Toxicology of 3-epi-deoxynivalenol,a deoxynivalenol-transformation product by Devosia mutans 17-2-E-8

Microbial detoxification of deoxynivalenol (DON) represents a new approach to treating DON-contaminated grains. A bacterium Devosia mutans 17-2-E-8 was capable of completely transforming DON into a major product 3-epi-DON and a minor product 3-keto-DON. Evaluation of toxicities of these DON-transformation products is an important part of hazard characterization prior to commercialization of the biotransformation application. Cytotoxicities of the products were demonstrated by two assays: a MTT bioassay assessing cell viability and a BrdU assay assessing DNA synthesis. Compared with DON, the IC₅₀ values of 3-epi-DON and 3-keto-DON were respectively 357 and 3.03 times higher in the MTT bioassay, and were respectively 1181 and 4.54 times higher in the BrdU bioassay. Toxicological effects of 14-day oral exposure of the B6C3F₁ mouse to DON and 3-epi-DON were also investigated. Overall, there were no differences between the control (free of toxin) and the 25 mg/kg bw/day or 100 mg/kg bw/day 3-epi-DON treatments in body and organ weights, hematology and organ histopathology. However, in mice exposed to DON (2 mg/kg bw/day), white blood cell numbers and serum immunoglobulin levels were altered relative to controls, and lesions were observed in adrenals, thymus, stomach, spleen and colon. Taken together, in vitro and in vivo studies indicate that 3-epi-DON is substantially less toxic than DON.     

Estimation of in vivo and in vitro exposure to bisphenol A as food contaminant

The goal of this cross-sectional study was to examine the occurrence of bisphenol A (BPA) in the morning spot urine taken from 145 female volunteers of various ages. Total urine BPA concentration was detected in 38.6% samples in the 0.92–70.96 μg/g Cr range. The majority of BPA + women belonged to the 25 + body mass index (BMI) group (54.5% were overweight and 43.4% were obese women). Occurrence of BPA in the urine samples was higher at 40 + ages. The maximum BPA concentration of 70.96 μg/g Cr was detected in the urine sample of an obese woman. It is known that BPA is highly toxic in vitro. In this study BPA impaired significantly the growth of all investigated cell lines, i.e. the EC₅₀ values were reached at very low concentrations, in the range from 3.24 to 34.85 μg/mL. The obtained in vivo results suggest that a higher exposure to BPA could contribute to weight problems in women and the absence of the BPA in vitro selective toxicity studies indicates to its general toxic mode of action and raises awareness of the health risks associated with its ubiquitous presence in the environment.     

Lipid-soluble green tea extract: Genotoxicity and subchronic toxicity studies

To assess the potential safety of lipid soluble green tea extract, also referred to as lipid soluble tea polyphenols (LSTP), a series of genotoxicity tests were conducted, including an Ames, in vivo mouse micronucleus, and in vivo mouse sperm abnormality test. The toxicity of LSTP was evaluated in 90- and 30-day feeding studies. LSTP did not show mutagenic activity in the Ames test and no genotoxic potential in the in vivo assays at doses up to 10 g/kg body weight (bw). In the 90-day feeding study, LSTP was given in the diet at levels providing 0, 0.125, 0.25, or 0.50 g/kg bw/day. No significant effects were noted on body weight, food consumption, hematology, clinical chemistry, organ weights, and histopathological examination. The no-observed-adverse-effect level (NOAEL) was therefore considered to be 0.50 g/kg bw/day, the highest dose tested. Likewise, dosing of SD rats by gavage for 30 days also showed no adverse effects of growth, hematology, clinical chemistry, organ weights, or histopathology at doses of 0.58, 1.17, and 2.33 g/kg bw/day. The NOAEL in the 30-day study was considered to be the highest dose tested. These data provide evidence to support the safe use of LSTP in food.     

