Evaluation of cytotoxicity,genotoxicity and embryotoxicity of insecticide propoxur using flounder gill (FG) cells and zebrafish embryos

Cytotoxicity, genotoxicity and embryotoxicity of carbamate insecticide propoxur were evaluated using flounder gill (FG) cells and zebrafish embryos. The cytotoxicity of propoxur in FG cells was analyzed by MTT, neutral red uptake (NRU), lactate dehydrogenase (LDH) release and Hoechst 33342 and propidium iodide double staining, and acute cytotoxic effects were observed in a concentration-dependent manner. The 24 h-IC₅₀ values of 89.96 ± 1.04, 103.4 ± 1.14 and 86.59 ± 1.13 μg/ml propoxur were obtained by MTT, NRU and LDH assays, respectively. The lethal effects were induced in FG cells mainly through necrosis but not apoptosis as evidenced by double fluorescence staining. Comet assay showed weak genotoxic effects and statistically significant DNA damages were recorded in the cells exposed to highest tested concentration of 75 μg/ml propoxur (p < 0.05). Propoxur exerted obvious acute toxic effects on the survival, spontaneous movement, hatching and heart rate, and development (yolk and pericardial sac edema) of zebrafish embryos in both time- and concentration-dependent manner only at ⩾100 μg/ml. The corresponding 24 h-, 48 h- and 96 h-LC₅₀ values of propoxur in zebrafish embryos were 166.4 ± 1.06, 146.3 ± 1.07 and 134.8 ± 1.06 μg/ml, respectively. The above data obtained suggest a low acute toxicity of propoxur to the in vitro cultured FG cells and zebrafish embryos.     

The in vitro and in vivo genotoxicity of isotretinoin assessed by cytokinesis blocked micronucleus assay and comet assay

Isotretinoin is a retinoic acid frequently used in monotherapy or combined with narrow-band ultraviolet B (NBUVB) irradiation to treat patients with acne and psoriasis vulgaris. As both diseases need frequent and/or prolonged therapeutic interventions, the study of the genotoxicity of retinoids becomes important.Our aim was to study the genotoxic effects of isotretinoin alone or combined with NBUVB. In vitro studies were performed in the absence of S9 metabolic activation using blood from five healthy volunteers, incubated 72 h with isotretinoin (1.2–20 μM) (i.e., at concentrations usually achieved in blood with therapeutic doses as well as at higher concentrations). In vivo studies were also performed using blood from two patients with acne and three patients with psoriasis vulgaris treated with isotretinoin in monotherapy (8 or 20 mg/day) or combined with NBUVB (20 mg isotretinoin/day + NBUVB). The genotoxic effect was evaluated by the cytokinesis-blocked micronucleus and the comet assays.Our studies showed that isotretinoin alone was not genotoxic when tested in human lymphocytes in vitro and in vivo. There was no clear genotoxic effect in psoriatic patients treated with isotretinoin and NBUVB. The in vitro studies showed that isotretinoin induced apoptosis and necrosis in human lymphocytes at higher doses.     

Leaf extracts from Teucrium ramosissimum protect against DNA damage in human lymphoblast cell K562 and enhance antioxidant,antigenotoxic and antiproliferative activity

The in vitro antioxidant, antigenotoxic and antiproliferative activities of Teucrium ramosissimum extracts were investigated. The antioxidant activities of the tested extracts were evaluated through three chemical assays: The Cupric reducing antioxidant capacity, the reducing power and the ferric reducing antioxidant power. TR1 fraction from methanol extract showed the best antioxidant activity evaluated by the CUPRAC, RP and FRAP assays with TEAC values of 4.04, 1.77 and 1.48 μM respectively compared to control. Yet, TR2 fraction exhibited the lowest antioxidant effect with a TEAC values of 1.97, 0.408 and 0.35 μM respectively. All the tested extracts were also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals. Furthermore, the effects of T. ramosissimum extracts on cell proliferation were also examined. The cytotoxic study revealed that methanol extract significantly inhibited the proliferation of K562 cells (IC50 = 150 μg/mL). The antigenotoxic properties of these extracts were investigated by assessing the induction and inhibition of the genotoxicity induced by the direct-acting mutagen, hydrogen peroxide (H₂O₂), using an eukaryotic system; the “Comet assay.” The results showed that all the extracts inhibited the genotoxicity induced by H₂O₂, and particularly TR2 fraction (96.99%) and methanol extract (96.64%).The present study has demonstrated that T. ramosissimum extract possess potent antioxidant, antiproliferative and antigenotoxic activities, which could be derived from compounds such as flavonoids and polyphenols.     

