Breheny D.,British American Tobacco |
Oke O.,British American Tobacco |
Pant K.,Bioreliance Corporation |
Gaca M.,British American Tobacco
Environmental and Molecular Mutagenesis | Year: 2017
In vitro cell transformation assays (CTA) are used to assess the carcinogenic potential of chemicals and complex mixtures and can detect nongenotoxic as well as genotoxic carcinogens. The Bhas 42 CTA has been developed with both initiation and promotion protocols to distinguish between these two carcinogen classes. Cigarette smoke is known to be carcinogenic and is positive in in vitro genotoxicity assays. Cigarette smoke also contains nongenotoxic carcinogens and is a tumour promoter and cocarcinogen in vivo. We have combined a suite of in vitro assays to compare the relative biological effects of new categories of tobacco and nicotine products with traditional cigarettes. The Bhas promotion assay has been included in this test battery to provide an in vitro surrogate for detecting tumor promoters. The activity of an electronic cigarette (e-cigarette; Vype ePen) was compared to that of a reference cigarette (3R4F) in the promotion assay, using total particulate matter (TPM)/aerosol collected matter (ACM) and aqueous extracts (AqE) of product aerosol emissions. 3R4F TPM was positive in this assay at concentrations ≥6 µg/mL, while e-cigarette ACM did not have any promoter activity. AqE was found to be a lesssuitable test matrix in this assay due to high cytotoxicity. This is the first study to use the Bhas assay to compare tobacco and nicotine products and demonstrates the potential for its future application as part of a product assessment framework. These data add to growing evidence suggesting that e-cigarettes may provide a safer alternative to traditional cigarettes. Environ. Mol. Mutagen. 58:190–198, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
Paranjpe M.G.,BioReliance Corporation |
Elbekaei R.H.,BioReliance Corporation |
Shah S.A.,BioReliance Corporation |
Hickman M.,BioReliance Corporation |
And 2 more authors.
International Journal of Toxicology | Year: 2013
The Tg.rasH2 mouse is a hemizygous transgenic mouse, approved by regulatory agencies for carcinogenicity assessment. However, the absence of a historical database for the incidence of spontaneous neoplasms has subsequently led to reluctance by some pharmaceutical companies to adopt the use of this short-term carcinogenicity assay. Our laboratory has generated a database summarizing the mortality, body weights, and the incidence of spontaneous tumors in 1420 male and female mice assigned to 26 studies conducted at our facility. In addition, we present the incidence of tumors in positive control mice treated with urethane from these studies. Mortality in the vehicle-treated Tg.rasH2 mouse was low (average of 1% in each study). The most common spontaneous tumors in the Tg.rasH2 mice were alveolar bronchiolar adenoma of the lungs (10.14% in males and 5.77% in females) and hemangiosarcoma of the spleen (3.66% in both males and females). The incidence of all other tumors was generally very low. In the positive control, urethane-treated animals, the incidence of alveolar bronchiolar adenomas and alveolar bronchiolar carcinomas in the lungs was 93.69% and 42.88% in males and 92.43% and 72.79% in females, respectively. In addition, the incidence of splenic hemangiosarcomas in urethane-treated males was 89.18% and 92.25% in females. The 6-month Tg.rasH2 assay is more precise, faster, and more economical than the conventional 2-year mouse assays because of the low incidence of background tumors, very high survival, shorter duration, and the lower number of animals used. © 2013 The Author(s).
Ilchmann A.,BioReliance Ltd |
Armstrong A.A.,BioReliance Ltd |
Clayton R.F.,BioReliance Ltd
Biologicals | Year: 2017
Emerging viruses, as potential contaminants of raw materials used in the manufacture of biologicals represent a challenge in the safety testing of biopharmaceutical products intended for human or veterinary use. Here, we report the challenge of an in vitro adventitious virus platform used in safety testing of biologicals, where a broad panel of detector cell lines was challenged to provide evidence that Schmallenberg virus is detectable by a classical reporting endpoint of cytopathic effect with Vero, BHK-21 and CHO-K1 detector cells, within typical in vitro assay timescales. We conclude that Schmallenberg virus is robustly detectable by classical in vitro viral biosafety assays. © 2017 International Alliance for Biological Standardization.
