Evaluation of the genotoxic potential of apoptosis inducers with the γH2AX assay in human cells.

Human risk assessment of genotoxic chemicals is an important area of research. However, the specificity of in vitro mammalian genotoxicity assays is sometime low, as they yield to misleading positive results that are not observe in in vivo studies. Apoptosis can be a confounding factor in the interpretation of the results. Recently, a new strategy for genotoxicity screening, based on the combined analysis of phosphorylated histones H2AX (γH2AX) and H3 (pH3), was proposed to discriminate efficiently aneugenic from clastogenic compounds. However, γH2AX biomarker could also be induce by apoptosis. The aim of the present study was to investigate the specificity of this genotoxic biomarker. For this purpose, we analyzed 26 compounds inducing apoptosis by different mechanism of action, with the γH2AX assay in three human cell lines after 24 h treatment. Most of the tested chemicals were negative in the assay, whatever the cell line tested. The few compounds that generated positive data have also been report positive in other genotoxicity assays. The data presented here demonstrate that the γH2AX assay is not vulnerable to the generation of misleading positive results by apoptosis inducers. Currently, no formal guidelines have been approve for the γH2AX assay for regular genotoxicity studies, but we suggest that this biomarker could be used as a new standard genotoxicity assay.

Assessment of the DNA damaging potential of environmental chemicals using a quantitative high-throughput screening approach to measure p53 activation.

Genotoxicity potential is a critical component of any comprehensive toxicological profile. Compounds that induce DNA or chromosomal damage often activate p53, a transcription factor essential to cell cycle regulation. Thus, within the US Tox21 Program, we screened a library of ∼10,000 (∼8,300 unique) environmental compounds and drugs for activation of the p53-signaling pathway using a quantitative high-throughput screening assay employing HCT-116 cells (p53+/+ ) containing a stably integrated ß-lactamase reporter gene under control of the p53 response element (p53RE). Cells were exposed (-S9) for 16 hr at 15 concentrations (generally 1.2 nM to 92 µM) three times, independently. Excluding compounds that failed analytical chemistry analysis or were suspected of inducing assay interference, 365 (4.7%) of 7,849 unique compounds were concluded to activate p53. As part of an in-depth characterization of our results, we first compared them with results from traditional in vitro genotoxicity assays (bacterial mutation, chromosomal aberration); ∼15% of known, direct-acting genotoxicants in our library activated the p53RE. Mining the Comparative Toxicogenomics Database revealed that these p53 actives were significantly associated with increased expression of p53 downstream genes involved in DNA damage responses. Furthermore, 53 chemical substructures associated with genotoxicity were enriched in certain classes of p53 actives, for example, anthracyclines (antineoplastics) and vinca alkaloids (tubulin disruptors). Interestingly, the tubulin disruptors manifested unusual nonmonotonic concentration response curves suggesting activity through a unique p53 regulatory mechanism. Through the analysis of our results, we aim to define a role for this assay as one component of a comprehensive toxicological characterization of large compound libraries. Environ. Mol. Mutagen. 58:494-507, 2017. © 2017 Wiley Periodicals, Inc.

Carbamates and ICH M7 classification: Making use of expert knowledge.

Carbamates are widely used in the chemical industry so understanding their toxicity is important to safety assessment. Carbamates have been associated with certain toxicities resulting in publication of structural alerts, including alerts for mutagenicity. Structural alerts for bacterial mutagenicity can be used in combination with statistical systems to enable ICH M7 classification, which allows assessment of the genotoxic risk posed by pharmaceutical impurities. This study tested a hypothetical bacterial mutagenicity alert for carbamates and examined the impact it would have on ICH M7 classifications using (Q)SAR predictions from the expert rule-based system Derek Nexus and the statistical-based system Sarah Nexus. Public datasets have a low prevalence of mutagenic carbamates, which highlighted that systems containing an alert for carbamates perform poorly for achieving correct ICH M7 classifications. Carbamates are commonly used as protecting groups and proprietary datasets containing such compounds were also found to have a low prevalence of mutagenic compounds. Expert review of the mutagenic compounds established that mutagenicity was often only observed under certain (non-standard) conditions and more generally that the Ames test may be a poor predictor for the risk of carcinogenicity posed by chemicals in this class. Overall a structural alert for the in vitro bacterial mutagenesis of carbamates does not benefit workflows for assigning ICH M7 classification to impurities.