Assessment of DNA damage,cytotoxicity, and apoptosis in human hepatoma (HepG2) cells after flurochloridone herbicide exposure

In vitro effects of flurochloridone (FLC) and its formulations Twin Pack Gold® [25% active ingredient (a.i.)] and Rainbow® (25% a.i.) were evaluated in HepG2 cells. Whereas cytokinesis-blocked micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were employed for genotoxicity, MTT, neutral red, and apoptosis detections were used for cytotoxicity evaluation. Activities were tested within the concentration range of 0.25–15 μg/ml FLC. Results demonstrated that neither FLC nor Rainbow® was able to induce MNs. On the other hand, 5 μg/ml Twin Pack Gold® only increased MN frequency. Furthermore, 10 and 15 μg/ml of both formulations resulted in cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. A marked increase in the genetic damage index was observed after treatment with all compounds. SCGE assay appeared to be more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of FLC than did MN. Our results reveal that FLC and its two formulations trigger apoptosis on HepG2 cells. The results represent the first experimental evidence of the in vitro apoptogenic role exerted on mammalian cells by FLC and the FLC-based formulations Rainbow® and Twin Pack Gold®, at least on HepG2 cells.     

Teratogenic effects of organic extracts from the Pearl River sediments on Xenopus laevis embryos

Toxicity of organic extracts from the Pearl River sediments was investigated with Xenopus laevis embryos. The effects of sediment organic extracts on the mortality, body length and malformation of X. laevis embryos were tested by the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). The 96-h LC₅₀ values for X. laevis embryos ranged from 62 to 137 g/L (g extracted sediment per L), and the toxicity effect on body length of larvae was not significant under 20 g/L. However, the teratogenic effects produced by sediment organic extracts were diverse, including edema, hypopigmentation, cardiac and ocular malformations, abdomen recurved and curved spine. The percentage of malformations increased with increasing sediment organic extracts, and even reached almost 100% at 10 and 20 g/L in Guangzhou district. A gradient of pollution in the Pearl River sediments was discerned from the teratogenic toxicity. Guangzhou district showed higher teratogenic toxicity compared with Panyu and Nansha districts as a possible consequence of high levels of PAHs, PCBs, OCPs and NP in the sediments. The teratogenic effects of organic extracts from the Pearl River sediments were successfully assessed which indicated the feasibility of teratogenic potential studies of sediments using X. laevis embryos.     

Snakehead-fish cell line,SSN-1 (Ophicephalus striatus) as a model for cadmium genotoxicity testing

The purpose of this study is to evaluate the potential of snakehead-fish cell line (SSN-1 cells) derived from a striped snakehead (Ophicephalus striatus) as a model in the genotoxic assessment of cadmium (Cd). The first approach employed was to determine the contaminated Cd levels in commercial snakehead fish by the Graphite Atomic Absorption Spectrophotometer. In the second approach, the sensitivity of SSN-1 cells to cytotoxicity and genotoxicity of Cd was assessed by Trypan blue and micronucleus assays following 24, 48, and 72 h of incubation period. Exposure of SSN-1 cells to four increasing Cd concentrations ranging from 0.005 to 5 ppm for 72 h did not affect their survival as compared to the control cells. The Cd uptake by SSN-1 cells showed a concentration-dependent increase in intracellular Cd levels. Three non-cytotoxic Cd concentrations (0.05, 0.5, and 5 ppm) showed a concentration-dependent genotoxic effect, compared to relevant control cells. Both micronucleus frequencies and cadmium uptake levels by SSN-1 cells depended on exposure concentrations. These results showed that the SSN-1 cells are suitable for use as a model for in vitro Cd genotoxicity testing.     

