Exogenous Metabolic Activation Research Articles

Optimizing the detection of N-nitrosamine mutagenicity in the Ames test

Accurately determining the mutagenicity of small-molecule N-nitrosamine drug impurities and nitrosamine drug substance-related impurities (NDSRIs) is critical to identifying mutagenic and cancer hazards. In the current study we have evaluated several approaches for enhancing assay sensitivity for evaluating the mutagenicity of N-nitrosamines in the bacterial reverse mutagenicity (Ames) test. Preincubation assays were conducted using five activation conditions: no exogenous metabolic activation and metabolic activation mixes employing both 10% and 30% liver S9 from hamsters and rats pretreated with inducers of enzymatic activity. In addition, preincubations were conducted for both 60 min and 30 min. These test variables were evaluated by testing 12 small-molecule N-nitrosamines and 17 NDSRIs for mutagenicity in Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537, and Escherichia coli strain WP2 uvrA (pKM101). Eighteen of the 29 N-nitrosamine test substances tested positive under one or more of the testing conditions and all 18 positives could be detected by using tester strains TA1535 and WP2 uvrA (pKM101), preincubations of 30 min, and S9 mixes containing 30% hamster liver S9. In general, the conditions under which NDSRIs were mutagenic were similar to those found for small-molecule N-nitrosamines.

Mutagenic and genotoxic in silico QSAR prediction of dimer impurity of gliflozins; canagliflozin, dapaglifozin, and emphagliflozin and in vitro evaluation by Ames and micronucleus test

Canagliflozin, Dapagliflozin, and Empagliflozin, glucagon-like peptide-1 receptor agonists, are indicated for managing type II diabetes. Although the genotoxicity profiles of these drugs are well-explored, limited information exists regarding the genotoxic potential of their impurities. In this investigation, the dimer impurities of Canagliflozin, Dapagliflozin, and Empagliflozin underwent both in silico and in vitro assessments for mutagenic potential. Tester strains of Salmonella typhimurium and Escherichia coli were subjected to the Ames test, utilizing concentrations of up to 1 µg per plate, with and without the presence of metabolic activation. Evaluation of micronucleus induction in TK6 cells was conducted through a micronucleus test, exploring concentrations up to 500 µg/mL, with or without the presence of exogenous metabolic activation. Under the specific test conditions, the dimer impurities of Canagliflozin, Dapagliflozin, and Empagliflozin showed no evidence of mutagenicity or clastrogenicity, establishing their in vitro classification as nonmutagenic. These findings align with negative in silico predictions from quantitative structure–activity relationship (QSAR) analyses for mutagenicity and genotoxicity of the dimer impurities. Collectively, these studies contribute clinically relevant safety information by confirming that the dimer impurities of Canagliflozin, Dapagliflozin, and Empagliflozin are nonmutagenic and nongenotoxic, emphasizing the consistency between in silico and in vitro data.

Assessing Genotoxicity of Bovine Pericardium in Guided Tissue Regeneration: Bacterial Reverse Mutation Assay With Exogenous Metabolic Activation.

Introduction Guided tissue regeneration (GTR) is integral to periodontal therapy, facilitating the repair of osseous defects. Due to the widespread use of bovine pericardium (BP) in GTR, a thorough investigation into its genotoxicity is essential for patient safety and treatment efficacy. This study aimed to evaluate the genotoxic effects of local BP in GTR for periodontal osseous defects. Materials and methods The Bacterial Reverse Mutation Assay (Ames test) was used to assess the genotoxic potential of local BP. An exogenous metabolic activation system was employed to evaluate the direct effects of the material on bacterial cells. Results The study investigated the mutagenic effects of local BP across multiple strains of Salmonella typhimurium, utilizing concentrations ranging from 0.3125 mg/plate to 5 mg/plate. While some variability was observed in revertant counts, the generally low SDs suggest a consistent response to the test substance. The maximum revertant count for each strain did not significantly exceed the mean values, indicating the absence of notable outliers or exceptionally high revertant counts at any specific concentration. Based on the data and toxicity assessment criteria, there is insufficient evidence to suggest that the experimental material induces genotoxic effects in the tested bacterial strains under the provided experimental conditions. Conclusion This study assessed the mutagenic potential of local BP membranes used in GTR with the Ames test. Results showed no evidence of mutagenicity, as revertant counts did not exceed twice the negative control in all bacterial strains with exogenous metabolic activation. This suggests that bovine pericardium membranes are safe for medical use under the test conditions. The study highlights the biocompatibility and non-mutagenic nature of BP membranes in GTR for periodontal therapy.