Principles and procedures for implementation of ICH M7 recommended (Q)SAR analyses.

The ICH M7 guideline describes a consistent approach to identify, categorize, and control DNA reactive, mutagenic, impurities in pharmaceutical products to limit the potential carcinogenic risk related to such impurities. This paper outlines a series of principles and procedures to consider when generating (Q)SAR assessments aligned with the ICH M7 guideline to be included in a regulatory submission. In the absence of adequate experimental data, the results from two complementary (Q)SAR methodologies may be combined to support an initial hazard classification. This may be followed by an assessment of additional information that serves as the basis for an expert review to support or refute the predictions. This paper elucidates scenarios where additional expert knowledge may be beneficial, what such an expert review may contain, and how the results and accompanying considerations may be documented. Furthermore, the use of these principles and procedures to yield a consistent and robust (Q)SAR-based argument to support impurity qualification for regulatory purposes is described in this manuscript.

An evaluation of 25 selected ToxCast chemicals in medium-throughput assays to detect genotoxicity.

ToxCast is a multiyear effort to develop a cost-effective approach for the US EPA to prioritize chemicals for toxicity testing. Initial evaluation of more than 500 high-throughput (HT) microwell-based assays without metabolic activation showed that most lacked high specificity and sensitivity for detecting genotoxicants. Thus, EPA initiated a pilot project to investigate the use of standard genotoxicity endpoints using medium-throughput genotoxicity (MTG) assays in the context of a large testing program. Twenty-five chemicals were selected from the ToxCast program based in part on their known genotoxicity. The two MTG assays used were the Ames II(™) assay and 96-well In Vitro MicroFlow(®) Micronucleus (MN) assay. The Ames II assay showed a reasonable correlation with published Ames test data and industry submissions, though specificity was much better than sensitivity due to restraints on top concentrations as prescribed by ToxCast. Overall concordance was 73% both with and without metabolic activation. The flow MN assay had concordances of 71% and 58% with and without metabolic activation, respectively, when compared to published data and submissions. Importantly, a comparison of results without S9 from the MTG assays to an HT ToxCast p53 activation assay showed a fairly good degree of concordance (67%). The results reported here indicate that assays for genotoxicity endpoints can be conducted in a MT format and have the potential to add to the interpretation of results from large-scale testing programs such as EPA's ToxCast program. Inherent limitations such as the top concentrations used in large scale testing programs are discussed. Environ. Mol. Mutagen. 56:468-476, 2015. © 2014 Wiley Periodicals, Inc.

Evaluation of toxic endpoints for a set of cosmetic ingredients with CAESAR models.

The randomly selected set of 558 chemicals from Cosmetic inventory was studied with internet accessible program package CAESAR. Four toxic endpoints were considered: mutagenicity, carcinogenicity, developmental toxicity and skin sensitization. The CAESAR program provides beside the predictions comprehensive information on applicability domain and the similarity between the considered compound and the compounds from model's training set. This information was used to implement for clustering and classification of chemicals. As the technique the Self Organizing Maps was applied. This technique also enables us to define to each cluster the cluster indicator, i.e., the characteristic compound, which is considered as a representative for a cluster.

The predominant role of apoptosis in γH2AX formation induced by aneugens is useful for distinguishing aneugens from clastogens.

