A multiparametric and orthogonal approach indicates low toxicity for zein nanoparticles in a repellent formulation.

The incidence of viruses such as Zika, Dengue, and Chikungunya affects human health worldwide, and insect repellents are recommended for individual protection. Formulations incorporating nanotechnology should be carefully assessed for toxicity, particularly regarding the security levels established for human health and the environment. This study evaluates the cytotoxicity of a repellent formulation containing zein nanoparticles (NP) loading geraniol (Ger) and icaridin (Ica) in three cell lines: NIH/3T3, HaCaT, and SIRC. To address formulation hazards, IC50 values were determined by MTT and Calcein-AM assays. In both NIH/3T3 and HaCaT, the IC50 values for NP + Ger + Ica formulation were around 0.2%. For risk assessment, cell viability was also determined after a single exposure and repeated exposure to the formulation. No evidence of cytotoxicity was observed for NP + Ger + Ica formulation-treated cells. The risk assessment for eye damage revealed cytotoxicity in SIRC cells when exposed to a 5% concentration, which may be attributed to ocular geraniol toxicity, because zein nanoparticles alone did not exhibit any signs of toxicity. Cell internalization indicated low uptake in NIH/3T3 and HaCaT cells. Phenotypic profiling resulted in similar phenotypes for untreated cells and cells exposed to NP + Ger + Ica formulation. The toxicological profile outlined by the multiparametric and orthogonal approach suggests that the NP + Ger + Ica formulation poses no significant risk to the topical application under the tested conditions. Adopting an orthogonal approach brings robustness to our findings.

Geraniol and Carvacrol in Essential Oil Bearing Thymus pulegioides: Distribution in Natural Habitats and Phytotoxic Effect.

Phenolic and non-phenolic chemotypes of Thymus pulegioides L. are common in Europe. Essential oils of these chemotypes, as various compositions of allelochemicals, can have different phytotoxic effects on neighboring plants in natural habitats. The aim of this study was to establish the distribution of carvacrol and geraniol in T. pulegioides, growing wild in Lithuania, and compare phytotoxity of essential oils of carvacrol and geraniol chemotypes on selected plant species. In investigating 131 T. pulegioides habitats, essential oils were isolated by hydrodistillation and analyzed by GC-FID and GC-MS. Phytotoxity of essential oils extracted from carvacrol and geraniol chemotypes transmitted through water and air to selected plants was determined under laboratory conditions. Pharmacologically valuable Hypericum perforatum L. and the important forage grass Phleum pratense L. were respectively selected for experimentation from among 35 medicinal plants and 10 feed Poaceae species, growing in T. pulegioides habitats. Field results showed that carvacrol is common throughout Lithuania's territory, whereas the geraniol is predominantly located under the continental climatic conditions of the eastern region of the country. In the laboratory experiment, it was established that there was stronger inhibition of P. pratense seed germination by the essential oil of the geraniol chemotype than the carvacrol chemotype. None of the H. perforatum seeds germinated after exposure to the essential oil of the geraniol chemotype. In general, this study builds on previous studies by providing further evidence that different T. pulegioides chemotypes have contrasting phytotoxic effects on neighboring plants within their natural habitats.

Chemical Composition and Preliminary Toxicity Evaluation of the Essential Oil from Peperomia circinnata Link var. circinnata. (Piperaceae) in Artemia salina Leach.

Peperomia Ruiz and Pav, the second largest genus of the Piperaceae, has over the years shown potential biological activities. In this sense, the present work aimed to carry out a seasonal and circadian study on the chemical composition of Peperomia circinata essential oils and aromas, as well as to evaluate the preliminary toxicity in Artemia salina Leach and carry out an in silico study on the interaction mechanism. The chemical composition was characterized by gas chromatography (GC/MS and GC-FID). In the seasonal study the essential oil yields had a variation of 1.2-7.9%, and in the circadian study the variation was 1.5-5.6%. The major compounds in the seasonal study were ß-phellandrene and elemicin, in the circadian they were ß-phellandrene and myrcene, and the aroma was characterized by the presence of ß-phellandrene. The multivariate analysis showed that the period and time of collection influenced the essential oil and aroma chemical composition. The highest toxicity value was observed for the essential oil obtained from the dry material, collected in July with a value of 14.45 ± 0.25 µg·mL-1, the in silico study showed that the major compounds may be related to potential biological activity demonstrated by the present study.

