40 results on '"Aardema, Marilyn"'
Search Results
2. Toxicology and Genetic Toxicology in the New Era of 'Toxicogenomics': Impact of '-omics' Technologies
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Aardema, Marilyn J., MacGregor, James T., Inoue, Tohru, editor, and Pennie, William D., editor
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- 2003
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3. Use of the EpiDermTM 3D reconstructed skin micronucleus assay for fragrance materials.
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Thakkar, Yax, Moustakas, Holger, Aardema, Marilyn, Roy, Shambhu, Pfuhler, Stefan, and Api, Anne Marie
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NUCLEOLUS ,ANIMAL experimentation ,CHEMICAL potential ,GENETIC toxicology ,ODORS - Abstract
In order to evaluate the utility of the 3D reconstructed skin micronucleus assay (3DRSMN) to assess clastogenic/aneugenic potential of the fragrance chemicals, a set of 22 fragrance materials were evaluated in 3DRSMN assay. These materials evaluated were also evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per Organisation for Economic Co-operation and Development guidelines. The results of the RSMN assay were in 100% agreement with the in vivo micronucleus assay results. From this dataset, 18 materials were positive in an in vitro micronucleus assay but were negative in an in vivo micronucleus assay. All these 18 materials were also concluded to be negative in 3DRSMN assay, stressing the importance of the assay to help minimize misleading positive outcomes from the in vitro assay. Since the highest exposure for fragrances is through the dermal route, the RSMN assay fits the applicability domain for testing. Thus, RSMN assay is an important alternative to animal testing for characterization of the genotoxicity potential of fragrance materials. [ABSTRACT FROM AUTHOR]
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- 2022
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4. Validation of the 3D reconstructed human skin micronucleus (RSMN) assay: an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays.
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Pfuhler, Stefan, Downs, Thomas R, Hewitt, Nicola J, Hoffmann, Sebastian, Mun, Greg C, Ouedraogo, Gladys, Roy, Shambhu, Curren, Rodger D, and Aardema, Marilyn J
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GENETIC toxicology ,NUCLEOLUS ,GENETIC mutation ,CELL culture ,ANIMAL experimentation ,HUMAN beings - Abstract
In vitro test batteries have become the standard approach to determine the genotoxic potential of substances of interest across industry sectors. While useful for hazard identification, standard in vitro genotoxicity assays in 2D cell cultures have limited capability to predict in vivo outcomes and may trigger unnecessary follow-up animal studies or the loss of promising substances where animal tests are prohibited or not desired. To address this problem, a team of regulatory, academia and industry scientists was established to develop and validate 3D in vitro human skin-based genotoxicity assays for use in testing substances with primarily topical exposure. Validation of the reconstructed human skin micronucleus (RSMN) assay in MatTek Epi-200™ skin models involved testing 43 coded chemicals selected by independent experts, in four US/European laboratories. The results were analysed by an independent statistician according to predefined criteria. The RSMN assay showed a reproducibly low background micronucleus frequency and exhibited sufficient capacity to metabolise pro-mutagens. The overall RSMN accuracy when compared to in vivo genotoxicity outcomes was 80%, with a sensitivity of 75% and a specificity of 84%, and the between- and within-laboratory reproducibility was 77 and 84%, respectively. A protocol involving a 72-h exposure showed increased sensitivity in detecting true positive chemicals compared to a 48-h exposure. An analysis of a test strategy using the RSMN assay as a follow-up test for substances positive in standard in vitro clastogenicity/aneugenicity assays and a reconstructed skin Comet assay for substances with positive results in standard gene mutation assays results in a sensitivity of 89%. Based on these results, the RSMN assay is considered sufficiently validated to establish it as a 'tier 2' assay for dermally exposed compounds and was recently accepted into the OECD's test guideline development program. [ABSTRACT FROM AUTHOR]
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- 2021
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5. Simulation of mouse and rat spermatogenesis to inform genotoxicity testing using OECD test guideline 488.