The phosphorylated form of the histone protein H2AX, called γH2AX, is recognized as a useful biomarker not only for DNA double-strand breaks but also for a wide range of other DNA damage. An increasing number of publications propose γH2AX to be measured when determining genotoxicity, phototoxicity, and the effectiveness of cancer therapy. Because γH2AX is also generated by apoptosis, a γH2AX-assay might assess genotoxic risk incorrectly. The aim of this study was to elucidate the influence of apoptosis on measurements of γH2AX by flow cytometry, with the clastogens mitomycin C (MMC) and etoposide (ETP), and the aneugens vinblastine (VB) and paclitaxel (PT), which do not react directly with DNA. TK6 human lymphoblastoid cells were treated with the clastogens and the aneugens, stained for the apoptotic biomarker caspase-3 and for γH2AX, and then analyzed by flow cytometry. All the test compounds caused a dose-dependent increase of γH2AX-positive (γH2AX+) cells. The γH2AX+ cell population included both caspase-3-positive (γH2AX+/caspase-3+) and caspase-3-negative (γH2AX+/caspase-3-) cells. The increase in γH2AX+ cells after treatment with the aneugens corresponded to the increase in caspase-3+ cells. The increase in γH2AX+/caspase-3- cells after treatment with the clastogens was significant, but there was only a slight increase after treatment with the aneugens. This reflects the fact that the apoptotic pathway of a clastogen starts from DNA damage, whereas that of an aneugen starts from cell-cycle arrest in the M-phase. Therefore, the two pathways contribute differently to apoptosis. Double staining for γH2AX and caspase-3 provided helpful information for the different mechanistic effects of aneugens and clastogens that induce γH2AX.

A proposal to improve clarity and communication in the EU Classification process for chemicals for carcinogenicity and reproductive and developmental toxicity.

There is an issue in the EU classification of substances for carcinogenicity and for reproductive or developmental toxicity which has brought difficulties to those involved in the process. The issue lies in the inability of the classification system to distinguish between carcinogens and reproductive toxicants with different levels of concern. This has its origins in the early years of toxicology when it was thought that a relatively small number of chemicals would be either carcinogens or reproductive toxicants, but this has turned out not to be the case. This can cause problems in communicating to the users of chemicals, including the public, the nature of the hazard presented by chemicals. Processes have been developed within the classification system for setting specific concentration limits which assess the degree of hazard for carcinogens and reproductive toxicants as high, medium or low. However these categories are not otherwise used in classification. It is proposed that their wider use would bring the advantages of transparency, clarity of communication, certainty of the process and would allow chemicals with a high degree of hazard to be identified and managed in an appropriate way.

In vivo murine hepatic microRNA and mRNA expression signatures predicting the (non-)genotoxic carcinogenic potential of chemicals.

There is a high need to improve the assessment of, especially non-genotoxic, carcinogenic features of chemicals. We therefore explored a toxicogenomics-based approach using genome-wide microRNA and mRNA expression profiles upon short-term exposure in mice. For this, wild-type mice were exposed for seven days to three different classes of chemicals, i.e., four genotoxic carcinogens (GTXC), seven non-genotoxic carcinogens (NGTXC), and five toxic non-carcinogens. Hepatic expression patterns of mRNA and microRNA transcripts were determined after exposure and used to assess the discriminative power of the in vivo transcriptome for GTXC and NGTXC. A final classifier set, discriminative for GTXC and NGTXC, was generated from the transcriptomic data using a tiered approach. This appeared to be a valid approach, since the predictive power of the final classifier set in three different classifier algorithms was very high for the original training set of chemicals. Subsequent validation in an additional set of chemicals revealed that the predictive power for GTXC remained high, in contrast to NGTXC, which appeared to be more troublesome. Our study demonstrated that the in vivo microRNA-ome has less discriminative power to correctly identify (non-)genotoxic carcinogen classes. The results generally indicate that single mRNA transcripts do have the potential to be applied in risk assessment, but that additional (genomic) strategies are necessary to correctly predict the non-genotoxic carcinogenic potential of a chemical.

A metabolomics investigation of non-genotoxic carcinogenicity in the rat.