Genotoxicity,acute and subchronic toxicity evaluation of savory food ingredients

The potential toxicity of two savory food ingredients produced by fermentation of enzymatically hydrolyzed corn starch (Savory Base 100 and Savory Base 200) was evaluated individually in a bacterial reverse mutation assay, an in vitro mammalian cell gene mutation assay, an acute oral study and as a mixture in a 90-day dietary study. In the bacterial reverse mutation and in vitro mammalian cell gene mutation assays, neither ingredient was mutagenic at concentrations up to 5000 μg/plate and 5000 μg/mL, respectively in the presence and absence of metabolic activation. In the acute study, the no-observed-adverse-effect level (NOAEL) for each Savory Base 100 and Savory Base 200 in male and female rats was 2000 mg/kg body weight. In the 90-day study, the hematology and clinical chemistry findings and histopathological changes noted in the liver, heart and kidneys were deemed to be of no toxicological significance, as the mean values were within the historical control range, were not dose-dependent, occurred at a similar frequency in control groups, or only occurred in the control group. Considering these findings, the NOAEL for Savory Base 100 and Savory Base 200 was 2333 and 1167 mg/kg body weight, respectively, the highest dose tested in each case.     

CSAHi study-2: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information

With the aim of reconsidering ICH S7B and E14 guidelines, a new in vitro assay system has been subjected to worldwide validation to establish a better prediction platform for potential drug-induced QT prolongation and the consequent TdP in clinical practice. In Japan, CSAHi HEART team has been working on hiPS-CMs in the MEA (hiPS-CMs/MEA) under a standardized protocol and found no inter-facility or lot-to-lot variability for proarrhythmic risk assessment of 7 reference compounds. In this study, we evaluated the responses of hiPS-CMs/MEA to another 31 reference compounds associated with cardiac toxicities, and gene expression to further clarify the electrophysiological characteristics over the course of culture period.The hiPS-CMs/MEA assay accurately predicted reference compounds potential for arrhythmogenesis, and yielded results that showed better correlation with target concentrations of QTc prolongation or TdP in clinical setting than other current in vitro and in vivo assays. Gene expression analyses revealed consistent profiles in all samples within and among the testing facilities.This report would provide CiPA with informative guidance on the use of the hiPS-CMs/MEA assay, and promote the establishment of a new paradigm, beyond conventional in vitro and in vivo assays for cardiac safety assessment of new drugs.     

A comprehensive study on in vitro and in vivo toxicological evaluation of Artemisia capillaris

Artemisia capillaris (AC) has been used as an alternative therapy in obesity, atopic dermatitis, and liver diseases through several biological activity including anti-steatotic, antioxidant, and anti-inflammatory activities. Despite its ethnomedicinal benefits, no sufficient background information is available about the long-term safety and genotoxicity of the AC extract. Therefore, the present study was carried out to investigate the 13-week subchronic toxicity and genotoxicity of the AC extract according to the test guidelines published by the Organization for Economic Cooperation and Development. In the 13-week toxicity study using doses of 25, 74, 222, 667, and 2000 mg/kg body weight, oral administration of the AC extract in male and female rats did not result in any significant adverse effects in food/water consumption, body weight, mortality, hematology, serum biochemistry, organ weight and histopathology. Accordingly, the no-observed-adverse-effect level in rats of both genders was established for the AC extract at 2000 mg/kg/day, the highest dose level tested. In addition, the AC extract was not genotoxic in a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. In conclusion, we demonstrated that the AC extract is considered as a safe traditional medicine for human consumption.     

Fish cell lines as a tool for the ecotoxicity assessment and ranking of engineered nanomaterials

Risk assessment of engineered nanomaterials (ENMs) is being hindered by the sheer production volume of these materials. In this regard, the grouping and ranking of ENMs appears as a promising strategy. Here we sought to evaluate the usefulness of in vitro systems based on fish cell lines for ranking a set of ENMs on the basis of their cytotoxicity. We used the topminnow (Poeciliopsis lucida) liver cell line (PLHC-1) and the rainbow trout (Oncorhynchus mykiss) fibroblast-like gonadal cell line (RTG-2). ENMs were obtained from the EU Joint Research Centre repository. The size frequency distribution of ENM suspensions in cell culture media was characterized. Cytotoxicity was evaluated after 24 h of exposure. PLHC-1 cells exhibited higher sensitivity to the ENMs than RTG-2 cells. ZnO-NM was found to exert toxicity mainly by altering lysosome function and metabolic activity, while multi-walled carbon nanotubes (MWCNTs) caused plasma membrane disruption at high concentrations. The hazard ranking for toxicity (ZnO-NM > MWCNT ≥ CeO₂-NM = SiO₂-NM) was inversely related to the ranking in size detected in culture medium. Our findings reveal the suitability of fish cell lines for establishing hazard rankings of ENMs in the framework of integrated approaches to testing and assessment.     