Genotoxicity and cytotoxicity evaluation of a heat-not-burn product

‘Heat-not-burn’ products (HnBP) contain lower levels of harmful substances than traditional cigarettes, but the use of these products warrants further toxicological evaluation. We have compared the cytotoxicity and genotoxicity of a heat-not burn product with conventional cigarettes, in vivo and in vitro. Male Sprague Dawley rats were exposed to mainstream smoke from conventional cigarettes or a HnBP, for 4 or 28 days, followed by isolation of bone marrow polychromatic erythrocytes (PCE) and histological examination of the testes. Chinese hamster lung fibroblast cells were exposed in vitro to total particulate matter from cigarette smoke obtained through Cambridge filters. The cytotoxicity and genotoxicity of total particulate matter were assessed by the neutral red uptake assay, chromosome aberration assay, in vitro micronucleus test, comet assay, and Ames assay. In the short-term exposure rat models, only the conventional-cigarettes group showed a significant increase in the ratio of micronuclei to total PCE. There was no significant difference in rat testis histology in the long-term exposure models. In vitro, in the neutral red uptake assay, the HnBP product showed lower cytotoxicity than conventional cigarettes. Conventional cigarettes showed greater genotoxicity in the chromosome aberration assay, high-dose Ames tests with exogenous metabolic activation, and micronucleus tests. In summary, our results suggest that HnBP have lower cytotoxicity and genotoxicity than conventional cigarettes.

Estrogenic, androgenic, and genotoxic activities of zearalenone and deoxynivalenol in in vitro bioassays including exogenous metabolic activation

Zearalenone (ZEN) and deoxynivalenol (DON) and their derivatives are well-known mycotoxins, which can occur not only in crops but also in water bodies, including drinking water sources. In vitro bioassays can be used to detect biological effects of hazardous compounds in water. To this, when studying biological effects and toxicity in vitro, metabolism is important to consider. In this study, ZEN, α-zearalenol (α-ZEL), DON, 3-acetyl DON, and 15-acetyl DON were evaluated in vitro for hormone receptor–mediated effects (estrogen receptor [ER] and androgen receptor [AR]) and genotoxicity (micronucleus assay) in the presence of an exogenous metabolic activation system (MAS). The ER bioassay proved to be a highly sensitive method to detect low concentrations of the ZEN compounds (EC10 values of 31.4 pM for ZEN, 3.59 pM for α-ZEL) in aqueous solutions. In the presence of the MAS, reduced estrogenic effects were observed for both ZEN compounds (EC10 values of 6.47 × 103 pM for ZEN, 1.55 × 102 pM for α-ZEL). Of the DON compounds, only 3-acetyl DON was estrogenic (EC10 of 0.31 µM), and the effect was removed in the presence of the MAS. Anti-androgenic effects of the ZEN compounds and androgenic effects of the DON compounds were detected in the micromolar range. No induction of genotoxicity was detected for ZEN or DON in the presence of the MAS. Our study highlighted that inclusion of exogenous MAS is a useful tool to detect biological effects of metabolites in in vitro bioassays.

Genotoxicity Evaluation of The Novel Psychoactive Substance MTTA.