Cytotoxicity and mutagenicity studies on migration extracts from nanocomposites with potential use in food packaging

Clays are used in the food packaging industry to obtain nanocomposites. The use of these new materials is a concern, because they could reach consumers by oral exposure through possible migration, and potential toxic effects could be derived. In the present study, several in vitro basal cytotoxicity and mutagenicity tests on migration extracts obtained from a nanocomposite material with poly (lactic) acid (PLA) and two modified clays, Clay1 and Clay2, are shown. Migration extracts in distilled water showed values of 0.1 ± 0.2 mg/dm² in all samples. Also, the content of characteristic metals of the clays structure (Al, Ca, Mg, Fe, Si) was studied and no statistical differences were observed. For the cytotoxicity assays, the human intestinal Caco-2 and human liver HepG2 cells were selected. Cells were exposed to concentrations between 2.5% and 100% extracts determining three different biomarkers of cellular viability. No significant differences were observed in the cytotoxicity assays. Finally, mutagenicity was evaluated by the Ames test and resulted in the absence of mutagenic response at all the concentrations assayed. Taking in account all above mentioned, these new materials show a good profile for their use in food packaging although further research is still needed.     

Impact of assay selection and study design on the outcome of cytotoxicity testing of medical devices: The case of multi-purpose vision care solutions

Medical device biocompatibility testing usually includes a cytotoxicity component. Assay selection and protocol design often depend on a specific testing standard rather than on the characteristics of the medical device. To better understand the impact of assay selection on study outcome of unstructured medical devices, we evaluated contact lens multi-purpose solutions (MPS) in the agar diffusion, direct contact and two elution cytotoxicity assays. To simulate the conditions of use, MPS were evaluated alone and in combination with contact lenses. All MPS passed the agar diffusion assay (n = 3) and extracts prepared from contact lenses soaked in MPS passed the USP elution assay (n = 3). Both the duration of contact and MPS concentration impacted the outcome of a modified elution assay. When tested at 25% strength for 48 h, all MPS evaluated were non-cytotoxic (n > 3). Test article movement and mechanical damage were significant issues with the direct contact assay. Movement was effectively controlled by manipulating contact lens orientation while using 0.8 mL culture medium. All MPS passed the USP direct contact cytotoxicity test when evaluated using this optimized methodology (n = 3). These data are consistent with MPS results in ocular irritation studies in rabbits (n = 3).     

Cytotoxicity and genotoxicity of phenazine in two human cell lines

Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9–123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2′-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC₅₀: 11 μM; 48 h IC₅₀: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC₅₀: 47 μM; 48 h IC₅₀: 17 μM). IC₅₀ values for HepG2 proliferation and viability were 54–77% lower compared to T24 cells. In both cell lines, IC₅₀ values for proliferation were 66–90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9–30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7–123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.     

Genotoxicity and oxidative stress of the mutagenic compounds formed in fumes of heated soybean oil,sunflower oil and lard

This study was to investigate the genotoxicity and cytotoxicity of the oil fumes formed from heating three common commercial cooking oils (soybean oil, sunflower oil, and lard) on human lung carcinoma pulmonary type II-like epithelium cell (A-549 cell). The major alkenal mutagenic compounds (trans-trans-2,4-decadienal, t-t-2,4-DDE; trans-trans-2,4-nonadienal, t-t-2,4-NDE; trans-2-decenal, t-2-DCA and trans-2-undecenal, t-2-UDA) contained in three oil fumes and their effects on the induction of reactive oxygen species (ROS) were also studied. It was found that the most potent mutagenic compound (t-t-2,4-DDE) of oil fumes was 66.4, 35.9 and 40.3 μg/g in soybean oil, sunflower oil and lard, respectively. The results indicated that the methanolic extracts of oil fumes could apparently lead to cytotoxicity and oxidative DNA damage. Glutathione (GSH) contents and the activities of antioxidant enzymes such as GSH reductase, and GSH S-transferase were adversely reduced by the methanolic extracts of oil fumes. When human A-549 cells were exposed to the methanolic extracts of oil fumes for 30 min, there was an increase in the formation of intracellular ROS, which was determined by dichlorofluorescein assay. Moreover, the methanolic extracts of oil fumes caused significant (p < 0.05) oxidative damage through the 8-hydroxy-2′-deoxyguanosine formation in A-549 cells at the concentrations from 50 to 200 μg/ml. These results demonstrated that the DNA damage in A-549 cells, induced by cooking oil fumes, was related to the ROS formation. It is inferred that women exposed to emitted fumes from cooking oil were at higher risk of contracting lung cancer.     