Sahu S.C.,U.S. Food and Drug Administration |
Roy S.,Bioreliance Corporation |
Zheng J.,U.S. Food and Drug Administration |
Yourick J.J.,U.S. Food and Drug Administration |
Sprando R.L.,U.S. Food and Drug Administration
Journal of Applied Toxicology | Year: 2014
As a consequence of the increased use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics to prevent fungal and bacterial growth, there is a need for validated rapid screening methods to assess the safety of nanoparticle exposure. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential genotoxicity of 20-nm nanosilver. The average silver nanoparticle size as determined by transmission electron microscopy (TEM) was 20.4 nm. Dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The silver concentration in a 20-nm nanosilver solution determined by the inductively coupled plasma-mass spectrometry (ICP-MS) analysis was 0.962 mg ml-1. Analysis by ICP-MS and TEM demonstrated the uptake of 20-nm silver by both HepG2 and Caco2 cells. Genotoxicity was determined by the cytochalasin B-blocked micronucleus assay with acridine orange staining and fluorescence microscopy. Concentration- and time-dependent increases in the frequency of binucleated cells with micronuclei induced by the nanosilver was observed in the concentration range of 0.5 to 15 μg ml-1 in both HepG2 and Caco2 cells compared with the control. Our results indicated that HepG2 cells were more sensitive than Caco2 cells in terms of micronuclei formation induced by nanosilver exposure. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, represent potential screening models for prediction of genotoxicity of silver nanoparticles by in vitro micronucleus assay. © 2014. This article is a U.S. Government work and is in the public domain in the USA.
Wang S.,J. Craig Venter Institute |
Sundaram J.P.,BioReliance Corporation |
Stockwell T.B.,J. Craig Venter Institute
Nucleic Acids Research | Year: 2012
A gene prediction program, VIGOR (Viral Genome ORF Reader), was developed at J. Craig Venter Institute in 2010 and has been successfully performing gene calling in coronavirus, influenza, rhinovirus and rotavirus for projects at the Genome Sequencing Center for Infectious Diseases. VIGOR uses sequence similarity search against custom protein databases to identify protein coding regions, start and stop codons and other gene features. Ribonucleicacid editing and other features are accurately identified based on sequence similarity and signature residues. VIGOR produces four output files: a gene prediction file, a complementary DNA file, an alignment file, and a gene feature table file. The gene feature table can be used to create GenBank submission. VIGOR takes a single input: viral genomic sequences in FASTA format. VIGOR has been extended to predict genes for 12 viruses: measles virus, mumps virus, rubella virus, respiratory syncytial virus, alphavirus and Venezuelan equine encephalitis virus, norovirus, metapneumovirus, yellow fever virus, Japanese encephalitis virus, parainfluenza virus and Sendai virus. VIGOR accurately detects the complex gene features like ribonucleicacid editing, stop codon leakage and ribosomal shunting. Precisely identifying the mat-peptide cleavage for some viruses is a built-in feature of VIGOR. The gene predictions for these viruses have been evaluated by testing from 27 to 240 genomes from GenBank. © 2012 The Author(s).