Detection of ECG effects of (2R,4R)-monatin,a sweet flavored isomer of a component first identified in the root bark of the Sclerochitin ilicifolius plant

Enzymatically-synthesized (2R,4R)-monatin has, due to its pure sweet taste, been evaluated for potential use in foods. Non-clinical studies have shown that (2R,4R)-monatin is well tolerated at high dietary concentrations, is not genotoxic/mutagenic, carcinogenic, or overtly toxic. In a pharmacokinetic and metabolism study involving 12 healthy males, consumption of a single oral dose (2 mg/kg) of (2R,4R)-monatin resulted in a small reduction of heart rate and prolongation of the QTcF interval of 20–24 ms, corresponding to the time of peak plasma levels (t_max). These findings were evaluated in a cross-over thorough QT/QTc study with single doses of 150 mg (2R,4R)-monatin, placebo and positive control (moxifloxacin) in 56 healthy males. Peak (2R,4R)-monatin plasma concentration (1720 ± 538 ng/mL) was reached at 3.1 h (mean t_max). The placebo-corrected, change-from-baseline QTcF (ΔΔQTcF) reached 25 ms three hours after dosing, with ΔΔQTcF of 23 ms at two and four hours. Using exposure response (QTc) analysis, a significant slope of the relationship between (2R,4R)-monatin plasma levels and ΔΔQTcF was demonstrated with a predicted mean QT effect of 0.016 ms per ng/mL. While similarly high plasma levels are unlikely to be achieved by consumption of (2R,4R)-monatin in foods, QTc prolongation at this level is a significant finding.     

Genotoxic and cytotoxic evaluation of Jatropha dioica Sessé ex Cerv. by the micronucleus test in mouse peripheral blood

Jatropha dioica Sessé ex Cerv. is a medicinal plant credited with low cytotoxicity in vitro. Thus, the objective of this work was to evaluate the possible genotoxic and cytotoxic effect in vivo of the J. dioica aqueous extract by means of micronucleus assay in mouse peripheral blood. Four different J. dioica aqueous extract dose-units were evaluated (30, 60, 100, and 300 mg/kg). The extract was administered orally to male Balb-C-strain mice every 24 h during 5 days. Blood samples were taken at 0, 24, 48, 72, 96, and 120 h from the mouse's tail and were performed in duplicate extensions. The number of Polychromatic Erythrocytes (PCE), Polychromatic Micronucleus Erythrocytes (PCEMN), and Micronucleus Erythrocytes (MNE) was determined at the different sampling times in the different study groups. Our results showed that the group that received 60 mg/kg of cyclophosphamide (positive control) presented a significant decrease in the PCE (p = 0.044) proportion and a significant increase in MNE (p = 0.032, p = 0.0001). The groups that received the different J. dioica aqueous extract doses did not present either a PCE decrease or an increase in PCEMN and MNE. J. dioica exerts neither a genotoxic nor a cytotoxic effect on mouse peripheral blood at high doses.     

Flow cytometry evaluation of in vitro cellular necrosis and apoptosis induced by silver nanoparticles

Particles possess unique properties in the nanoscale, e.g., enhanced catalytic activity, high surface area, and light emission/absorption properties, that might result in interference with colorimetric in vitro cytotoxicity assays such as MTT, XTT or MTS. Alternatively, assays that do not use spectrophotometric detection, such as trypan blue exclusion or flow cytometry (FC) based assays, are less likely to be influenced by nanoparticle interference. The aim of this study was to evaluate FC assays to assess the cytotoxicity of three different sizes (10, 100, or 200 nm) of silver nanoparticles (AgNPs) at different mass concentrations (1, 25, or 50 ug/ml) in L-929 fibroblast cells. After 4 h and 24 h exposure, cell necrosis and apoptosis were assessed using 7-AAD and Annexin V dyes, respectively, with FC. The data indicate that cell necrosis and apoptosis in AgNP-exposed fibroblasts depends on dose, exposure time, and AgNP size. The data indicate that AgNPs produced a dose- and time-dependent decrease in cell viability; however, 10 nm AgNPs were significantly more toxic than larger-sized particles. Thus, standard FC assays can be utilized to assess apoptosis and necrosis in response to nanomaterial exposure.     