Non-genotoxic carcinogens (NGCs) promote tumor growth by altering gene expression, which ultimately leads to cancer without directly causing a change in DNA sequence. As a result NGCs are not detected in mutagenesis assays. While there are proposed biomarkers of carcinogenic potential, the definitive identification of non-genotoxic carcinogens still rests with the rat and mouse long-term bioassay. Such assays are expensive and time-consuming and require a large number of animals, and their relevance to human health risk assessments is debatable. Metabolomics and lipidomics in combination with pathology and clinical chemistry were used to profile perturbations produced by 10 compounds that represented a range of rat non-genotoxic hepatocarcinogens (NGC), non-genotoxic non-hepatocarcinogens (non-NGC), and a genotoxic hepatocarcinogen. Each compound was administered at its maximum tolerated dose level for 7, 28, and 91 days to male Fisher 344 rats. Changes in liver metabolite concentration differentiated the treated groups across different time points. The most significant differences were driven by pharmacological mode of action, specifically by the peroxisome proliferator activated receptor alpha (PPAR-α) agonists. Despite these dominant effects, good predictions could be made when differentiating NGCs from non-NGCs. Predictive ability measured by leave one out cross validation was 87% and 77% after 28 days of dosing for NGCs and non-NGCs, respectively. Among the discriminatory metabolites we identified free fatty acids, phospholipids, and triacylglycerols, as well as precursors of eicosanoid and the products of reactive oxygen species linked to processes of inflammation, proliferation, and oxidative stress. Thus, metabolic profiling is able to identify changes due to the pharmacological mode of action of xenobiotics and contribute to early screening for non-genotoxic potential.

Toxicology and carcinogenesis studies of Ginkgo biloba extract (CAS No. 90045-36-6) in F344/N rats and B6C3F1/N mice (Gavage studies).