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Marchetti, Francesco, Aardema, Marilyn, Beevers, Carol, van Benthem, Jan, Douglas, George R., Godschalk, Roger, Yauk, Carole L., Young, Robert, and Williams, Andrew
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SPERMATOGENESIS , *GENETIC toxicology , *GENETIC mutation , *LABORATORY rats - Abstract
The Organisation for Economic Co-operation and Development Test Guideline (TG) 488 for the transgenic rodent (TGR) mutation assay recommends two sampling times for assessing germ cell mutagenicity following the required 28-day exposure period: 28 + > 49 days for mouse sperm and 28 + >70 days for rat sperm from the cauda epididymis, or three days (i.e., 28 + 3d) for germ cells from seminiferous tubules (hereafter, tubule germ cells) plus caudal sperm for mouse and rat. Although the latter protocol is commonly used for mutagenicity testing in somatic tissues, it has several shortcomings for germ cell testing because it provides limited exposure of the proliferating phase of spermatogenesis when mutations are fixed in the transgene. Indeed, analysis of sperm at 28 + 3d has generated negative results with established germ cell mutagens, while the analysis of tubule germ cells has generated both positive and either negative or equivocal results. The Germ Cell workgroup of the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute modelled mouse and rat spermatogenesis to better define the exposure history of the cell population collected from seminiferous tubules. The modelling showed that mouse tubule germ cells at 28 + 3d receive, as a whole, 42% of the total exposure during the proliferating phase. This percentage increases to 99% at 28 + 28d and reaches 100% at 28 + 30d. In the rat, these percentages are 22% and 80% at 28 + 3d and 28 + 28d, reaching 100% at 28 + 44d. These results show that analysis of tubule germ cells at 28 + 28d may be an effective protocol for assessing germ cell mutagenicity in mice and rats using TG 488. Since TG 488 recommends the 28 + 28d protocol for slow dividing somatic tissues, this appears to be a better compromise than 28 + 3d when slow dividing somatic tissues or germ cells are the critical tissues of interest. [ABSTRACT FROM AUTHOR]
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- 2018
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6. Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods.
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Elespuru, Rosalie, Pfuhler, Stefan, Aardema, Marilyn J, Chen, Tao, Doak, Shareen H, Doherty, Ann, Farabaugh, Christopher S, Kenny, Julia, Manjanatha, Mugimane, and Mahadevan, Brinda
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GENETIC toxicology ,NANOSTRUCTURED materials ,CHROMOSOME abnormalities - Abstract
Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute’s Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing. [ABSTRACT FROM AUTHOR]
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- 2018
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7. A review of the carcinogenic potential of glyphosate by four independent expert panels and comparison to the IARC assessment.
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Williams, Gary M., Aardema, Marilyn, Acquavella, John, Berry, Sir Colin, Brusick, David, Burns, Michele M., de Camargo, Joao Lauro Viana, Garabrant, David, Greim, Helmut A., Kier, Larry D., Kirkland, David J., Marsh, Gary, Solomon, Keith R., Sorahan, Tom, Roberts, Ashley, and Weed, Douglas L.