Genotoxicity induced by nerol, an essential oil present in citric plants using human peripheral blood mononuclear cells (PBMC) and HepG2/C3A cells as a model.

Nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) is a monoterpene widely used in cosmetic products, household detergents and cleaners, as well as a flavoring in several food products. Despite the high level of human exposure to nerol, an absence of studies regarding potential genetic toxicity in human cells exists. The aim of this investigation was to examine the cytotoxic and genotoxic potential of this monoterpene on human peripheral blood mononuclear cells as well as hepatic metabolizing HepG2/C3A human cell line. Cytotoxicity was assessed using trypan blue staining and MTT assay while genotoxicity was determined utilizing the comet and micronucleus test. Cytotoxicity tests showed cell viability greater than 70% for concentrations between 2.5 and 500 µg/ml. Both cell types exhibited significant DNA damage and chromosomal mutations after medium and high concentration incubation with nerol indicating that the safety of use of this monoterpene in various formulations to which humans are exposed needs to be monitored and requires more comprehensive investigations.

Persistence and ingestion characteristics of phytochemical volatiles as bio-fumigants in Sitophilus oryzae adults.

Fumigant toxicity of phytochemical volatiles has been widely reported against stored product insect pests. Such volatiles are considered as natural fumigants and bio-fumigants in post-harvest food protection research. In the present study, persistence and ingestion of diallyl disulfide, citral, eucalyptol, eugenol and menthol were investigated in Sitophilus oryzae adults in comparison with fumigant toxicity and microstructural impact in elytra. The fumigant toxicity bioassay was performed with increasing concentrations of phytochemical volatiles at 25, 125, 250 and 500 µL/L air against S. oryzae adults in 50 mL glass vials. The phytochemical residues were examined from the treated adults by Gas Chromatography coupled with Flame Ionization Detector (GC-FID) and their pathological impacts on the elytral surface was observed under Scanning Electron Microscopy (SEM). After 72 h of fumigation, diallyl disulfide and eucalyptol were identified as potential fumigants with 5.24 and 8.30 µL/L air LC50 values, respectively. GC-FID analyses showed that diallyl disulfide and eucalyptol molecules persistence (1.29 and 2.60 ppb persistence with 0.94 and 0.90 r2 values respectively at 72 h exposure) on the body surface of weevil was positively correlated with the fumigation exposure and toxicity. Whereas, phytochemical molecules ingestion into the body of weevils was not directly linked with the insect mortalities. The SEM observations indicated that diallyl disulfide and eucalyptol molecules caused severe microstructural impacts on the elytra of weevils compared to other molecules. As a result, the present study suggested that phytochemical fumigants are persisted on the body surface and caused insecticidal toxicities in S. oryzae adults. In addition, it was predicted that persisted molecules might be entered into the body of weevils via cuticular penetration.

How the 62-year old Delaney Clause continues to thwart science: Case study of the flavor substance ß-myrcene.

The Delaney Clause is a provision of the 1958 Food Additive Amendment to the Food, Drug and Cosmetic Act of 1938 which stipulates that if a substance is found by the Food and Drug Administration to be carcinogenic in any species of animal or in humans, then it cannot be used as a food additive. This paper presents a case study of ß-myrcene, one of seven synthetic substances that was challenged under the Delaney Clause, ultimately resulting in revocation of its regulatory approval as a food additive despite a lack of safety concern. While it is listed as a synthetic flavor in 21 CFR 172.515, ß-myrcene is also a substance naturally occurring in a number of dietary plants. The exposure level to naturally-occurring ß-myrcene is orders of magnitude higher (estimated to be 16,500 times greater) than the exposure via ß-myrcene added to food as a flavoring substance. The National Toxicology Program conducted genotoxicity testing (negative), a 13-week range-finding study, and a two-year cancer bioassay in B6C3F1 mice and F344/N rats. An increase in liver tumors was seen in male mice and kidney tumors in male rats, ultimately resulting in ß-myrcene being classified by IARC as a Class 2B carcinogen and being listed on California Proposition 65; in contrast, ß-myrcene is not classified as a carcinogen by any other regulatory authority. The doses administered in the NTP bioassay were five-six orders of magnitude higher than human exposures, and the FDA concluded after a thorough evaluation that there was no safety concern associated with the use of ß-myrcene as a flavor substance at the current use level. The Delaney Clause, however, does not consider the exposure potential or the human health relevance of effects observed in animals. The lack of options available to the US FDA led to the 2018 decision to remove ß-myrcene from the list of approved food additives. This revocation has contributed to the ongoing erosion of trust in regulatory agencies (and industry), which has both economic implications for food manufacturers and consumers alike, and implications for consumer perception of safety of the US food supply. It is time for us to reconsider the rationale behind any legislation that relies on classification alone, and whether there is, in fact, a reason to still classify nongenotoxic carcinogens at all.