Ginkgo biloba extract has been used primarily as a medicinal agent in the treatment or prevention of cardiovascular and cerebrovascular dysfunction. Ginkgo biloba extract was nominated for study by the National Cancer Institute because of its widespread use as an herbal supplement to promote mental function and the limited availability of toxicity and carcinogenicity data. Furthermore, one of the major ingredients in Ginkgo biloba extract, quercetin, is a known mutagen. The Ginkgo biloba extract used in the current studies was procured from a supplier known to provide material to United States companies and contained 31.2% flavonol glycosides, 15.4% terpene lactones (6.94% bilo-balide, 3.74% ginkgolide A, 1.62% ginkgolide B, 3.06% ginkgolide C), and 10.45 ppm ginkgolic acid. Male and female F344/N rats and B6C3F1/N mice were administered Ginkgo biloba extract in corn oil by gavage for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were administered 0, 62.5, 125, 250, 500, or 1,000 mg Ginkgo biloba extract/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) were administered the same doses, 5 days per week for 23 days. All rats survived to the end of the study. Mean body weights of all dosed groups were similar to those of the vehicle control groups. Liver weights of all dosed groups of males and females were significantly greater than those of the vehicle control groups. The incidences of hepatocyte hypertrophy in all dosed groups of males and in 500 and 1,000 mg/kg females were significantly greater than those in the vehicle control groups; there was a dose-related increase in severity of this lesion in males. Hepatocyte fatty change occurred in all dosed males. The incidences of thyroid gland follicular cell hypertrophy were significantly increased in 500 and 1,000 mg/kg males and in 1,000 mg/kg females. The incidences of pigmentation in the olfactory epithelium of the nose were significantly increased in 500 and 1,000 mg/kg males and in females administered 125 mg/kg or greater. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were administered 0, 125, 250, 500, 1,000, or 2,000 mg Ginkgo biloba extract/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. One female mouse in the 1,000 mg/kg group died of a dosing accident during week 11. Mean body weights of 2,000 mg/kg females were significantly less than those of the vehicle control group. Ruffled fur was observed in two 1,000 mg/kg males between weeks 7 and 8 and all 2,000 mg/kg males between weeks 5 and 9. Liver weights of 250 mg/kg or greater males and all dosed groups of females were significantly greater than those of the vehicle control groups. Kidney weights of 2,000 mg/kg males were significantly less than those of the vehicle control group. The Markov transition matrix analyses indicate female mice in the 2,000 mg/kg group had a significantly higher probability of extended estrus than did the vehicle control females. The incidences of hepatocytic hypertrophy were significantly increased in males and females in the 250 mg/kg or greater groups. Significantly increased incidences of focal hepatocytic necrosis occurred in 1,000 and 2,000 mg/kg males. The incidences of hyaline droplet accumulation in the respiratory epithelium of the nose were significantly increased in 500 mg/kg males and 1,000 and 2,000 mg/kg females. In the olfactory epithelium of the nose, the incidences of hyaline droplet accumulation were significantly increased in the 125 (female only), 500, and 1,000 mg/kg groups. Incidences of atrophy of the olfactory epithelium were significantly increased in the 1,000 mg/kg groups. The incidences of pigment accumulation in macrophages in the olfactory epithelium were significantly increased in males in the 500 mg/kg or greater groups and in 1,000 and 2,000 mg/kg females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0, 100, 300, or 1,000 mg Ginkgo biloba extract/kg body weight in corn oil by gavage, 5 days per week for 104 or 105 (females) weeks. Additional groups of 10 male and 10 female rats (special study) were administered the same doses, 5 days per week for 14 weeks. Survival of 1,000 mg/kg males was significantly less than that of the vehicle controls. At week 14, all dosed groups of males and 1,000 mg/kg females had increased levels of thyroid stimulating hormone compared to those of the vehicle control groups. There were no significant decreases in the levels of triiodothyronine or total thyroxine. Mean body weights of 300 mg/kg males and females were less (10% or more) than those of the vehicle controls after week 93, and those of 1,000 mg/kg males and females were less after week 89. Clinical findings included ruffled fur in seven, eight, and 10 males in the 100, 300, and 1,000 mg/kg groups, respectively, beginning at week 89; four vehicle control males also had ruffled fur. Liver weights were significantly increased in all dosed groups of special study rats at 14 weeks. In the liver at 2 years, incidences of hepatocellular adenoma were slightly increased in 100 and 300 mg/kg males. Significantly increased incidences of nonneoplastic lesions at 2 years included hepatocyte hypertrophy and bile duct hyperplasia in all dosed groups of males and females, focal fatty change in all dosed groups of females, cystic degeneration in 100 and 1,000 mg/kg males, and oval cell hyperplasia and necrosis in 1,000 mg/kg males. In the thyroid gland, incidences of follicular cell adenoma were slightly increased in 300 and 1,000 mg/kg males and 300 mg/kg females. Single incidences of follicular cell carcinoma occurred in the 300 and 1,000 mg/kg female groups. There were significantly increased incidences of follicular cell hypertrophy in all dosed groups of males and females and follicle hyperplasia in all dosed groups of males. In the nose, adenoma of the respiratory epithelium occurred in two females receiving 300 mg/kg. Except for respiratory epithelium hyperplasia in 100 mg/kg females, the incidences of transitional epithelium and respiratory epithelium hyperplasia were significantly increased in all dosed groups of males and females. Except for olfactory epithelium respiratory metaplasia in 100 mg/kg females, the incidences of atrophy, respiratory metaplasia, nerve atrophy, and pigmentation were significantly increased in the olfactory epithelium of all dosed groups of males and females. Incidences of goblet cell hyperplasia in the respiratory epithelium were significantly increased in 300 and 1,000 mg/kg males and females, and incidences of chronic active inflammation were significantly increased in 1,000 mg/kg males and females. The incidence of submucosa fibrosis was significantly increased in 1,000 mg/kg males. The incidences of mononuclear cell leukemia in 300 and 1,000 mg/kg males were significantly greater than that in the vehicle controls. Dose-related increased severity of kidney nephropathy was noted in all dosed groups of males. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 0, 200, 600, or 2,000 mg Ginkgo biloba extract/kg body weight in corn oil by gavage, 5 days per week for 104 weeks. Survival of 600 and 2,000 mg/kg males was significantly less than that of the vehicle controls; survival of 600 mg/kg females was significantly greater than that of the vehicle controls. Mean body weights of 600 and 2,000 mg/kg males were less (10% or more) than those of the vehicle controls after weeks 85 and 77, respectively; mean body weights of 2,000 mg/kg females were generally less than those of the vehicle controls between weeks 17 and 69 and after week 93. In the liver, there were significantly increased incidences of hepatocellular adenoma in all dosed groups of females, hepatocellular carcinoma in all dosed groups of males and 2,000 mg/kg females, and hepatoblastoma in all dosed groups of males and 600 and 2,000 mg/kg females. The increased incidences of these neoplasms were primarily due to increased incidences of multiple adenoma, carcinoma, and hepatoblastoma. Except for the incidences of hepatocellular carcinoma or hepatoblastoma (combined) in 200 and 600 mg/kg females, the incidences of hepatocellular adenoma or carcinoma (combined), hepatocellular carcinoma or hepatoblastoma (combined), and hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma (combined) were significantly increased in all dosed groups of males and females. Significantly increased incidences of nonneoplastic liver lesions included hypertrophy in all dosed groups of males and females, erythrophagocytosis in all dosed groups of males and in 600 and 2,000 mg/kg females, hematopoietic cell proliferation, inflammation, and necrosis in 600 and 2,000 mg/kg males, and cytoplasmic vacuolization, eosinophilic focus, and mixed cell focus in all dosed groups of females. In the thyroid gland, two incidences each of follicular cell adenoma occurred in the 600 and 2,000 mg/kg male groups. The incidence of follicle hyperplasia was significantly increased in 2,000 mg/kg males, and the incidences of follicular cell hypertrophy were significantly increased in 2,000 mg/kg males and 600 and 2,000 mg/kg females. In the forestomach, the incidences of inflammation, epithelium hyperplasia, and epithelium hyperkeratosis were significantly increased in all dosed groups of males and in 2,000 mg/kg females; the incidences of epithelium ulcer were significantly increased in 2,000 mg/kg males and females. GENETIC TOXICOLOGY Ginkgo biloba extract was mutagenic in S. typhimurium strains TA98 and TA100, and in E. coli strain WP2 uvrA/pKM101, with and without exogenous metabolic activation. (ABSTRACT TRUNCATED)