Shi J.,BioReliance Corporation |
Springer S.,BioReliance Corporation |
Escobar P.,BioReliance Corporation
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2010
There is considerable discussion within the scientific community as to the appropriate measures of cytotoxicity to use when deciding on the maximum concentration of a substance to test in vitro for its ability to induce DNA damage using the Comet assay. Conventional cytotoxicity assessment methods, such as trypan blue dye exclusion or relative cell number (cell counts) may not be the most biologically relevant measurement for cytotoxicity in this assay. Thus, we evaluated for decreased levels of adenosine triphosphate (ATP) and activation of Caspase-3/7 as well as relative cell number and trypan blue exclusion in order to understand the correlation among test compound concentration, cytotoxicity and genotoxicity outcomes in the Comet assay. We tested two non-genotoxic and non-cytotoxic compounds (d-glucose and ethanol), two non-genotoxic but cytotoxic compounds (2,4-dichlorophenol and tunicamycin) and four genotoxic and cytotoxic compounds (methyl methanesulfonate, ethyl methanesulfonate, etoposide and 4-nitroquinoline-N-oxide) in TK6 human lymphoblast cells. Our data show that measuring ATP and Caspase-3/7 levels provides more rapid and perhaps more biologically relevant measures of cytotoxicity compared with trypan blue dye exclusion and relative cell number. Furthermore, incorporating these two assays into the Comet assay also provided insight on the cytotoxic mode of action of the chemicals tested. By extrapolation, such assays may also be useful in other in vitro genotoxicity assays. © 2010 Elsevier B.V. All rights reserved.
Onions D.,BioReliance Corporation |
Cote C.,BioReliance Corporation |
Love B.,BioReliance Corporation |
Toms B.,BioReliance Corporation |
And 4 more authors.
Vaccine | Year: 2011
Massively parallel, deep, sequencing of the transcriptome coupled with algorithmic analysis to identify adventitious agents (MP-Seq™) is an important adjunct in ensuring the safety of cells used in vaccine production. Such cells may harbour novel viruses whose sequences are unknown or latent viruses that are only expressed following stress to the cells. MP-Seq is an unbiased and comprehensive method to identify such viruses and other adventitious agents without prior knowledge of the nature of those agents. Here we demonstrate its utility as part of an integrated approach to identify and characterise potential contaminants within commonly used virus and vaccine production cell lines. Through this analysis, in combination with more traditional approaches, we have excluded the presence of porcine circoviruses in the ATCC Vero cell bank (CCL-81), however, we found that a full length betaretrovirus related to SRV can be expressed in these cells, a factor that may be of importance in the production of certain vaccines. Similarly, insect cells are proving to be valuable for the production of virus like particles and sub-unit vaccines, but they can harbour a range of latent viruses. We show that following MP-Seq of the Trichoplusia ni (High Five cell line) transcriptome we were able to detect a contaminating, latent nodavirus and identify an expressed errantivirus genome. Collectively, these studies have reinforced the role of MP-Seq as an integral tool for the identification of contaminating agents in vaccine cell substrates. © 2011 Elsevier Ltd.
Shah S.A.,BioReliance Corporation |
Paranjpe M.G.,BioReliance Corporation |
Atkins P.I.,BioReliance Corporation |
Zahalka E.A.,BioReliance Corporation
International Journal of Toxicology | Year: 2012
The lack of a clear guidance on the adequate number of animals used for positive controls in the short-term (26-weeks) transgenic mouse carcinogenicity studies has resulted in the use of high number of animals. In our earlier Tg.rasH2 studies, 25 mice/sex were used in the urethane-positive control dose groups that were sacrificed by 18 weeks. Based on a robust response, several of our protocols for Tg.rasH2 studies with 15 mice/sex and terminal sacrifice at 17 ± 1 weeks were submitted and accepted by the Carcinogenicity Assessment Committee of the US Food and Drug Administration since we demonstrated close to 100% response for the development of lung and splenic tumors (target organs) in 500 mice/sex. These 500 mice/sex included 17 groups of 25 mice/sex and 5 groups of 15 mice/sex. The objective of this investigation was to determine whether the number of animals can be further reduced along with the shortened duration of exposure to urethane. Accordingly, 10 Tg.rasH2 mice/sex/group were administered a total of 3 intraperitoneal (IP) injections of urethane (1000 mg/kg per day) on study days 1, 3, and 5, and the presence of tumors in the lungs and spleen was evaluated after 8, 10, 12, 14, or 16 weeks. Our results demonstrate that 100% of the mice at 8 weeks had developed lung tumors, whereas close to 100% of the mice at 14 weeks had developed splenic tumors. Based on the development of lung tumors alone in 100% of the mice, we recommend that 10 mice/sex are sufficient and that these mice can also be sacrificed as early as 10 ± 1 weeks following the administration of urethane. © The Author(s) 2012.