MTTA, also known as mephtetramine, is a stimulant novel psychoactive substance characterized by a simil-cathinonic structure. To date, little has been studied on its pharmaco-toxicological profile, and its genotoxic potential has never been assessed. In order to fill this gap, the aim of the present work was to evaluate its genotoxicity on TK6 cells in terms of its ability to induce structural and numerical chromosomal aberrations by means of a cytofluorimetric protocol of the "In Vitro Mammalian Cell Micronucleus (MN) test". To consider the in vitro effects of both the parental compound and the related metabolites, TK6 cells were treated with MTTA in the absence or presence of an exogenous metabolic activation system (S9 mix) for a short-term time (3 h) followed by a recovery period (23 h). No statistically significant increase in the MNi frequency was detected. Specifically, in the presence of S9 mix, only a slight increasing trend was observable at all tested concentrations, whereas, without S9 mix, at 75 µM, almost a doubling of the negative control was reached. For the purposes of comprehensive evaluation, a long-term treatment (26 h) was also included. In this case, a statistically significant enhancement in the MNi frequency was observed at 50 µM.

Safety assessment of a solid lipid curcumin particle preparation: In vitro and in vivo genotoxicity studies.

Curcumin, one of the three principal curcuminoids found within turmeric rhizomes, has long been associated with numerous physiologically beneficial effects; however, its efficacy is limited by its inherently low bioavailability. Several novel formulations of curcumin extracts have been prepared in recent years to increase the systemic availability of curcumin; Longvida®, a solid lipid curcumin particle preparation, is one such formulation that has shown enhanced bioavailability compared with standard curcuminoid extracts. As part of a safety assessment of Longvida® for use as a food ingredient, a bacterial reverse mutation test (OECD TG 471) and mammalian cell erythrocyte micronucleus test (OECD TG 474) were conducted to assess its genotoxic potential. In the bacterial reverse mutation test, Longvida® did not induce base-pair or frame-shift mutations at the histidine locus in the genome of Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537, in the presence or absence of exogenous metabolic activation. Additionally, two gavage doses (24 h apart) of Longvida® to Swiss albino mice at 500, 1000, or 2000-mg/kg body weight/day did not cause structural or numerical chromosomal damage in somatic cells in the mammalian erythrocyte micronucleus test. It was therefore concluded that Longvida® is non-genotoxic.

Characterization of Lantana Camara Roots (Pentacyclic Triterpenoid) and Mutagenicity Testing of Extracted Oleanolic Acid Using Salmonella Typhimurium

The objective of the present study was to isolate oleanolic acid from the roots of Lantana camara, and characterize it chemically using DSC, HPLC, and FTIR methods, and additionally carrying out a short term assay for assessment of its mutagenic potential by conducting bacterial reverse mutation test to evaluate the ability of the “Oleanolic acid (Pentacyclic Triterpenoid)” to induce point mutations in tester strains of Salmonella typhimurium in both presence and absence of exogenous metabolic activation system (S9) containing microsomal enzymes. Differential scanning calorimetry (DSC), High Performance Liquid chromatography (HPLC), and Fourier transform infrared (FTIR) spectrometers were used to chemically analyze the isolated molecule. Oleanolic acid was utilized to carry out Preliminary Cytotoxicity and mutagenicity study. According to the results of spectrophotometric research, oleanolic acid extracted from Lantana camara roots exhibits identical spectrum characteristics to standard oleanolic acid also the mutagenic potential of Oleanolic acid (Pentacyclic Triterpenoid). Oleanolic acid was found to be non-mutagenic in all five test strains of Salmonella typhimurium—TA98, TA100, TA102, TA1535, and TA1537 employing plate incorporation assays. It may be determined that oleanolic acid isolated from Lantana camara roots gives identical, identifiable signals and absorbance like previously reported reference standard based on the results of DLC, HPLC, and FTIR spectra and their interpretation and was determined that oleanolic acid purified from Lantana camara roots is non-mutagenic in Salmonella typhimurium.

Hydromorphone impurity 2,2-bishydromorphone does not exert mutagenic and clastogenic properties via in silico QSAR prediction and in vitro Ames and micronucleus test