Determination of genotoxicity by the Comet assay applied to murine precision-cut lung slices

Precision-cut lung slices (PCLSs) are an organotypic lung model that is widely used in pharmacological, physiological, and toxicological studies. Genotoxicity testing, as a pivotal part of early risk assessment, is currently established in vivo in various organs including lung, brain, or liver, and in vitro in cell lines or primary cells. The aim of the present study was to provide the three-dimensional organ culture PCLS as a new ex vivo model for determination of genotoxicity using the Comet assay.Murine PCLS were exposed to increasing concentrations of ethyl methane sulfonate ‘EMS’ (0.03–0.4%) and formalin (0.5–5 mM). Tissue was subsequently dissociated, and DNA single-strand breaks were quantified using the Comet assay. Number of viable dissociated lung cells was between 4 × 10⁵ and 6.7 × 10⁵ cells/slice. Even treatment with EMS did not induce toxicity compared to untreated tissue control. As expected, DNA single-strand breaks were increased dose-dependently and significantly after exposure to EMS. Here, tail length rose from 24 μm to 75 μm. In contrast, formalin resulted in a significant induction of DNA cross-links.The effects induced by EMS and formalin demonstrate the usefulness of PCLS as a new ex vivo lung model for genotoxicity testing in the early risk assessment of airborne substances in the future.     

Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells

IntroductionPrestoBlue (PB) is a new, simple and extremely fast live assay to monitor cell viability and cytotoxicity.Herein, we compared two in vitro cytotoxicity assays, new (PB) and classic (MTT), in the assessment of viability of human umbilical vein endothelial cells (HUVECs) in the presence of selected plant extracts.MethodsThe anti-proliferative effects of two extracts from medicinal plants, i.e., walnut husk extract and spent hop extract, used at the concentration range of 1–200 μg/ml of gallic acid equivalent, were compared with the effects recorded for resveratrol — a natural polyphenolic compound. Reduction of dyes by endothelial cells was determined colorimetrically (MTT and PB) and fluorometrically (PB).ResultsAt higher concentrations, all tested compounds caused significant loss of cell viability. Regardless of plant compound, the PB assay, when measured colorimetrically, produced higher EC₅₀ values compared to other modes of measurement, however, the statistically significant differences in EC₅₀ values among the assays were revealed only for spent hop extract. Conversely, the EC₅₀ values for each plant compound obtained in MTT (colorimetric assay) and PB (fluorometric assay) were similar. According to EC₅₀ values, the cytotoxicity of plant compounds ranked as follows: spent hop extract > resveratrol > walnut husk extract. Furthermore, the MTT assay showed overall lower inter-assay variability and higher signal-to-noise ratio compared to PB assay.DiscussionIn conclusion, we recommend fluorometric PrestoBlue assay for cytotoxicity assessment in human endothelial cells. Due to substantial differences in EC₅₀ values and S/N ratios between spectrophotometric PB and MTT or fluorometric PB assays, colorimetric quantification of HUVECs' viability with the use of PB reagent should be avoided.     

The effect of long term storage on tobacco smoke particulate matter in in vitro genotoxicity and cytotoxicity assays

Particulate matter (PM) collected from mainstream tobacco smoke is a test article commonly used for in vitro genotoxicity and cytotoxicity testing of combustible tobacco products. However, little published data exists concerning the stability of PM. We completed a 2 year study to quantify the effect of PM storage at ?80 °C, on the genotoxicity and cytotoxicity of PM generated from 3R4F and M4A reference cigarettes. The Ames test, Micronucleus assay (MNvit), Mouse Lymphoma assay (MLA) and the Neutral Red Uptake assay (NRU) were used. The majority of M4A and 3R4F PMs were genotoxic and cytotoxic at the timepoints tested. Some minor but statistically significant differences were observed for stored versus freshly prepared PM, but the magnitude of changes were within the variability observed for repeat testing.     

