Risk assessment of feed components of botanical origin - Approaches taken in the European Union.

In view of a continuous trend in replacing synthetic feed additives and especially flavouring compounds by botanical preparations, different aspects of the safety evaluations of plants and plant-derived preparations and components in feed are discussed. This includes risk assessment approaches developed by the European Food Safety Authority (EFSA) for phytotoxins regarding unintentional exposure of target animals and of consumers to animal derived food via carry-over from feed. Relevant regulatory frameworks for feed additives and feed contaminants in the European Union are summarised and the essentials of existing guidelines used in the safety evaluation of botanicals and their preparations and components in feed are outlined. The examples presented illustrate how the safety of the botanicals, their preparations and components present in feed is assessed. An outlook on possible future developments in risk assessment by applying new in vitro and in silico methodologies is given.

Oral nicotine pouches with an aftertaste? Part 1: screening and initial toxicological assessment of flavorings and other ingredients.

Nicotine pouches are oral products that deliver nicotine without containing tobacco. Previous studies mainly focused on the determination of known tobacco toxicants, while yet no untargeted analysis has been published on unknown constituents, possibly contributing to toxicity. Furthermore, additives might enhance product attractiveness. We therefore performed an aroma screening with 48 different nicotine-containing and two nicotine-free pouches using gas chromatography coupled to mass spectrometry, following acidic and basic liquid-liquid extraction. For toxicological assessment of identified substances, European and international classifications for chemical and food safety were consulted. Further, ingredients listed on product packages were counted and grouped by function. Most abundant ingredients comprised sweeteners, aroma substances, humectants, fillers, and acidity regulators. 186 substances were identified. For some substances, acceptable daily intake limits set by European Food Safety Agency (EFSA) and Joint FAO/WHO Expert Committee on Food Additives are likely exceeded by moderate pouch consumption. Eight hazardous substances are classified according to the European CLP regulation. Thirteen substances were not authorized as food flavorings by EFSA, among them impurities such as myosmine and ledol. Three substances were classified by International Agency for Research on Cancer as possibly carcinogenic to humans. The two nicotine-free pouches contain pharmacologically active ingredients such as ashwagandha extract and caffeine. The presence of potentially harmful substances may point to the need for regulation of additives in nicotine-containing and nicotine-free pouches that could be based on provisions for food additives. For sure, additives may not pretend positive health effects in case the product is used.

A regulatory relic: After 60 years of research on cancer risk, the Delaney Clause continues to keep us in the past.

The Delaney Clause of the Federal Food, Drug, and Cosmetic Act became law in 1958 because of concerns that potentially harmful chemicals were finding their way into foods and causing cancer. It states, "[n]o additive shall be deemed to be safe if it is found to induce cancer when ingested by man or animal, or if it is found, after tests which are appropriate for the evaluation of the safety of food additives, to induce cancer in man or animal." The United States Food and Drug Administration (US FDA) and United States Environmental Protection Agency (US EPA, prior to implementation of the Food Quality Protection Act) were charged with implementing this clause. Over 60 years, advances in cancer research have elucidated how chemicals induce cancer. Significant advancements in analytical methodologies have allowed for accurate and progressively lower detection limits, resulting in detection of trace amounts. Based on current scientific knowledge, there is a need to revisit the Delaney Clause's utility. The lack of scientific merit to the Delaney Clause was very apparent when recently the US FDA had to revoke the food additive approvals of 6 synthetic flavoring substances because high dose testing in animals demonstrated a carcinogenic response. However, US FDA determined that these 6 synthetic flavoring substances do not pose a risk to public health under the conditions of intended use. The 7th substance, styrene, was de-listed because it is no longer used by industry. The scientific community is committed to improving public health by promoting relevant science in risk assessment and regulatory decision making, and this was discussed in scientific sessions at the American Association for the Advancement of Science (AAAS) 2020 Annual Meeting and the Society of Toxicology (SOT) 2019 Annual Meeting. Expert presentations included advances in cancer research since the 1950s; the role of the Delaney Clause in the current regulatory paradigm with a focus on synthetic food additives; and the impact of the clause on scientific advances and regulatory decision making. The sessions concluded with panel discussions on making the clause more relevant based on 21st-century science.

A Pilot Safety Assessment for Recombinant Epinephelus lanceolatus Piscidin Yeast Powder as a Drug Food Additive after Subacute and Subchronic Administration to SD Rats.

