxicity was observed for CNFs, or mDTEB-labeled CNFs, either in the triculture cells or in the zebrafish embryos.

Physical, chemical, and toxicological characterization of sulfated cellulose nanocrystals for food-related applications using in vivo and in vitro strategies.

Cellulose nanocrystals (CNCs) are a next-generation cellulose product with many unique properties including applications in the food industry as a food additive, food coating, and in food-contact packaging material. While CNC is anticipated to be safe due to its similarity to the many forms of cellulose currently used as food additives, special consideration is given to it as it is the first manufactured form of cellulose that is nanoscale in both length and width. A proactive approach to safety has been adopted by manufacturers to demonstrate CNC safety toward responsible commercialization. As part of the safety demonstration, in vivo and in vitro testing strategies were commissioned side-by-side with conventional cellulose, which has been safely used in food for decades. Testing included a 90-day rodent feeding study as well as additional physical, chemical, and biological studies in vitro that follow European Food Safety Authority (EFSA) guidance to demonstrate the safe use of novel food ingredients. The strategy includes assessment of neat materials side-by-side with simulated digestion, mimicking conditions that occur along the gastrointestinal tract as well as intracellularly. An intestinal co-culture model examined any potential toxicological effects from exposure to either pristine or digested forms of CNC including cytotoxicity, metabolic activity, membrane permeability, oxidative stress, and proinflammatory responses. None of the studies demonstrated any toxicity via oral or simulated oral exposure. These studies demonstrate that CNC produced by InnoTech Alberta is similarly safe by ingestion as conventional cellulose with a no-observed-adverse-effect level of 2085.3 (males) and 2682.8 (females) mg/kg/day.

A 90-day dietary study with fibrillated cellulose in Sprague-Dawley rats.

Novel forms of fibrillated cellulose offer improved attributes for use in foods. Conventional cellulose and many of its derivatives are already widely used as food additives and are authorized as safe for use in foods in many countries. However, novel forms have not yet been thoroughly investigated using standardized testing methods. This study assesses the 90-day dietary toxicity of fibrillated cellulose, as compared to a conventional cellulose, Solka Floc. Sprague Dawley rats were fed 2 %, 3 %, or 4 % fibrillated cellulose for 90 consecutive days, and parallel Solka Floc groups were used as controls. Survival, clinical observations, body weight, food consumption, ophthalmologic evaluations, hematology, serum chemistry, urinalysis, post-mortem anatomic pathology, and histopathology were monitored and performed. No adverse observations were noted in relation to the administration of fibrillated cellulose. Under the conditions of this study and based on the toxicological endpoints evaluated, the no-observed-adverse-effect level (NOAEL) for fibrillated cellulose was 2194.2 mg/kg/day (males) and 2666.6 mg/kg/day (females), corresponding to the highest dose tested (4 %) for male and female Sprague Dawley rats. These results demonstrate that fibrillated cellulose behaves similarly to conventional cellulose and raises no safety concerns when used as a food ingredient at these concentrations.

Physical, chemical, and toxicological characterization of fibrillated forms of cellulose using an in vitro gastrointestinal digestion and co-culture model.

Fibrillated cellulose is a next-generation material in development for a variety of applications, including use in food and food-contact materials. An alternative testing strategy including simulated digestion was developed to compare the physical, chemical, and biological characteristics of seven different types of fibrillated cellulose, following European Food Safety Authority guidance. Fibrillated forms were compared to a conventional form of cellulose which has been used in food for over 85 years and has Generally Recognized as safe regulatory status in the USA. The physical and chemical characterization of fibrillated celluloses demonstrate that these materials are similar physically and chemically, which composed of the same fundamental molecular structure and exhibit similar morphology, size, size distribution, surface charge, and low levels of impurities. Simulated gastrointestinal and lysosomal digestions demonstrate that these physical and chemical similarities remain following exposure to conditions that mimic the gastrointestinal tract or intracellular lysosomes. A toxicological investigation with an advanced intestinal co-culture model found that exposure to each of the fibrillated and conventional forms of cellulose, in either the pristine or digested form at 0.4% by weight, showed no adverse toxicological effects including cytotoxicity, barrier integrity, oxidative stress, or inflammation. The results demonstrate the physical, chemical, and biological similarities of these materials and provide substantive evidence to support their grouping and ability to read-across data as part of a food safety demonstration.

Risk Analysis of Cellulose Nanomaterials by Inhalation: Current State of Science.

Cellulose nanomaterials (CNs) are emerging advanced materials with many unique properties and growing commercial significance. A life-cycle risk assessment and environmental health and safety roadmap identified potential risks from inhalation of powdered CNs in the workplace as a key gap in our understanding of safety and recommended addressing this data gap to advance the safe and successful commercialization of these materials. Here, we (i) summarize the currently available published literature for its contribution to our current understanding of CN inhalation hazard and (ii) evaluate the quality of the studies for risk assessment purposes using published study evaluation tools for nanomaterials to assess the weight of evidence provided. Our analysis found that the quality of the available studies is generally inadequate for risk assessment purposes but is improving over time. There have been some advances in knowledge about the effects of short-term inhalation exposures of CN. The most recent in vivo studies suggest that short-term exposure to CNs results in transient inflammation, similarly to other poorly soluble, low toxicity dusts such as conventional cellulose, but is markedly different from fibers with known toxicity such as certain types of multiwalled carbon nanotubes or asbestos. However, several data gaps remain, and there is still a lack of understanding of the effects from long-term, low-dose exposures that represent realistic workplace conditions, essential for a quantitative assessment of potential health risk. Therefore, taking precautions when handling dry forms of CNs to avoid dust inhalation exposure is warranted.

Widespread nanoparticle-assay interference: implications for nanotoxicity testing.

The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. We tested the potential for: 1) nanoparticle intrinsic fluorescence/absorbance, 2) interactions between nanoparticles and assay components, and 3) the effects of adding both nanoparticles and analytes to an assay, to interfere with the accurate assessment of toxicity. Silicon, cadmium selenide, titanium dioxide, and helical rosette nanotubes each affected at least one of the six assays tested, resulting in either substantial over- or under-estimations of toxicity. Simulation of realistic assay conditions revealed that interference could not be predicted solely by interactions between nanoparticles and assay components. Moreover, the nature and degree of interference cannot be predicted solely based on our current understanding of nanomaterial behaviour. A literature survey indicated that ca. 95% of papers from 2010 using biochemical techniques to assess nanotoxicity did not account for potential interference of nanoparticles, and this number had not substantially improved in 2012. We provide guidance on avoiding and/or controlling for such interference to improve the accuracy of nanotoxicity assessments.