Mechanistic Insights on Skin Sensitization to Linalool Hydroperoxides: EPR Evidence on Radical Intermediates Formation in Reconstructed Human Epidermis and 13C NMR Reactivity Studies with Thiol Residues.

Linalool is one of the most commonly used fragrance terpenes in consumer products. While pure linalool is considered as non-allergenic because it has a very low skin sensitization potential, its autoxidation on air leads to allylic hydroperoxides that have been shown to be major skin sensitizers. These hydroperoxides have the potential to form antigens via radical mechanisms. In order to obtain in-depth insights of such reactivity, we first investigated the formation of free radicals derived from linalool hydroperoxides in situ in a model of human reconstructed epidermis by electron paramagnetic resonance combined with spin trapping. The formation of carbon- and oxygen-centered radical species derived from the hydroperoxides was especially evidenced in an epidermis model, mimicking human skin and thus closer to what may happen in vivo. To further investigate these results, we synthesized linalool hydroperoxides containing a 13C-substitution at positions precursor of carbon radicals to elucidate if one of these positions could react with cysteine, its thiol chemical function being one of the most labile groups prone to react through radical mechanisms. Reactions were followed by mono- and bidimensional 13C NMR. We validated that carbon radicals derived from allylic hydrogen abstraction by the initially formed alkoxyl radical and/or from its ß-scission can alter directly the lateral chain of cysteine forming adducts via radical processes. Such results provide an original vision on the mechanisms likely involved in the reaction with thiol groups that might be present in the skin environment. Consequently, the present findings are a step ahead toward the understanding of protein binding processes to allergenic allylic hydroperoxides of linalool through the involvement of free radical species and thus of their sensitizing potential.

Synthesis of New Quaternary Ammonium Salts with a Terpene Function and Evaluation of their Fungicidal and Herbicidal Activities.

A series of new quaternary ammonium salts (QASs) containing a terpenoid moiety derived from perillyl alcohol, citronellol, and geraniol was synthesized. Structures of all novel compounds were confirmed by spectral methods and elemental analyses. Fungicidal activity of the obtained compounds against six plant pathogens, against four fungi destroying wood and technical materials as well as herbicidal activity against ten species of temperate climate weeds has been examined. Several salts showed a higher antifungal and herbicidal activity than activity of the reference compounds.

Citral presents cytotoxic and genotoxic effects in human cultured cells.

Citral, 3,7-dimethyl-2,6-octadien-1-al, one of the main components of the essential oils obtained from several plants, is used as a food additive and as a fragrance for detergents, cosmetics and other toiletries. The literature shows disparity regarding citral genotoxicity. Thus, the main objective of our work was to evaluate the genotoxic effects of citral in human cell cultures, HepG2 and leukocytes. Cytotoxicity assays (trypan blue and MTT) showed citral toxic effects in HepG2 cells (with metabolizing liver enzymes), which contrasted with the absence of toxicity in leukocytes. After citral exposure, both cell types did not demonstrate clastogenic/aneugenic effects in the micronucleus test. However, for the comet assay, citral exposure lead to significant genotoxic effects in both HepG2 (even to citral low concentrations) and leukocytes. The use of citral must be viewed with caution due to its ability to induce DNA damages, especially after being metabolized by cells with active liver enzymes.

Model to design insecticides against Aedes aegypti using in silico and in vivo analysis of different pharmacological targets.