Juniperus oxycedrus L. subsp. oxycedrus and Juniperus oxycedrus L. subsp. macrocarpa (Sibth. & Sm.) Ball. “berries” from Turkey: Comparative evaluation of phenolic profile,antioxidant, cytotoxic and antimicrobial activities

This work aimed to evaluate and compare the phenolic profile and some biological properties of the ripe “berries” methanol extracts of Juniperus oxycedrus L. subsp. oxycedrus (Joo) and Juniperus oxycedrus L. subsp. macrocarpa (Sibth. & Sm.) Ball. (Jom) from Turkey. The total phenolic content resulted about 3-fold higher in Jom (17.89 ± 0.23 mg GAE/g extract) than in Joo (5.14 ± 0.06 mg GAE/g extract). The HPLC–DAD–ESI–MS analysis revealed a similar flavonoid fingerprint in Joo and Jom, whereas a difference in their quantitative content was found (4632 μg/g extract and 12644 μg/g extract). In addition, three phenolic acids were detected in Jom only (5765 μg/g extract), and protocatechuic acid was the most abundant one. The antioxidant capacity of the extracts was evaluated by different in vitro assays: in the DPPH and in the TBA tests a stronger activity in Jom was highlighted, while Joo exhibited higher reducing power and metal chelating activity. Joo and Jom did not affect HepG2 cell viability and both extracts resulted virtually non-toxic against Artemia salina. The extracts were also studied for their antimicrobial potential, displaying efficacy against Gram-positive bacteria.     

Pre-clinical safety assessment of the synthetic human milk,nature-identical,oligosaccharide Lacto-N-neotetraose (LNnT)

Lacto-N-neotetraose (LNnT) is a tetrasaccharide naturally occurring in human breast milk, but not in cow’s milk. The safety data generated on a potential new LNnT ingredient produced by chemical synthesis is presented. Standard in vitro genotoxicity tests were performed. LNnT was also administered via gavage in 14-, 28- and 90-day studies at levels corresponding to 0 (control), 1000, 2500 and 5000 mg/kg bw/day in juvenile rats. Fructooligosaccharide (FOS) currently approved for use in infant formulae was used as a reference control at one dose level of 5000 mg/kg bw/day. LNnT was non-mutagenic in in vitro assays. Oral administration up to 5000 mg/kg bw/day to rats over 90 days was not associated with any adverse effects, based on clinical observations, body weight gain, feed consumption, clinical pathology, organ weights and histopathology findings. Regarding gastrointestinal effects, LNnT was better tolerated than FOS during the first 2 weeks of treatment. A No Observed Adverse Effect Level (NOAEL) of 5000 mg/kg bw/day for both male and female rats was identified for LNnT when administered by gavage for 90 days. These findings in the juvenile rat support the safety of LNnT for possible use in infant foods and allow further investigation in clinical studies.     

Protective effect of dry olive leaf extract in adrenaline induced DNA damage evaluated using in vitro comet assay with human peripheral leukocytes

Excessive release of stress hormone adrenaline is accompanied by generation of reactive oxygen species which may cause disruption of DNA integrity leading to cancer and age-related disorders. Phenolic-rich plant product dry olive leaf extract (DOLE) is known to modulate effects of various oxidants in human cells. The aim was to evaluate the effect of commercial DOLE against adrenaline induced DNA damage in human leukocytes by using comet assay. Peripheral blood leukocytes from 6 healthy subjects were treated in vitro with three final concentrations of DOLE (0.125, 0.5, and 1 mg/mL) for 30 min at 37 °C under two different protocols, pretreatment and post-treatment. Protective effect of DOLE was assessed from its ability to attenuate formation of DNA lesions induced by adrenaline. Compared to cells exposed only to adrenaline, DOLE displayed significant reduction (P < 0.001) of DNA damage at all three concentrations and under both experimental protocols. Pearson correlation analysis revealed a significant positive association between DOLE concentration and leukocytes DNA damage (P < 0.05). Antigenotoxic effect of the extract was more pronounced at smaller concentrations. Post-treatment with 0.125 mg/mL DOLE was the most effective against adrenaline genotoxicity. Results indicate genoprotective and antioxidant properties in dry olive leaf extract, strongly supporting further explorations of its underlying mechanisms of action.     