In vitro assessment of the cytotoxic, apoptotic, and mutagenic potentials of isatin.

Isatin (1H-indole-2,3-dione) is a chemical found in various medicinal plant species and responsible for a broad spectrum of pharmacological and biological properties that may be beneficial to human health, as an anticonvulsant, antibacterial, antifungal, antiviral, and anticancer agent. The aim of the present study was to determine in vitro the cytotoxic, mutagenic, and apoptotic effects of isatin on CHO-K1 and HeLa cells using the MTT viability assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), micronucleus (MN) test, apoptosis index, and nuclear division index (NDI). The 5 isatin concentrations evaluated in the mutagenicity and apoptosis tests were 0.5, 1, 5, 10, and 50 µM, selected through a preliminary MTT assay. Positive (doxorubicin, DXR) and negative (phosphate buffered saline, PBS) control groups were also included in the analysis. Isatin did not exert a mutagenic effect on CHO-K1 after 3 and 24 h of treatment or on HeLa cells after 24 h. However, 10 and 50 µM concentrations inhibited cell proliferation and promoted apoptosis in both CHO-K1 and HeLa cells. Data indicate that the cytotoxic, apoptotic, and antiproliferative effects of isatin were concentration independent and cell line independent.

Evaluation of the mutagenicity and genotoxicity of Arrabidaea chica Verlot (Bignoneaceae), an Amazon plant with medicinal properties.

Arrabidaea chica Verlot (Bignoniaceae) is an important folk medicine plant native to the Amazon region and used to treat anemia, hemorrhage, inflammation, intestinal colic, hepatitis, and skin affections. Although studies showed its therapeutic properties, little knowledge regarding genotoxic properties of this plant is available. The aim of this study was to determine the potential mutagenic and genotoxic/antigenotoxic effects of an A. chica chloroformic fraction (Ac-CF) obtained from leaves containing bioactive metabolites. The mutagenic effects were evaluated using the Salmonella mutagenicity assay, with TA98, TA97a, TA100, TA102, and TA1535 strains, with and without metabolic activation. In vivo mutagenic and genotoxic/antigenotoxic effects were investigated using the micronucleus (MN) test in bone marrow and alkaline comet assay in blood and liver after administration of 100, 500, or 1000 mg/kg Ac-CF in CF-1 mice by gavage (once a day for 3 d). In vitro antioxidant potential was evaluated using DPPH and xanthine/hypoxanthine assays. Ac-CF was not mutagenic in any of the Salmonella typhimurium strains tested and showed negative responses for mutagenicity and genotoxicity in mice. Further, Ac-CF displayed antigenotoxic effects by decreasing the oxidative DNA damage induced by hydrogen peroxide by greater than 50% in blood and liver. The antioxidant action detected in the in vitro assays demonstrated IC50 of 0.838 mg/ml in the xanthine/hypoxanthine assay and IC50 of 28.17 µg/ml in the DPPH assay. In conclusion, Ac-CF did not induce mutagenic and genotoxic effects and was able to protect DNA against oxidative damage in vivo, suggesting that this fraction may not pose genetic risks, although further toxicology assays are necessary.