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GLYPHOSATE , *CARCINOGENICITY , *GENETIC toxicology , *OXIDATIVE stress - Abstract
The International Agency for Research on Cancer (IARC) published a monograph in 2015 concluding that glyphosate is “probably carcinogenic to humans” (Group 2A) based on limited evidence in humans and sufficient evidence in experimental animals. It was also concluded that there was strong evidence of genotoxicity and oxidative stress. Four Expert Panels have been convened for the purpose of conducting a detailed critique of the evidence in light of IARC’s assessment and to review all relevant information pertaining to glyphosate exposure, animal carcinogenicity, genotoxicity, and epidemiologic studies. Two of the Panels (animal bioassay and genetic toxicology) also provided a critique of the IARC position with respect to conclusions made in these areas. The incidences of neoplasms in the animal bioassays were found not to be associated with glyphosate exposure on the basis that they lacked statistical strength, were inconsistent across studies, lacked dose-response relationships, were not associated with preneoplasia, and/or were not plausible from a mechanistic perspective. The overall weight of evidence from the genetic toxicology data supports a conclusion that glyphosate (including GBFs and AMPA) does not pose a genotoxic hazard and therefore, should not be considered support for the classification of glyphosate as a genotoxic carcinogen. The assessment of the epidemiological data found that the data do not support a causal relationship between glyphosate exposure and non-Hodgkin’s lymphoma while the data were judged to be too sparse to assess a potential relationship between glyphosate exposure and multiple myeloma. As a result, following the review of the totality of the evidence, the Panels concluded that the data do not support IARC’s conclusion that glyphosate is a “probable human carcinogen” and, consistent with previous regulatory assessments, further concluded that glyphosate is unlikely to pose a carcinogenic risk to humans. [ABSTRACT FROM PUBLISHER]
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- 2016
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8. Genotoxicity Expert Panel review: weight of evidence evaluation of the genotoxicity of glyphosate, glyphosate-based formulations, and aminomethylphosphonic acid.
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Brusick, David, Aardema, Marilyn, Kier, Larry, Kirkland, David, and Williams, Gary
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GENETIC toxicology , *GLYPHOSATE , *PHOSPHONIC acids , *ANIMAL models of carcinogenesis , *OXIDATIVE stress , *LABORATORY mice - Abstract
In 2015, the International Agency for Research on Cancer (IARC) published a monograph concluding there was strong evidence for genotoxicity of glyphosate and glyphosate formulations and moderate evidence for genotoxicity of the metabolite aminomethylphosphonic acid (AMPA). These conclusions contradicted earlier extensive reviews supporting the lack of genotoxicity of glyphosate and glyphosate formulations. The IARC Monograph concluded there was strong evidence of induction of oxidative stress by glyphosate, glyphosate formulations, and AMPA. The Expert Panel reviewed the genotoxicity and oxidative stress data considered in the IARC Monograph, together with other available data not considered by IARC. The Expert Panel defined and used a weight of evidence (WoE) approach that included ranking of studies and endpoints by the strength of their linkage to events associated with carcinogenic mechanisms. Importantly, the Expert Panel concluded that there was sufficient information available from a very large number of regulatory genotoxicity studies that should have been considered by IARC. The WoE approach, the inclusion of all relevant regulatory studies, and some differences in interpretation of individual studies led to significantly different conclusions by the Expert Panel compared with the IARC Monograph. The Expert Panel concluded that glyphosate, glyphosate formulations, and AMPA do not pose a genotoxic hazard and the data do not support the IARC Monograph genotoxicity evaluation. With respect to carcinogenicity classification and mechanism, the Expert Panel concluded that evidence relating to an oxidative stress mechanism of carcinogenicity was largely unconvincing and that the data profiles were not consistent with the characteristics of genotoxic carcinogens. [ABSTRACT FROM PUBLISHER]
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- 2016
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9. A weight of evidence review of the genotoxicity of titanium dioxide (TiO2).
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Kirkland, David, Aardema, Marilyn J., Battersby, Rüdiger V., Beevers, Carol, Burnett, Karin, Burzlaff, Arne, Czich, Andreas, Donner, E. Maria, Fowler, Paul, Johnston, Helinor J., Krug, Harald F., Pfuhler, Stefan, and Stankowski, Leon F.