Compounds having insecticidal activity can be used to control Aedes aegypti mosquitoes, a major worldwide vector, and several plants have a source of such molecules. A principal component analysis (PCA) was carried out to determine the criterion to select larvicidal metabolites. The insecticidal activity of seven selected metabolites by PCA was validated by determining its lethal concentrations 50 (LC50) by probit analysis. Six of the seven evaluated molecules presented LC50 values <100 ppm. The effects of these six molecules on acetylcholinesterase and the respiratory chain complexes of the mitochondria of Ae. aegypti were evaluated. Four metabolites presenting the highest inhibition effects on these targets were mixed in 11 different combinations, and the percentage of mortality of each mixture on Ae. aegypti larvae were determined. Secondary metabolites such as geranyl acetate, α-humulene, ß-caryophyllene, geraniol, nerol, and n-octanol presented LC50 values under 100 ppm (44, 41, 48, 84, 87, and 98 ppm, respectively), whereas 1,8-cineole presented a LC50 value of 183 ppm. We found that, geranyl acetate, α-humulene, ß-caryophyllene, nerol, n-octanol, and geraniol inhibited at least one of the six targets with an efficiency between 25 and 41%. Overall, the evaluation of the different mixtures revealed a synergistic effect between geranyl acetate and geraniol, and an antagonistic effect between α-humulene and ß-caryophyllene compounds.

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.

An unbiased approach elucidates variation in (S)-(+)-linalool, a context-specific mediator of a tri-trophic interaction in wild tobacco.

Plant volatile organic compounds (VOCs) mediate many interactions, and the function of common VOCs is especially likely to depend on ecological context. We used a genetic mapping population of wild tobacco, Nicotiana attenuata, originating from a cross of 2 natural accessions from Arizona and Utah, separated by the Grand Canyon, to dissect genetic variation controlling VOCs. Herbivory-induced leaf terpenoid emissions varied substantially, while green leaf volatile emissions were similar. In a field experiment, only emissions of linalool, a common VOC, correlated significantly with predation of the herbivore Manduca sexta by native predators. Using quantitative trait locus mapping and genome mining, we identified an (S)-(+)-linalool synthase (NaLIS). Genome resequencing, gene cloning, and activity assays revealed that the presence/absence of a 766-bp sequence in NaLIS underlies the variation of linalool emissions in 26 natural accessions. We manipulated linalool emissions and composition by ectopically expressing linalool synthases for both enantiomers, (S)-(+)- and (R)-(-)-linalool, reported to oppositely affect M. sexta oviposition, in the Arizona and Utah accessions. We used these lines to test ovipositing moths in increasingly complex environments. The enantiomers had opposite effects on oviposition preference, but the magnitude of the effect depended strongly both on plant genetic background, and complexity of the bioassay environment. Our study reveals that the emission of linalool, a common VOC, differs by orders-of-magnitude among geographically interspersed conspecific plants due to allelic variation in a linalool synthase, and that the response of a specialist herbivore to linalool depends on enantiomer, plant genotype, and environmental complexity.

Genotoxic effects induced by beta-myrcene following metabolism by liver HepG2/C3A human cells.

Beta-myrcene [or myrcene (1,6-Octadiene, 7-methyl-3-methylene-)] and the essential oils containing this monoterpene have been widely used in cosmetics, detergents, and soaps, and as flavoring additives for food and beverages. Due to the potentially high level of human exposure to beta-myrcene, and absence of studies involving its genotoxicity in human cells, the aim of this study was to investigate the cytotoxic and genotoxic potential of this terpenoid in non-metabolizing cells (leukocytes) and liver metabolizing cells (HepG2/C3A cells). Prior to the genotoxic assessment by the comet and micronucleus (MN) assays, a range of beta-myrcene concentrations was tested in a preliminary MTT assay. Regarding the MTT assay, the results showed cytotoxic effects for leukocytes at 250 µg/ml and higher concentrations, while for HepG2/C3A cells, absence of cytotoxicity was noted relative to all tested concentrations (after 24 hr exposure). Thus, the concentrations of 2.5, 10, 25, 50, and 100 µg/ml for leukocytes, and 2.5, 100, and 1000 µg/ml for HepG2/C3A cells were selected for subsequent assays. Genotoxicity evaluation demonstrated significant DNA damage in the comet assay and significant chromosomal abnormalities including nucleoplasmic bridges and nuclear buds in HepG2/C3A cells at beta-myrcene concentrations of 100 and 1000 µg/ml. Under our experimental conditions, caution is recommended in the use of beta-myrcene, since this compound produced genotoxic effects especially after metabolic activation using human HepG2/C3A cells, which may be associated with carcinogenic and teratogenic effects previously reported in the literature.