Recombinant Epinephelus lanceolatus piscidin (RELP) was previously shown to improve growth performance and immune response when used as a feed additive for Gallus gallus domesticus. However, the long-term toxicity of RELP has not be thoroughly investigated. In the present study, we evaluated the subacute and subchronic oral toxicities of RELP in SD rats by hematological, biochemical, and histopathological analyses. To determine subacute and subchronic toxicities, male and female rats were fed with RELP 1000 mg/kg bodyweight/day for 28 and 90 days, respectively. Bodyweight and food intake were unchanged by RELP treatment over the course of the studies. After exposure, samples of blood, heart, lung, liver, and kidney were collected and analyzed. Results demonstrated that RELP exposure did not cause any observable hematological, biochemical, or histological abnormalities in SD rats. Thus, RELP may be a safe feed additive for use in agriculture and aquaculture.

Potential of ToxCast Data in the Safety Assessment of Food Chemicals.

Tox21 and ToxCast are high-throughput in vitro screening programs coordinated by the U.S. National Toxicology Program and the U.S. Environmental Protection Agency, respectively, with the goal of forecasting biological effects in vivo based on bioactivity profiling. The present study investigated whether mechanistic insights in the biological targets of food-relevant chemicals can be obtained from ToxCast results when the chemicals are grouped according to structural similarity. Starting from the 556 direct additives that have been identified in the ToxCast database by Karmaus et al. [Karmaus, A. L., Trautman, T. D., Krishan, M., Filer, D. L., and Fix, L. A. (2017). Curation of food-relevant chemicals in ToxCast. Food Chem. Toxicol. 103, 174-182.], the results showed that, despite the limited number of assays in which the chemical groups have been tested, sufficient results are available within so-called "DNA binding" and "nuclear receptor" target families to profile the biological activities of the defined chemical groups for these targets. The most obvious activity identified was the estrogen receptor-mediated actions of the chemical group containing parabens and structurally related gallates, as well the chemical group containing genistein and daidzein (the latter 2 being particularly active toward estrogen receptor ß as a potential health benefit). These group effects, as well as the biological activities of other chemical groups, were evaluated in a series of case studies. Overall, the results of the present study suggest that high-throughput screening data could add to the evidence considered for regulatory risk assessment of food chemicals and to the evaluation of desirable effects of nutrients and phytonutrients. The data will be particularly useful for providing mechanistic information and to fill data gaps with read-across.

In vitro genotoxicity assessment of monopotassium glutamate and magnesium diglutamate.

Food additives are approved chemicals used for various purposes in foods; to provide nutritional safety, increase flavor, extend shelf life, reduce nutrient losses etc. In this study, the in vitro genotoxic effects of flavor enhancers, Monopotassium glutamate (MPG) and Magnesium diglutamate (MDG) were investigated in human peripheral blood lymphocytes by using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), cytokinesis-block micronucleus cytome (CBMN-Cyt), and comet assays. Four concentrations of MPG (125, 250, 500, and 1000 µg/mL) and MDG (93.75, 187.5, 375, and 750 µg/mL) were used. Both food additives significantly reduced mitotic index and increased the frequency of CAs at high concentrations. MPG and MDG (except 93.75 µg/mL) significantly increased SCEs/Cell in concentration-dependent manner. In the CBMN-Cyt test, both MPG and MDG increased the formation of micronucleus, nuclear buds, and nucleoplasmic bridges compared to control in a concentration-dependent manner. However, these increases were statistically significant at higher concentrations. MPG (at 500 and 1000 µg/mL) and MDG (except 93.75 µg/mL) significantly increased DNA damages observed by comet assay. It is concluded from these results that MPG and MDG have clastogenic, mutagenic, aneugenic, and cytotoxic effects, particularly at high concentrations in human lymphocytes in vitro.

Safety Assessment of Food Additives: Case Example With Myrcene, a Synthetic Flavoring Agent.

In the United States, the Food and Drug Administration (FDA) regulates the safe use of food ingredients, including food additives. Food additives are subject to FDA premarket review and approval, a process conducted by FDA scientists to evaluate the additive's safety for the intended conditions of use. Typically, an acceptable daily intake level is established by toxicologists based on the highest no observable adverse effect level for the most sensitive noncancer toxicity end point determined from a pivotal nonclinical study with application of an appropriate safety factor. Utilizing other information, including the additive's use and exposure levels, a safety determination (reasonable certainty of no harm) is made. During ongoing safety assessments, pathologists are often consulted by toxicologists for case-specific reasons, which may include verifying that an observed pathological effect is treatment related and adverse, confirming the determination of the pivotal study, endorsing a mode of action, or evaluating the human relevance of a toxicological effect found in experimental animals. Last year, the FDA took regulatory action to no longer allow the use of the food additive myrcene, a synthetic flavoring agent, based on results from National Toxicology Program carcinogenicity studies. The cancer and noncancer end points from the rat studies are discussed.

Incorporating new approach methodologies in toxicity testing and exposure assessment for tiered risk assessment using the RISK21 approach: Case studies on food contact chemicals.