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TITANIUM dioxide , *GENETIC toxicology , *CHROMOSOME abnormalities , *PHYSIOLOGICAL stress , *REACTIVE oxygen species , *GENETIC mutation - Abstract
Titanium dioxide is a ubiquitous white material found in a diverse range of products from foods to sunscreens, as a pigment and thickener, amongst other uses. Titanium dioxide has been considered no longer safe for use in foods (nano and microparticles of E171) by the European Food Safety Authority (EFSA) due to concerns over genotoxicity. There are however, conflicting opinions regarding the safety of Titanium dioxide. In an attempt to clarify the situation, a comprehensive weight of evidence (WoE) assessment of the genotoxicity of titanium dioxide based on the available data was performed. A total of 192 datasets for endpoints and test systems considered the most relevant for identifying mutagenic and carcinogenic potential were reviewed and discussed for both reliability and relevance (by weight of evidence) and in the context of whether the physico-chemical properties of the particles had been characterised. The view of an independent panel of experts was that, of the 192 datasets identified, only 34 met the reliability and quality criteria for being most relevant in the evaluation of genotoxicity. Of these, 10 were positive (i.e. reported evidence that titanium dioxide was genotoxic), all of which were from studies of DNA strand breakage (comet assay) or chromosome damage (micronucleus or chromosome aberration assays). All the positive findings were associated with high cytotoxicity, oxidative stress, inflammation, apoptosis, necrosis, or combinations of these. Considering that DNA and chromosome breakage can be secondary to physiological stress, it is highly likely that the observed genotoxic effects of titanium dioxide, including those with nanoparticles, are secondary to physiological stress. Consistent with this finding, there were no positive results from the in vitro and in vivo gene mutation studies evaluated, although it should be noted that to definitively conclude a lack of mutagenicity, more robust in vitro and in vivo gene mutation studies would be useful. Existing evidence does not therefore support a direct DNA damaging mechanism for titanium dioxide (nano and other forms). • EFSA have recently banned titanium dioxide in foods due to concerns over genotoxicity. • A tiered weight of evidence analysis was performed on genotoxicity data for TiO 2 , according to relevance and reliability. • TiO 2 was positive for chromosome damage mainly at levels where reactive oxygen or other cellular toxicity were prevalent. • TiO 2 was negative for point mutations in vivo , the panel noted more data would be required to make definitive conclusions. [ABSTRACT FROM AUTHOR]
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- 2022
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10. An evaluation of 25 selected Tox Cast chemicals in medium-throughput assays to detect genotoxicity.
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Kligerman, Andrew D., Young, Robert R., Stankowski, Leon F., Pant, Kamala, Lawlor, Tim, Aardema, Marilyn J., and Houck, Keith A.
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GENETIC toxicology ,NUCLEOLUS ,BIOTRANSFORMATION (Metabolism) ,CHEMICAL processes - Abstract
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. [ABSTRACT FROM AUTHOR]- Published
- 2015
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11. Approaches for identifying germ cell mutagens: Report of the 2013 IWGT workshop on germ cell assays☆.
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Yauk, Carole L., Aardema, Marilyn J., Benthem, Jan van, Bishop, Jack B., Dearfield, Kerry L., DeMarini, David M., Dubrova, Yuri E., Honma, Masamitsu, Lupski, James R., Marchetti, Francesco, Meistrich, Marvin L., Pacchierotti, Francesca, Stewart, Jane, Waters, Michael D., and Douglas, George R.
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MUTAGEN analysis , *GERM cells , *BIOLOGICAL assay , *GENETIC toxicology , *TOXICITY testing , *MUTAGENICITY testing - Abstract
This workshop reviewed the current science to inform and recommend the best evidence-based approaches on the use of germ cell genotoxicity tests. The workshop questions and key outcomes were as follows. (1) Do genotoxicity and mutagenicity assays in somatic cells predict germ cell effects? Limited data suggest that somatic cell tests detect most germ cell mutagens, but there are strong concerns that dictate caution in drawing conclusions. (2) Should germ cell tests be done, and when? If there is evidence that a chemical or its metabolite(s) will not reach target germ cells or gonadal tissue, it is not necessary to conduct germ cell tests, notwithstanding somatic outcomes. However, it was recommended that negative somatic cell mutagens with clear evidence for gonadal exposure and evidence of toxicity in germ cells could be considered for germ cell mutagenicity testing. For somatic mutagens that are known to reach the gonadal compartments and expose germ cells, the chemical could be assumed to be a germ cell mutagen without further testing. Nevertheless, germ cell mutagenicity testing would be needed for quantitative risk assessment. (3) What new assays should be implemented and how? There is an immediate need for research on the application of whole genome sequencing in heritable mutation analysis in humans and animals, and integration of germ cell assays with somatic cell genotoxicity tests. Focus should be on environmental exposures that can cause de novo mutations, particularly newly recognized types of genomic changes. Mutational events, which may occur by exposure of germ cells during embryonic development, should also be investigated. Finally, where there are indications of germ cell toxicity in repeat dose or reproductive toxicology tests, consideration should be given to leveraging those studies to inform of possible germ cell genotoxicity. [ABSTRACT FROM AUTHOR]
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- 2015
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12. Opportunities to integrate new approaches in genetic toxicology: An ILSI-HESI workshop report.