The opioid agonist hydromorphone is indicated for the management of severe acute and chronic pain given that alternate treatments are insufficient. While the genotoxicity profile of hydromorphone is well investigated, little is known about the genotoxic potential of its impurities. In this study, 2,2-bishydromorphone was tested in silico and in vitro for both its mutagenic potential in an Ames test performed with Salmonella typhimurium and Escherichia coli tester strains up to a maximum concentration of 5 mg per plate in the absence and presence of metabolic activation. Furthermore, it was tested for its ability to induce micronuclei in TK6 cells in a micronucleus test up to a maximum concentration of 500 µg/mL with or without an exogenous metabolic activation system. 2,2-Bishydromorphone did not reveal any potential for inducing mutagenicity or clastogenicity under the conditions of the respective tests and is therefore considered non-mutagenic and non-clastogenic/aneugenic in vitro. These results are in line with negative in silico quantitative structure-activity relationship (QSAR) prediction for 2,2-bishydromorphone mutagenicity and clastogenicity and provide evidence of good correlation of in silico and in vitro data. Conclusively, these studies add important new clinically relevant information on the safety of hydromorphone as the impurity of 2,2-bishydromorphone is proven to be non-mutagenic and non-clastogenic.

Anthraquinones: Genotoxic until Proven Otherwise? A Study on a Substance-Based Medical Device to Implement Available Data for a Correct Risk Assessment.

A genotoxicological study was carried out on a substance-based medical device (SMD) containing anthraquinones in order to evaluate its potential mutagenic effect. The “In Vitro Mammalian Cell Micronucleus Test” was performed on human TK6 cells by flow cytometry. Cultures were treated with concentrations of SMD tested in the range of 0–2 mg/mL for short treatment time (3 h) both in the absence and presence of an exogenous metabolic activation system, followed by a recovery period in fresh medium (23 h) and for extended treatment time (26 h) without an exogenous metabolic activation system. At the end of both treatment times, cytotoxicity, cytostasis, apoptosis and micronuclei (MNi) frequency were analysed in treated cultures and then compared with those measured in concurrent negative control cultures. The SMD did not induce a statistically significant increase MNi frequency under any of experimental conditions tested. The negative outcome shows that the SMD is non-mutagenic in terms of its ability to induce chromosomal aberrations both in the absence and presence of an exogenous metabolic activation system. The study ended by analyzing intracellular ROS levels to exclude the pro-oxidant ability, typically linked to DNA damage. On the contrary, our results demonstrated the ability the SMD to counteract oxidative stress.

Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment\xa0of orally ingested substances

The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.

Pyrene-polyethylene glycol-modified multi-walled carbon nanotubes: Genotoxicity in V79-4 fibroblast cells

The genotoxicity of pyrene-polyethylene glycol-modified multi-walled carbon nanotubes (MWCNT-PyPEG), engineered as a nanoplatform for bioapplication, was evaluated. Toxicity was assessed in hamster lung fibroblast cells (V79–4). MTT and Cell Titer Blue methods were used to evaluate cell viability. Genotoxicity was measured by the comet assay and the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay, and fluorescence in situ hybridization (FISH) was used to test induction of structural chromosome aberrations (clastogenic activity) and/or numerical chromosome changes (aneuploidogenic activity). Exogenous metabolic activation enzymes were used in the CBMN-Cyt and FISH tests. Only with metabolic activation, the hybrids caused chromosomal damage, by both clastogenic and aneugenic processes.

The expression of Phase II drug-metabolizing enzymes in human B-lymphoblastoid TK6 cells

In vitro genotoxicity testing plays an important role in chemical risk assessment. The human B-lymphoblastoid cell line TK6 is widely used as a standard cell line for regulatory safety evaluations. Like many other mammalian cell lines, TK6 cells have limited metabolic capacity; therefore, usually require a source of exogenous metabolic activation for use in genotoxicity testing. Previously, we developed a set of TK6-derived cell lines that individually express one of fourteen cytochrome P450s (CYPs). In the present study, we surveyed a panel of major Phase II drug-metabolizing enzymes to characterize their baseline expression in TK6 cells. These results may serve as a reference enzymatic profile of this commonly used cell line.

In vitro mutagenicity of selected environmental carcinogens and their metabolites in MutaMouse FE1 lung epithelial cells.