Programs including the ToxCast project have generated large amounts of in vitro high‒throughput screening (HTS) data, and best approaches for the interpretation and use of HTS data, including for chemical safety assessment, remain to be evaluated. To fill this gap, we conducted case studies of two indirect food additive chemicals where ToxCast data were compared with in vivo toxicity data using the RISK21 approach. Two food contact substances, sodium (2-pyridylthio)-N-oxide and dibutyltin dichloride, were selected, and available exposure data, toxicity data, and model predictions were compiled and assessed. Oral equivalent doses for the ToxCast bioactivity data were determined by in-vitro in-vivo extrapolation (IVIVE). For sodium (2-pyridylthio)-N-oxide, bioactive concentrations in ToxCast assays corresponded to low- and no-observed adverse effect levels in animal studies. For dibutyltin dichloride, the ToxCast bioactive concentrations were below the dose range that demonstrated toxicity in animals; however, this was confounded by the lack of toxicokinetic data, necessitating the use of conservative toxicokinetic parameter estimates for IVIVE calculations. This study highlights the potential utility of the RISK21 approach for interpretation of the ToxCast HTS data, as well as the challenges involved in integrating in vitro HTS data into safety assessments.

Risk Assessment Paradigm for Glutamate.

BACKGROUND: Re-evaluation of the use of glutamic acid and glutamate salts (referred to as glutamate hereafter) by the European Food Safety Authority (EFSA) proposed a group acceptable daily intake (ADI) of 30 mg/kg body weight (bw)/day. SUMMARY: This ADI is below the normal dietary intake, while even intake of free glutamate by breast-fed babies can be above this ADI. In addition, the pre-natal developmental toxicity study selected by EFSA, has never been used by regulatory authorities worldwide for the safety assessment of glutamate despite it being available for nearly 40 years. Also, the EFSA ignored that toxicokinetic data provide support for eliminating the use of an uncertainty factor for interspecies differences in kinetics. Key Messages: A 3-generation reproductive toxicity study in mice that includes extensive brain histopathology, provides a better point of departure showing no effects up to the highest dose tested of 6,000 mg/kg bw/day. Furthermore, kinetic data support use of a compound-specific uncertainty factor of 25 instead of 100. Thus, an ADI of at least 240 mg/kg bw/day would be indicated. In fact, there is no compelling evidence to indicate that the previous ADI of "not specified" warrants any change.

Review of toxicological assessment of d-limonene, a food and cosmetics additive.

R-(+)-limonene (d-limonene) is a commonly used flavor additive in food, beverages and fragrances for its pleasant lemon-like odor. Considering its increasing applications, it's necessary to understand toxicological effects and risk associated with its use. R-(+)-limonene is rapidly absorbed in experimental animals and human beings following oral administration. In humans, it gets distributed to liver, kidney, and blood resulting in the formation of metabolites like perillic acid, dihydroperillic acid, limonene-1,8-diol and limonene-1,2 diol. Important toxic effects primarily reported in rodents are severe hyaline droplet nephrotoxicity (only in male rats due to specific protein α2u-globulin; however, this effect isn't valid for humans), hepatotoxicity and neurotoxicity. R-(+)-limonene does not show genotoxic, immunotoxic and carcinogenic effects. Substantial data is available about limonene's stability after treatment with thermal and non-thermal food processing techniques; however, information about toxicity of metabolites formed and their safe scientific limits is not available. In addition, toxicity of limonene degradation products formed during storage of citrus juices isn't known. Based on all available toxicological considerations, R-(+)-limonene can be categorized as low toxic additive. More detailed studies are required to better understand interaction of limonene with modern food processing techniques as well as degradation products generated and toxicity arising from such products.

Assessment of hepato-renal damage and genotoxicity induced by long-term exposure to five permitted food additives in rats.

The present study assessed the long-term daily administration of benzoic acid (BA), potassium sorbate (PS), chlorophyll (CPL), tartrazine (TAZ), and butylated hydroxyanisole (BHA) on hepato-renal changes and DNA damage in rats. Animals were orally administered with the 10 times of the acceptable daily intake (ADI) from each tested substance daily for 60 consecutive days. Blood, liver, and kidney samples were collected to evaluate hematological, biochemical, histopathological, and genotoxic alterations. The extent of liver and kidney damage was evaluated by comet assay and histopathologically. Significant reduction of leukocyte numbers and lymphocytes % in CPL- and TAZ-treated rats. However, significant increases in platelet count in all treated groups after 60 days were detected. The levels of serum transaminases enzymes (ALT, AST), alkaline phosphatase (ALP), and creatinine were significantly increased in all treatments except with BHA group, but no substantial differences were found in urea after 60 days. Aside from BHA, results of DNA damage revealed significant increases in tailed nuclei, tail moment, DNA% in the tail, and tail length in liver and kidney at different degrees. Moreover, the histopathological figures of liver and kidneys affirmed destructive and degenerative changes. The study indicates that most of the tested food additives may provoke genotoxicity and hepato-nephropathy, which could be serious for human health. Therefore, it is necessary to be informed about the hazardous effects of food additives and more attention should be focused towards using natural substitutes.