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Zeiger, Errol, Gollapudi, Bhaskar, Aardema, Marilyn J., Auerbach, Scott, Boverhof, Darrell, Custer, Laura, Dedon, Peter, Honma, Masamitsu, Ishida, Seiichi, Kasinski, Andrea L., Kim, James H., Manjanatha, Mugimane G., Marlowe, Jennifer, Pfuhler, Stefan, Pogribny, Igor, Slikker, William, Stankowski, Leon F., Tanir, Jennifer Y., Tice, Raymond, and van Benthem, Jan
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GENETIC toxicology ,EPIGENETICS ,CARCINOGENS ,DNA adducts ,DNA repair ,DNA mutational analysis ,LABORATORY rodents - Abstract
Genetic toxicity tests currently used to identify and characterize potential human mutagens and carcinogens rely on measurements of primary DNA damage, gene mutation, and chromosome damage in vitro and in rodents. The International Life Sciences Institute Health and Environmental Sciences Institute (ILSI-HESI) Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity Testing held an April 2012 Workshop in Washington, DC, to consider the impact of new understanding of biology and new technologies on the identification and characterization of genotoxic substances, and to identify new approaches to inform more accurate human risk assessment for genetic and carcinogenic effects. Workshop organizers and speakers were from industry, academe, and government. The Workshop focused on biological effects and technologies that would potentially yield the most useful information for evaluating human risk of genetic damage. Also addressed was the impact that improved understanding of biology and availability of new techniques might have on genetic toxicology practices. Workshop topics included (1) alternative experimental models to improve genetic toxicity testing, (2) Biomarkers of epigenetic changes and their applicability to genetic toxicology, and (3) new technologies and approaches. The ability of these new tests and technologies to be developed into tests to identify and characterize genotoxic agents; to serve as a bridge between in vitro and in vivo rodent, or preferably human, data; or to be used to provide dose response information for quantitative risk assessment was also addressed. A summary of the workshop and links to the scientific presentations are provided. Environ. Mol. Mutagen. 56:277-285, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
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- 2015
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13. Achieving professional success in US government, academia, and industry: An EMGS commentary.
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Poirier, Miriam C., Schwartz, Jeffrey L., and Aardema, Marilyn J.
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CAREER development ,POLITICAL science ,ACADEMIC programs ,INDUSTRIES ,GENOMICS ,PREDICTION of occupational success ,GENETIC toxicology ,JOB security ,SOCIETIES - Abstract
One of the goals of the EMGS is to help members achieve professional success in the fields they have trained in. Today, there is greater competition for jobs in genetic toxicology, genomics, and basic research than ever before. In addition, job security and the ability to advance in one's career is challenging, regardless of whether one works in a regulatory, academic, or industry environment. At the EMGS Annual Meeting in Monterey, CA (September, 2013), the Women in EMGS Special Interest Group held a workshop to discuss strategies for achieving professional success. Presentations were given by three speakers, each representing a different employment environment: Government (Miriam C. Poirier), Academia (Jeffrey L. Schwartz), and Industry (Marilyn J. Aardema). Although some differences in factors or traits affecting success in the three employment sectors were noted by each of the speakers, common factors considered important for advancement included networking, seeking out mentors, and developing exceptional communication skills. Environ. Mol. Mutagen. 55:525-529, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
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- 2014
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14. The holy grail in genetic toxicology: Follow-up approaches for positive results in the Ames assay.