Cyto-genotoxicity and oxidative stress induced by zinc oxide nanoparticle in human lymphocyte cells in vitro and Swiss albino male mice in vivo

ZnO-np has immense potential and application in cosmetic and health care sectors. Hence it was imperative to assess the toxicity/safety of these nanoparticles. In this study, we have evaluated the effects of ZnO-np in human peripheral blood mononuclear cells (PBMCs) in vitro and in Swiss albino male mice in vivo for cyto-genotoxicity and oxidative damage. In vitro results showed that ZnO-nps were weakly genotoxic, induced significant decrease in mitochondrial membrane potential and was capable of ROS generation, leading to apoptosis. In bone marrow cells in vivo, reduction of mitochondrial membrane potential (MMP), increased oxidative stress and G0/G1 cell cycle arrest was observed along with chromosome aberrations and micronuclei formation. In liver cells DNA damage and induction of oxidative stress with concurrent decrease in inhibition of antioxidant enzymes were noted. These in vitro and in vivo results demonstrated that ZnO-np induced genotoxic response and ROS production leading to apoptotic cell death and established a good co-relation between the two biological systems. More importantly, the results stress on the need of multiple endpoint assay-approaches, with an in vitro-in vivo study design to assess nanoparticle toxicology.     

A dietary two-generation reproductive toxicity study of (2R,4R)-monatin salt in Crl:CD(SD) rats

(2R,4R)-Monatin salt [sodium/potassium 2R,4R-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate] was fed at 5000, 15,000, or 35,000 ppm to Crl:CD(SD) rats over two generations. Reduced body weights were observed at all dose levels. Sustained effect on body weight gain at 35,000 ppm in the F₀ and F₁ parental animals was associated with lower feed efficiency, soft stool, and slightly lower numbers of implantation sites. Lower numbers of pups born and live litter size at 35,000 ppm were considered secondary to slightly lower numbers of former implantation sites in the dams. Spermatogenic endpoints, estrous cyclicity, reproductive performance, mean gestation length, and parturition were unaffected in the F₀ and F₁ generations. There were no effects on F₁ and F₂ generation postnatal survival. Reduced pre-weaning pup body weights at 35,000 ppm resulted in lower F₁ and F₂ body weights at study termination. Slight delays in pubertal landmarks in the F₁ offspring were considered secondary to the reduced pup body weights. The no-observed-adverse-effect level (NOAEL) was 15,000 ppm for systemic, reproductive, and neonatal effects based on test article-related effects on body weight and food efficiency, slight decrease in maternal implantation sites and corresponding reduction in live litter size, and reductions in pre-weaning pup body weights at 35,000 ppm.     

Assessment of oxidative stress responses and the cytotoxic and genotoxic potential of the herbicide tembotrione in HepG2 cells

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 μg/mL), residential exposure level (0.002 μg/mL) and acceptable operator exposure level (0.0012 μg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus “cytome” assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.     

Delamanid is not metabolized by Salmonella or human nitroreductases: A possible mechanism for the lack of mutagenicity