Toxicological Assessment of Mineral Hydrocarbons in Foods: State of Present Discussions.

The evaluation of mineral oils by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) provided high acceptable daily intakes for classes largely falling into the mass range strongly accumulated by humans. Because they are roughly 2 orders of magnitude above the present exposure, they authorize strongly increased exposure. An approach based on accumulation seems more adequate. Increased organ weights might be more critical than granulomas. Aromatic hydrocarbons with 1-2 aromatic rings should be distinguished from those with at least 3 aromatic rings. If mineral oil saturated hydrocarbon limits were low, no limit might be needed for the 1-2 ring aromatics. It should be considered to phase out substantial use of mineral oils in food application.

Critical review of the safety assessment of titanium dioxide additives in food.

Nanomaterial engineering provides an important technological advance that offers substantial benefits for applications not only in the production and processing, but also in the packaging and storage of food. An expanding commercialization of nanomaterials as part of the modern diet will substantially increase their oral intake worldwide. While the risk of particle inhalation received much attention, gaps of knowledge exist regarding possible adverse health effects due to gastrointestinal exposure. This problem is highlighted by pigment-grade titanium dioxide (TiO2), which confers a white color and increased opacity with an optimal particle diameter of 200-300 nm. However, size distribution analyses showed that batches of food-grade TiO2 always comprise a nano-sized fraction as inevitable byproduct of the manufacturing processes. Submicron-sized TiO2 particles, in Europe listed as E 171, are widely used as a food additive although the relevant risk assessment has never been satisfactorily completed. For example, it is not possible to derive a safe daily intake of TiO2 from the available long-term feeding studies in rodents. Also, the use of TiO2 particles in the food sector leads to highest exposures in children, but only few studies address the vulnerability of this particular age group. Extrapolation of animal studies to humans is also problematic due to knowledge gaps as to local gastrointestinal effects of TiO2 particles, primarily on the mucosa and the gut-associated lymphoid system. Tissue distributions after oral administration of TiO2 differ from other exposure routes, thus limiting the relevance of data obtained from inhalation or parenteral injections. Such difficulties and uncertainties emerging in the retrospective assessment of TiO2 particles exemplify the need for a fit-to-purpose data requirement for the future evaluation of novel nano-sized or submicron-sized particles added deliberately to food.

Safety assessment of sodium acetate, sodium diacetate and potassium sorbate food additives.

Cytotoxicity and genotoxicity of sodium acetate (SA), sodium diacetate (SDA), and potassium sorbate (PS) was tested on Human Umbilical Vein Endothelial Cells (HUVEC). Cytotoxicity was investigated by MTT assay and flow cytometry analysis, while genotoxicity was evaluated using DNA fragmentation and DAPI staining assays. The growth of treated HUVECs with various concentrations of SA, SDA and PS decreased in a dose-and time-dependent manner. The IC50 of 487.71, 485.82 and 659.96 µM after 24 h and IC50 of 232.05, 190.19 and 123.95 µM after 48 h of treatment were attained for SA, SDA and PS, respectively. Flow cytometry analysis showed that early and late apoptosis percentage in treated cells was not considerable. Also neither considerable DNA fragmentation nor DNA smear was observed using DAPI staining and DNA ladder assays. Overall, it can be concluded that the aforementioned food additives can be used as safe additives at low concentration in food industry.

Consideration of interaction between nanoparticles and food components for the safety assessment of nanoparticles following oral exposure: A review.

Nanoparticles (NPs) are increasingly used in food, and the toxicity of NPs following oral exposure should be carefully assessed to ensure the safety. Indeed, a number of studies have shown that oral exposure to NPs, especially solid NPs, may induce toxicological responses both in vivo and in vitro. However, most of the toxicological studies only used NPs for oral exposure, and the potential interaction between NPs and food components in real life was ignored. In this review, we summarized the relevant studies and suggested that the interaction between NPs and food components may exist by that 1) NPs directly affect nutrients absorption through disruption of microvilli or alteration in expression of nutrient transporter genes; 2) food components directly affect NP absorption through physico-chemical modification; 3) the presence of food components affect oxidative stress induced by NPs. All of these interactions may eventually enhance or reduce the toxicological responses induced by NPs following oral exposure. Studies only using NPs for oral exposure may therefore lead to misinterpretation and underestimation/overestimation of toxicity of NPs, and it is necessary to assess the synergistic effects of NPs in a complex system when considering the safety of NPs used in food.