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Aardema, Marilyn J.
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GENETIC toxicology ,AMES test ,ESCHERICHIA coli ,MUTAGENICITY testing ,DRUG development - Abstract
Positive results in the Ames/ E. coli bacterial mutagenicity assay create a significant hurdle for the development of new products/drugs and as a result, most companies drop mutagenic ingredients from further development. One important consequence of this is that the understanding of the human relevance of chemicals that are positive in the Ames assay is not increasing at the pace seen with ingredients that are positive in the other in vitro genotoxicity assays. Recent advances in defining thresholds for mutagenic carcinogens, along with new assays for measuring mutagenicity in vivo suggests it is time to direct more attention to the holy grail of clearly defining practical approaches to address positive results in the Ames assay. To stimulate further discussion and research in this area, a review of the most current thinking on approaches for dealing with Ames positive results is provided along with some suggestions for the way forward. Environ. Mol. Mutagen. 54:617-620, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
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- 2013
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15. Genotoxicity of nanomaterials: Refining strategies and tests for hazard identification.
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Pfuhler, Stefan, Elespuru, Rosalie, Aardema, Marilyn J., Doak, Shareen H., Maria Donner, E., Honma, Masamitsu, Kirsch‐Volders, Micheline, Landsiedel, Robert, Manjanatha, Mugimane, Singer, Tim, and Kim, James H.
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NANOSTRUCTURED materials ,GENETIC toxicology ,CENTROMERE ,NUCLEOLUS ,GENETIC mutation ,MAMMALIAN cell cycle - Abstract
A workshop addressing strategies for the genotoxicity assessment of nanomaterials (NMs) was held on October 23, 2010 in Fort Worth Texas, USA. The workshop was organized by the Environmental Mutagen Society and the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute. The workshop was attended by more than 80 participants from academia, regulatory agencies, and industry from North America, Europe and Japan. A plenary session featured summaries of the current status and issues related to the testing of NMs for genotoxic properties, as well as an update on international activities and regulatory approaches. This was followed by breakout sessions and a plenary session devoted to independent discussions of in vitro assays, in vivo assays, and the need for new assays or new approaches to develop a testing strategy for NMs. Each of the standard assays was critiqued as a resource for evaluation of NMs, and it became apparent that none was appropriate without special considerations or modifications. The need for nanospecific positive controls was questioned, as was the utility of bacterial assays. The latter was thought to increase the importance of including mammalian cell gene mutation assays into the test battery. For in-vivo testing, to inform the selection of appropriate tests or protocols, it was suggested to run repeated dose studies first to learn about disposition, potential accumulation, and possible tissue damage. It was acknowledged that mechanisms may be at play that a standard genotoxicity battery may not be able to capture. Environ. Mol. Mutagen. 54:229-239, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
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- 2013
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16. Evaluation of chemicals requiring metabolic activation in the EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay
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Aardema, Marilyn J., Barnett, Brenda B., Mun, Greg C., Dahl, Erica L., Curren, Rodger D., Hewitt, Nicola J., and Pfuhler, Stefan
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CHEMICALS , *BIOTRANSFORMATION (Metabolism) , *EPIDERMIS , *NUCLEOLUS , *BIOLOGICAL assay , *GENETIC toxicology , *COSMETICS - Abstract