Genotoxicity and carcinogenicity studies of bronchodilators and antiasthma drugs.

This survey is a compendium of genotoxicity and carcinogenicity information of bronchodilators and antiasthma drugs. Data from 46 marketed drugs were collected. Of these 46 drugs, 25 (54.3%) did not have retrievable genotoxicity or carcinogenicity data. The remaining 21 (45.7%) had at least one genotoxicity or carcinogenicity test result. Of these 21 drugs, 10 had at least one positive finding: three tested positive in at least one genotoxicity assay, eight in at least one carcinogenicity assay, and one of them gave positive results in both genotoxicity assay and carcinogenicity assay. Concerning the predictivity of genetic toxicology findings for the result(s) of long-term carcinogenesis assays, 15 drugs had both genotoxicity and carcinogenicity data: seven of them (46.6%) were neither genotoxic nor carcinogenic, 6 (40.0%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, 1 (6.7%) tested positive in genotoxicity assay but was non-carcinogenic, and 1 (6.7%) gave positive responses in both genotoxicity and carcinogenicity assay. Only 11 (23.9%) of the 46 drugs considered had all data required by current guidelines for testing of pharmaceuticals, but a large fraction of them were developed and marketed prior to the present regulatory climate.

Genotoxicity assessment in smokeless tobacco users: a case-control study.

India has a long history of tobacco, which includes chewing tobacco and smoking tobacco in various forms. Initially, the smokeless tobacco chewing habit was seen among the majority of the farmers who cultivated tobacco; but in recent years, smokeless tobacco is available in many forms and is cheaper as well and hence it is widely being used among literate and illiterate people. The subjects of our study are living in hilly regions of Yerkaud in Salem district, South India. Most of the inhabitants of our study area are illiterate and more particularly they are unaware of the health effects due to tobacco use. Recent epidemiological reports have strongly indicated the association of cancer risk with usage of smokeless tobacco. The prime aim of our study is to evaluate the genotoxic effects of tobacco use by analysing the cytogenetic end points such as chromosome aberrations in peripheral blood and micronucleus in peripheral blood and buccal cells. About 85 smokeless tobacco users were enrolled for the study and same numbers of age- and sex-matched nontobacco users were also enrolled to serve as controls. The result of our study revealed that tobacco users displayed varied levels of elevated chromosomal damage and micronucleated cells than nontobacco users. The variation in the extent of genetic damage was dependent on the duration of the tobacco use. In conclusion, this study might be helpful in creating awareness on the hazards of the smokeless tobacco products among the global population as a whole for those who chose such products as a cheap alternative to tobacco smoke.

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.

Prediction of mutagenicity, carcinogenicity, developmental toxicity, and skin sensitisation with Caesar program for a set of conazoles.

This article presents models to predict mutagenicity, carcinogenicity, developmental toxicity, and skin sensitisation for a set of 27 conazoles. The predictions were performed with the program package CAESAR, which is available on the Internet. The CAESAR programs were developed to support the European Community Regulation on chemicals and their safe use (REACH) and follow the OECD principles for (Q)SAR models used for regulatory purposes. The programs provide a number of information, including a binary classification of a compound as toxic or non-toxic and information on similar compounds from the model's training sets (similarity sets). In this study we analysed conazole sets using principal component analysis (PCA). The predictions were compared to the currently valid classification of these substances in the European Union (EU) or to the classification proposed at expert meetings of the Pesticide Risk Assessment and Peer Review (PRAPeR) group. The predicted classification for mutagenicity was in good agreement with regulatory classification, the predictions for carcinogenicity and developmental toxicity showed some discrepancy in particular cases, while the predictions for skin sensitisation showed even greater discrepancy.

Enzymes in cleaning products: an overview of toxicological properties and risk assessment/management.