Nitro-containing compounds such as nitrofuran and nitroimidazole are drugs used for the treatment of infectious diseases. However, many of these nitro-containing drugs are positive in the bacterial reverse mutation assay (Ames test). The recently approved anti-multidrug-resistant tuberculosis (MDR-TB) drug, delamanid (Deltyba™; OPC-67683), a derivative of 4-nitroimidazole, was negative for mutagenicity in the Ames assay. In Salmonella typhimurium, mutagenicity of nitro compounds has been closely associated with the ability of nitroreductase to metabolize (degradation)these compounds. To explore the lack of mutagenicity for delamanid, we examined the initial metabolic rate and mutagenic-specific activity of a series of nitro compounds in S. typhimurium TA100. The order of maximum mutagenic-activity was nitrofuran > 2-nitroimidazole > 5-nitroimidazole ≥ 4-nitroimidazole, which is very similar to the order of initial metabolic rate, i.e., the Pearson's correlation coefficient (r = 0.85) showed a correlation between metabolic rate and mutagenic-activity. No metabolism of delamanid was detected even after 60 h of treatment. In addition, delamanid was not reduced by two human nitroreductases. These facts may explain the absence of genotoxicity of delamanid in both in vitro and in vivo tests.     

Cytotoxic,genotoxic and biochemical markers of insecticide toxicity evaluated in human peripheral blood lymphocytes and an HepG2 cell line

This study evaluated the cyto- and genotoxic effects of three pesticides: α-cypermethrin, chlorpyrifos and imidacloprid applied in vitro to human lymphocytes and HepG2 cells for exposure times of 4 and 24 h at concentrations corresponding to OEL, ADI and REL. Assessments were made using oxidative stress biomarkers and the alkaline comet, cytokinesis-block micronucleus cytome and cell viability assays. Low doses of all three pesticides displayed DNA damaging potential, both in lymphocytes and HepG2 cells. At the tested concentrations, all three compounds induced lymphocyte apoptosis, though α-cypermethrin and chlorpyrifos were generally more cyto- and genotoxic than imidacloprid. At the tested concentrations, oxidative stress biomarkers were not significantly altered, and the effects mediated indirectly through free radicals may not have a key role in the formation of DNA damage. It is likely that the DNA damaging effects were caused by direct interactions between the tested compounds and/or their metabolites that destabilized the DNA structure. The tested pesticides had the potential for MN, NB and NPB formation and to disturb cell cycle kinetics in both cell types. There were also indications that exposure to α-cypermethrin led to the formation of crosslinks in DNA, though this would require more detailed study in the future.     

Genotoxicity of monosodium glutamate

Monosodium glutamate (MSG) is one of the most widely used flavor enhancers throughout the world. The aim of this study is to investigate the genotoxic potential of MSG by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), cytokinesis-blocked micronucleus (CBMN), and random amplified polymorphic DNA-polimerase chain reaction (RAPD-PCR) in cultured human lymphocytes and alkaline comet assays in isolated human lymphocytes, which were incubated with six concentrations (250, 500, 1000, 2000, 4000 and 8000 μg/mL) of MSG. The result of this study indicated that MSG significantly and dose dependently increased the frequencies of CAs, SCE and MN in all treatments and times, compared with control. However, the replication (RI) and nuclear division indices (NDI) were not affected. In this paper, in vitro genotoxic effects of the MSG was also investigated on human peripheral lymphocytes by analysing the RAPD-PCR with arbitrary 10-mer primers. The changes occurring in RAPD profiles after MSG treatment include increase or decrease in band intensity and gain or loss of bands. In the comet assay, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1-h in vitro exposure. Our results demonstrate that MSG is genotoxic to the human peripheral blood lymphocytes in vitro.     

FEMA expert panel review of p-mentha-1,8-dien-7-al genotoxicity testing results

p-Mentha-1,8-dien-7-al is a naturally occurring cyclic alpha,beta-unsaturated aldehyde that is used as a flavoring substance throughout the world. Due to the chemical structure and the potential DNA reactivity of the alpha,beta-unsaturated carbonyl moiety, a battery of genotoxicity assays was requested by the European Food Safety Authority. Previous genotoxicity studies on the substance gave mixed results, but both positive and negative results were hampered by not always being performed to any standard guideline. The new test battery data indicated some evidence of mutagenicity in vitro, but an in vivo comet/micronucleus combination assay performed in rats was concluded by the study directors to not result in any biologically relevant positive responses. However, EFSA concluded that the in vivo assay gave evidence that p-mentha-1,8-dien-7-al was of potential genotoxic concern. The Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) has reviewed the newly available data and considered its interpretation relative to standard guidelines such as that established by the Organization for Economic Cooperation and Development, and has concluded that the results in the comet/micronucleus combination assay are consistent with the interpretation by the study directors; namely, that p-mentha-1,8-dien-7-al does not appear to have any in vivo genotoxic potential.     