Shi J.,BioReliance Corporation |
Bezabhie R.,BioReliance Corporation |
Szkudlinska A.,BioReliance Corporation
Mutagenesis | Year: 2010
The in vitro micronucleus (MN) assay is widely used to assess genotoxic potential of the test substances by measuring frequency of MN in cultured mammalian cells. Traditionally, MN frequency has been determined by microscopy. In recent years, a flow cytometric method for enumeration of MN has been developed, which significantly shortens analysis time and enhances assay throughput. However, a major concern has been raised that the MN results obtained from flow cytometry can be impacted by chromatin bodies produced during apoptosis or necrosis. In this work, we further evaluated this flow cytometry-based in vitro MN assay with CHO-K1 cells in a 24-well platform. Our results showed that the MN frequency determined using the flow cytometric method was highly correlated with the microscopy results. Importantly, several non-genotoxic apoptosis inducers or cytotoxins that have been previously reported to produce 'artificial positives' in various in vitro genotoxicity tests were evaluated in this system. As a result, these non-genotoxic cytotoxins did not produce false-positive MN response in the flow cytometric system in CHO-K1 cells when cytotoxicity was <50±10%. Moreover, a total of 21 compounds were evaluated in this work, including direct or indirect clastogens, aneugens and non-genotoxic chemicals. A sensitivity of 83.3% and a specificity of 100% were obtained from the compounds we tested. Finally, significant increase of incidents in the hypodiploid region, an aneugenic signature, was confirmed in our evaluation. In conclusion, the flow cytometric in vitro MN assay is a reliable method that can be used to detect clastogenic or aneugenic potential of the test substances in CHO-K1 cells.
Pant K.,BioReliance Corporation
Mutation research | Year: 2010
The SHE cell transformation assay has traditionally been conducted with a feeder layer of X-ray irradiated cells to provide growth support to the target cells seeded in low numbers. The need for an X-ray irradiated feeder cell layer necessitates the maintenance of an X-ray machine and the additional step to seed feeder cells prior to plating target cells. This laboratory has previously reported a method allowing target cells to be seeded in conditioned media prepared from the stock culture flasks in lieu of plating them on a feeder layer (Pant et al. [1,2,4]). In order to expand the data base for chemicals tested using this method, we describe in this paper the results obtained testing Di(2-ethylhexyl)phthalate (DEHP) and N-nitroso-N-methylnitroguanidine (MNNG) which are known to give positive responses in the standard SHE cell transformation assay. With freshly prepared conditioned medium (used within 2 weeks of preparation), there was essentially no difference in the number of target cell colonies in the conditioned medium and in the plates with the X-ray irradiated feeder cell layer. The plating efficiencies of the vehicle controls were within the historical range for the standard SHE cell transformation assay. In more than ten experiments the positive control benzo(a)pyrene [B(a)P] elicited a significant increase in morphological transformation frequency (MTF), with or without X-ray irradiated feeder cells. Compounds, DEHP and MNNG, were tested in the SHE cell transformation assay with and without an X-ray irradiated feeder layer and using a 7-day exposure regimen. The results were comparable between experiments performed using either method. These results demonstrate the feasibility of conducting the SHE cell transformation assay without the use of an X-ray irradiated feeder layer, thereby simplifying the test procedure and assisting the scoring of morphologically transformed colonies. Copyright 2010 Elsevier B.V. All rights reserved.