Enzymes used in cleaning products have an excellent safety profile, with little ability to cause adverse responses in humans. For acute toxicity, genotoxicity, sub-acute and repeated dose toxicity, enzymes are unremarkable. Reproductive toxicity and carcinogenicity are also not endpoints of concern. Exceptions are the ability of some proteases to produce irritating effects at high concentrations and more importantly, the intrinsic potential of these bacterial/fungal proteins to act as respiratory sensitizers. It is a reasonable assumption that the majority of enzyme proteins possess this hazard. However, methods for characterising the respiratory sensitisation hazard of enzymes are lacking and the information required for risk assessment and risk management, although sufficient, remains limited. Previously, most data was generated in animal models and in in vitro immunoassays that assess immunological cross-reactivity. Nevertheless, by the establishment of strict limits on airborne exposure (based on a defined minimal effect limit of 60ng active enzyme protein/m(3)) and air and health monitoring, occupational safety can be assured. Similarly, by ensuring that airborne exposure is kept similarly low, coupled with knowledge of the fate of these enzymes on skin and fabrics, it has proven possible to establish a long history of safe consumer use of enzyme containing products.

Assessment of genotoxic potential of Cr(VI) in the mouse duodenum: an in silico comparison with mutagenic and nonmutagenic carcinogens across tissues.

In vitro studies on hexavalent chromium [Cr(VI)] indicate that reduced forms of this metal can interact with DNA and cause mutations. Recently, Cr(VI) was shown to induce intestinal tumors in mice; however, Cr(VI) elicited redox changes, cytotoxicity and hyperplasia - suggesting involvement of tissue injury rather than direct mutagenesis. Moreover, toxicogenomic analyses indicated limited evidence for DNA damage responses. Herein, we extend these toxicogenomic analyses by comparing the gene expression patterns elicited by Cr(VI) with those of four mutagenic and four nonmutagenic carcinogens. To date, toxicogenomic profiles for mutagenic and nonmutagenic duodenal carcinogens do not exist, thus duodenal gene changes in mice were compared to those elicited by hepatocarcinogens. Specifically, duodenal gene changes in mice following exposure to Cr(VI) in drinking water were compared to hepatic gene changes previously identified as potentially discriminating mutagenic and nonmutagenic hepatocarcinogens. Using multivariate statistical analyses (including logistic regression classification), the Cr(VI) gene responses clustered apart from mutagenic carcinogens and closely with nonmutagenic carcinogens. These findings are consistent with other intestinal data supporting a nonmutagenic mode of action (MOA). These findings may be useful as part of a full weight of evidence MOA evaluation for Cr(VI)-induced intestinal carcinogenesis. Limitations to this analysis will also be discussed.

Classification of test agent-specific effects in the Syrian hamster embryo assay (pH 6.7) using infrared spectroscopy with computational analysis.

The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has utility in the assessment of potential chemical carcinogenicity (both genotoxic and non-genotoxic mechanisms of action). The assay uses morphological transformation as an end point and has a reported sensitivity of 87%, specificity of 83% and overall concordance of 85% with in vivo rodent bioassay data. However, the scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with benzo[a]pyrene, 3-methylcholanthrene, anthracene, N-nitroso-N-methylnitroguanidine, ortho-toluidine HCl, 2,4-diaminotoluene or D-mannitol for 7 days before fixation with methanol. Identified colonies were interrogated by acquiring a minimum of five infrared (IR) spectra per colony using attenuated total reflection Fourier-transform IR spectroscopy. Individual IR spectra were acquired over a spatial area of approximately 250 × 250 µm. Resultant data were analysed using Fisher's linear discriminant analysis and feature histogram algorithms to extract classifying biomarkers of test agent-specific effects or transformation in SHE cells. Clustering of spectral points suggested co-segregation or discrimination of test agent categories based on mechanism of action. Towards transformation, unifying alterations were associated with alterations in the Amide I and Amide II peaks; these were consistently major classifying biomarkers for transformed versus non-transformed SHE cells. Our approach highlights a novel method towards objectively screening and classifying SHE cells, be it to ascertain test agent treatment based on mechanism of action or transformation.