Chemicals in commerce or under development must be assessed for genotoxicity; assessment is generally conducted using validated assays (e.g. Tk mouse lymphoma assay) as part of a regulatory process. Currently, the MutaMouse FE1 cell mutagenicity assay is undergoing validation for eventual use as a standard in vitro mammalian mutagenicity assay. FE1 cells have been shown to be metabolically competent with respect to some cytochrome P450 (CYP) isozymes; for instance, they can convert the human carcinogen benzo[a]pyrene into its proximate mutagenic metabolite. However, some contradictory results have been noted for other genotoxic carcinogens that require two-step metabolic activation (e.g. 2-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoxaline). Here, we examined three known or suspected human carcinogens, namely acrylamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP), together with their proximate metabolites (i.e. glycidamide, N-OH-PhIP and N-OH-4-ABP), to aid in the validation of the FE1 cell mutagenicity assay. Assessments of the parent compounds were conducted both in the presence and absence of an exogenous metabolic activation mixture S9; assessments of the metabolites were in the absence of S9. The most potent compound was N-OH-PhIP -S9, which elicited a mutant frequency (MF) level 5.3-fold over background at 5 µM. There was a 4.3-fold increase for PhIP +S9 at 5 µM, a 1.7-fold increase for glycidamide -S9 at 3.5 mM and a 1.5-fold increase for acrylamide +S9 at 4 mM. Acrylamide -S9 elicited a marginal 1.4-fold MF increase at 8 mM. Treatment with PhIP -S9, 4-ABP ±S9 and N-OH-4-ABP -S9 failed to elicit significant increases in lacZ MF with any of the treatment conditions tested. Gene expression of key CYP isozymes was quantified by RT-qPCR. Cyp1a1, 1a2 and 1b1 are required to metabolise PhIP and 4-ABP. Results showed that treatment with both compounds induced expression of Cyp1a1 and Cyp1b1 but not Cyp1a2. Cyp2e1, which catalyses the bioactivation of acrylamide to glycidamide, was not induced after acrylamide treatment. Overall, our results confirm that the FE1 cell mutagenicity assay has the potential for use alongside other, more traditional in vitro mutagenicity assays.

Structure-Activity Relationship (SAR) and in vitro Predictions of Mutagenic and Carcinogenic Activities of Ixodicidal Ethyl-Carbamates.

Ethyl-4-bromophenyl-carbamate (LQM 919) and Ethyl-4-chlorophenyl-carbamate (LQM 996) are compounds that inhibit egg-laying and hatching of tick larvae that are resistant to conventional ixodicides. The structure-activity relationship (SAR) to get the endpoint predictions of mutagenicity and carcinogenicity of the LQM 919 and LQM 996 was performed and the absence of mutagenicity was confirmed by Ames test. SAR analysis show no structural alerts indicating the ability of ethyl-carbamates to bind biomolecules or estrogen receptors. Endpoint of mutagenicity with and without metabolic activation showed that the ethyl-carbamates were negative (p <0.05) for mutagenicity induction in strains TA97, TA98, TA102, TA1535, TA1537 and TA1538 of Salmonella typhimurium. Pre-incubation with different ethyl-carbamate concentrations did not increase the number of spontaneously reverting colonies; moreover, the compounds did not induce a concentration-dependent increase in the number of reverting colonies in any of the strains used. This confirmed the absence of mutagenic activity in this test system. Exogenous metabolic activation did not modify these observations; suggesting that no metabolites with mutagenic activity were present. The endpoint of carcinogenicity in rats were negative for LQM 919 (p <0.05,) and LQM 996 (p <0.001). The results of the present study strongly suggest that ethyl-carbamates do not represent a risk for cancer in mammals.

Combining Different In Vitro Bioassays to Evaluate Genotoxicity of Water-Accommodated Fractions from Petroleum Products.

Genotoxicity assessment is of high relevance for crude and refined petroleum products, since oil compounds are known to cause DNA damage with severe consequences for aquatic biota as demonstrated in long-term monitoring studies. This study aimed at the optimization and evaluation of small-scale higher-throughput assays (Ames fluctuation, micronucleus, Nrf2-CALUX®) covering different mechanistic endpoints as first screening tools for genotoxicity assessment of oils. Cells were exposed to native and chemically dispersed water-accommodated fractions (WAFs) of three oil types varying in their processing degree. Independent of an exogenous metabolic activation system, WAF compounds induced neither base exchange nor frame shift mutations in bacterial strains. However, significantly increased chromosomal aberrations in zebrafish liver (ZF-L) cells were observed. Oxidative stress was indicated for some treatments and was not correlated with observed DNA damage. Application of a chemical dispersant increased the genotoxic potential rather by the increased bioavailability of dissolved and particulate oil compounds. Nonetheless, the dispersant induced a clear oxidative stress response, indicating a relevance for general toxic stress. Results showed that the combination of different in vitro assays is important for a reliable genotoxicity assessment. Especially, the ZF-L capable of active metabolism and DNA repair seems to be a promising model for WAF testing.