Inhibitory effects of wild dietary plants on lipid peroxidation and on the proliferation of human cancer cells

Thirteen hydroalcoholic extracts of edible plants from Southern Italy were evaluated for their in vitro antioxidant and antiproliferative activity on three human cancer cell lines: breast cancer MCF-7, hepatic cancer HepG2 and colorectal cancer LoVo. After 48 h of incubation the most antiproliferative plant extract was rosemary (Rosmarinus officinalis L.) on LoVo cell line with IC₅₀ of 16.60 µg/ml. Oregano (Origanum vulgare L. subsp. viridulum) showed a selective antiproliferative activity on hepatic cancer with IC₅₀ of 32.59 µg/ml.All the extracts, with the exception of Diplotaxis tenuifolia (L.) DC., exerted antioxidant properties, the most active plants being dewberry (Rubus caesius L.) and “laprista” (Rumex conglomerates Murray) with IC₅₀ of 4.91 and 5.53 µg/ml, respectively. Rumex conglomeratus contained the highest amount of flavonoids (15.5 mg/g) followed by Portulaca oleracea L. (11.8 mg/g). Rosmarinus officinalis contained the highest number of terpenes. Among them ketoursene (14.7%) and aristolone (11.3%) were found to be the major constituents. P. oleracea and Raphanus raphanistrum L. subsp. landra contained the highest number of sterols.     

Effects of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites on DNA damage and repair under in vitro conditions

3-monochloropropane-1,2-diol (3-MCPD) is a food contaminant that occurs during industrial production processes and can be found mainly in fat and salt containing products. 3-MCPD has exhibited mutagenic activity in vitro but not in vivo, however, a genotoxic mechanism for the occurrence of kidney tumors has not so far been excluded. The main pathway of mammalian 3-MCPD metabolism is via the formation of β – chlorolactatic acid and formation of glycidol has been demonstrated in bacterial metabolism. The aim of this study was to investigate genotoxic and oxidative DNA damaging effects of 3-MCPD and its metabolites, and to provide a better understanding of their roles in DNA repair processes. DNA damage was assessed by alkaline comet assay in target rat kidney epithelial cell lines (NRK-52E) and human embryonic kidney cells (HEK-293). Purine and pyrimidine base damage, H₂O₂ sensitivity and DNA repair capacity were assessed via modified comet assay. The results revealed in vitro evidence for increased genotoxicity and H₂O₂ sensitivity. No association was found between oxidative DNA damage and DNA repair capacity with the exception of glycidol treatment at 20 μg/mL. These findings provide further insights into the mechanisms underlying the in vitro genotoxic potential of 3-MCPD and metabolites.     

Toxicological potential of acyl glucuronides and its assessment

Idiosyncratic drug toxicity (IDT) is a serious problem in drug development. Reactive metabolites are postulated to be one of the causes for IDT. Conjugated metabolites are generally non-reactive except for acyl glucuronides (AGs), which are sufficiently reactive to covalently bind to endogenous proteins. Thus, it has been suggested that AGs would contribute to IDT caused by carboxylic acid-containing drugs. Glucuronidation of a carboxylate residue is catalyzed by UDP-glucuronosyltransferase 1A and 2B isoforms. Unstable AGs undergo intramolecular rearrangements as well as non-enzymatic and enzymatic hydrolysis. The instability and reactivity toward proteins have been well studied for a large number of AGs. Moreover, the half-life of AGs in neutral buffer is becoming a common marker for the prediction of toxicity caused by carboxylic acid-containing drugs in the screening of new chemical entities; however, the underlying mechanisms of the toxicity are not elucidated. Recently, an immunostimulation assay has been proposed for the assessment of the toxicological potential of AGs, which may have a better predictability compared with half-life and peptide adduct assays. In addition to in vitro studies, studies in model animals indicate the in vivo toxicological potential of AGs and help understand the mechanisms of the AG toxicity.