Comparative study of cytotoxic and genotoxic effects induced by herbicide S-metolachlor and its commercial formulation Twin Pack Gold® in human hepatoma (HepG2) cells

The in vitro effects of S-metolachlor and its formulation Twin Pack Gold^® (96% a.i.) were evaluated in human hepatoma (HepG2) cells. Cytokinesis-blocked micronucleus cytome (CBMN-cyt) and MTT assays as well as Neutral Red uptake were employed for genotoxicity and cytotoxicity evaluation. Activities were tested within the concentration range of 0.25–15 μg/ml S-metolachlor for 24 h of exposure. Both compounds rendered a minor reduction in the NDI although not reaching statistical significance. Results demonstrated that the S-metolachlor was not able to induce MNs. On the other hand, 0.5–6 μg/ml Twin Pack Gold^® increased the frequency of MNs. When cytotoxicity was estimated, S-metolachlor was not able to induce either a reduction of lysosomal or mitochondrial activity. Contrarily, whereas 1–15 μg/ml Twin Pack Gold^® induced a significant reduction of mitochondrial activity, all tested concentrations of the formulated product induced a significant decrease of lysosomal performance as a function of the concentration of the S-metolachlor-based formulation titrated into cultures. Genotoxicity and cytotoxicity differences obtained with pure S-metolachlor and the commercial S-metolachlor-based formulation indicate that the latter may contain additional unsafe xenobiotics and support the concept of the importance of evaluating not only the active principle but also the commercial formulation when estimating the real hazard from agrochemicals.     

Safety evaluation of a natural eggshell membrane-derived product

Natural Eggshell Membrane (NEM®) is a novel dietary ingredient that contains naturally occurring glycosaminoglycans and proteins essential for maintaining healthy joint and connective tissues. NEM® was evaluated for safety via in vitro and in vivo toxicological studies. This included testing for cytotoxicity, genotoxicity, acute oral toxicity, and 90-day repeated-dose oral toxicity. NEM® did not exhibit any cytotoxic effects at a dose of 100 μg in an in vitro human cell viability assay after incubation for up to 20 h. NEM® did not exhibit any genotoxic effects in an in vitro assay of four strains of histidine-dependent Salmonella typhimurium and one strain of tryptophan-dependent Escherichia coli at a dose of up to 5000 μg/plate. NEM® did not exhibit any signs of acute toxicity in rats at a single oral dose of up to 2000 mg/kg body weight, nor signs of toxicity (via urinalysis, hematology, clinical chemistry, or histopathological evaluation) in rats at a repeated oral dose of up to 2000 mg/kg body weight per day for 90 days. The results of these studies suggest that NEM® may be safe for human consumption.     

Oxidative stress,genotoxicity and cytotoxicity of 1-methyl-3-octylimidazolium chloride on Paramisgurnus dabryanus

This study evaluated the toxicity of 1-methyl-3-octylimidazolium chloride ([C₈mim]Cl) on Paramisgurnus dabryanus by enzyme analysis, comet assay, and apoptosis analysis. The study showed that [C₈mim]Cl had an obvious toxic effect inducing oxidative stress, genotoxicity, and cytotoxicity to fish liver cells. [C₈mim]Cl also induced changes in the activities of superoxide dismutase and catalase, and the glutathione content and malondialdehyde level in fish exposed at 20–80 mg L⁻¹. With increased exposure concentration and time, the four antioxidant enzyme activities, three different comet parameters and apoptosis rates of tested cells were significantly increased, with significant differences (P < 0.05 or P < 0.01) observed between control group and each treatment group. This study shows that [C₈mim]Cl could be a threat to aquatic organism health when accidentally released into aquatic ecosystems.     

Safety evaluation of oleic-rich triglyceride oil produced by a heterotrophic microalgal fermentation process

Numbers of macro- and microalgae have been used as food sources in various cultures for centuries. Several microalgae are currently being developed as modern food ingredients. The dietary safety of oleic-rich microalgal oil produced using a heterotrophic fermentation process was assessed in a 13-week feeding trial in rats with genotoxic potential evaluated using in vitro and in vivo assays. In the genotoxicity assays, the test oil was not mutagenic in Salmonella typhimurium or Escherichia coli tester strains (⩽5000 μg/plate) with or without metabolic activation. Further, no clastogenic response occurred in chromosome aberration assays in the bone marrow of mice administered a single intraperitoneal dose (2000 mg/kg). In the subchronic study, rats consumed feed containing 0, 25,000, 50,000 or 100,000 ppm oleic-rich oil for 90 days. No treatment-related mortalities or adverse effects occurred in general condition, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights or histopathology. Although several endpoints exhibited statistically significant effects, none were dose-related or considered adverse. Taking all studies into consideration, the NOAEL for the oleic-rich oil was 100,000 ppm, the highest concentration tested and equivalent to dietary NOAELs of 5200 mg/kg bw/day and 6419 mg/kg bw/day in male and female rats, respectively.