3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing.

MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/β-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.

Preclinical safety assessment of pathogen reduced red blood cells treated with amustaline and glutathione.

ABSTRACTBACKGROUNDThe nucleic acid targeted pathogen reduction (PR) system utilizing amustaline (S‐303) and glutathione (GSH) is designed to inactivate blood‐borne pathogens and leukocytes in red blood cell concentrates (PR‐RBCC). Inactivation is attained after amustaline intercalates and forms covalent nucleic acid adducts preventing replication, transcription, and translation. After pathogen inactivation, amustaline spontaneously hydrolyzes to S‐300, the primary negatively charged reaction product; amustaline is below quantifiable levels in PR‐RBCC. GSH quenches free unreacted amustaline.STUDY DESIGN AND METHODSThe genotoxic and carcinogenic potential of PR‐RBCC, the reaction by‐products, and S‐300 were assessed in accordance with the International Conference on Harmonization (ICH) guidelines and performed in compliance with the Food and Drug Administration (FDA) good laboratory practice standards, 21 CFR Part 58. in vitro bacterial reverse mutagenicity and chromosomal aberration assays were performed with and without exogenous S9 metabolic activation, and in in vivo clastogenicity and carcinogenic assays using validated murine models.RESULTSPR‐RBCCs were not genotoxic in vitro and in vivo and were non‐carcinogenic in p53+/− transgenic mice transfused over 26 weeks. Estimated safety margins for human exposure ranged from >90 to >36 fold for 2 to 5 PR‐RBCCs per day, respectively. PR‐RBCCs and S‐300 did not induce chromosome aberration in the in vivo murine bone marrow micronucleus assay at systemically toxic doses.CONCLUSIONSPR‐RBCCs did not demonstrate genotoxicity in vitro or in vivo and were not carcinogenic in vivo. These studies support the safety of PR‐RBCCs and suggest that there is no measurable genotoxic hazard associated with transfusion of PR‐RBCCs.

Absence of mutagenic activity in the bacterial reverse mutation assay with pulegone and peppermint oil

The essential oil of peppermint and one of its natural constituents, (R)-(+)-pulegone, are approved flavorings added to food worldwide. (R)-(+)-Pulegone and peppermint oil were tested separately in two independent bacterial reverse mutation assays according to Organisation for Economic Co-operation and Development Guideline 471. Both flavorings did not produce any evidence of mutagenicity up to cytotoxic concentrations in either the presence or the absence of exogenous metabolic activation.

In vitro cytogenetic assessment and comparison of vildagliptin and sitagliptin.

Vildagliptin and sitagliptin are commonly used antidiabetic drugs. Chromosomal aberration (CA), sister chromatid exchange (SCE) and cytokinesis-block micronucleus (CBMN) assays were employed to assess and compare cytotoxic and genotoxic effects of these drugs. Peripheral lymphocytes were exposed to 125μg/ml, 250μg/ml and 500μg/ml of vildagliptin and 250μg/ml, 500μg/ml and 1000μg/ml of sitagliptin for 24h and 48h with and without exogenous metabolic activation. At the end of the study, it was determined that these drugs and their metabolites had no genotoxic effects on CA, SCE and CBMN. On the other hand, parallel to the increase in dose, vildagliptin showed weak cytotoxicity on the mitotic index, and depending on its increase in dose; sitagliptin caused potential cytotoxicity and cytostatic effect on the mitotic index, nuclear division index and proliferation index. Due to their cytotoxic and cytostatic potential, these drugs inhibit cell proliferation.