Abstract: The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm™ is a promising new assay for evaluating genotoxicity of dermally applied chemicals. A global pre-validation project sponsored by the European Cosmetics Association (Cosmetics Europe - formerly known as COLIPA), and the European Center for Validation of Alternative Methods (ECVAM), is underway. Results to date demonstrate international inter-laboratory and inter-experimental reproducibility of the assay for chemicals that do not require metabolism [Aardema et al., Mutat. Res. 701 (2010) 123–131]. We have expanded these studies to investigate chemicals that do require metabolic activation: 4-nitroquinoline-N-oxide (4NQO), cyclophosphamide (CP), dimethylbenzanthracene (DMBA), dimethylnitrosamine (DMN), dibenzanthracene (DBA) and benzo(a)pyrene (BaP). In this study, the standard protocol of two applications over 48h was compared with an extended protocol involving three applications over 72h. Extending the treatment period to 72h changed the result significantly only for 4NQO, which was negative in the standard 48h dosing regimen, but positive with the 72h treatment. DMBA and CP were positive in the standard 48h assay (CP induced a more reproducible response with the 72h treatment) and BaP gave mixed results; DBA and DMN were negative in both the 48h and the 72h dosing regimens. While further work with chemicals that require metabolism is needed, it appears that the RMSN assay detects some chemicals that require metabolic activation (4 out of 6 chemicals were positive in one or both protocols). At this point in time, for general testing, the use of a longer treatment period in situations where the standard 48h treatment is negative or questionable is recommended. [Copyright &y& Elsevier]
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- 2013
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17. Summary of in vitro genetic toxicology assay results: Expected and unexpected effects of recent study design modifications.
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Clarke, Jane J., Lawlor, Timothy E., Madraymootoo, Wannie, Pant, Kamala, Young, Robert R., Wagner, Valentine O., and Aardema, Marilyn J.
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GENETIC toxicology ,CHROMOSOME abnormalities ,GENETIC mutation ,LYMPHOMAS ,LYMPHOCYTES - Abstract
Key modifications to in vitro genetic toxicology testing have been made in the last 5 years including the use of optimization approaches such as structure-activity relationships and screening assays to identify and eliminate potentially genotoxic chemicals from further consideration, better guidance on cytotoxicity assessment and dose selection, and greater use of p53-competent human cells. To determine the effect of these changes on testing outcomes, the pattern of positive results across assays conducted by BioReliance from 2005 to 2010 was examined. Data were tabulated for good laboratory practice (GLP)-compliant Ames, mouse lymphoma (MLA), chromosome aberration in Chinese hamster ovary (CHO) cells, and in human peripheral blood lymphocytes (HPBL) assays along with non-GLP screening Ames assays. A decrease in percentage of positive results in MLA and CHO chromosome aberration assays was observed, whereas the percentage of positive Ames assays remained consistent. This was not unexpected because MLA and CHO cytogenetic assays have undergone the most substantive changes (e.g., the establishment of the Global Evaluation Factor for the MLA and the use of the relative increase in cell counts in CHO chromosome aberration assays). Over the last 5 years, there has been an increase in the percentage of positive results observed in the chromosome aberration assay in HPBL. It is speculated that this may have led to an increase in HPBL-positive results if the chemicals routed to HPBL had previous positive genotoxicity results. Another factor may be the lack of a reliable cytotoxicity measurement in the HPBL assay. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
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- 2012
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18. ECVAM prevalidation study on in vitro cell transformation assays: General outline and conclusions of the study
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Corvi, Raffaella, Aardema, Marilyn J., Gribaldo, Laura, Hayashi, Makoto, Hoffmann, Sebastian, Schechtman, Leonard, and Vanparys, Philippe
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CELL transformation , *ANIMAL models of carcinogenesis , *GENETIC toxicology , *CARCINOGEN dose-response relationship , *CELL lines , *DIMETHYL sulfoxide , *GENETIC mutation - Abstract
Abstract: The potential for a compound to induce carcinogenicity is a key consideration when ascertaining hazard and risk assessment of chemicals. Among the in vitro alternatives that have been developed for predicting carcinogenicity, in vitro cell transformation assays (CTAs) have been shown to involve a multistage process that closely models important stages of in vivo carcinogenesis and have the potential to detect both genotoxic and non-genotoxic carcinogens. These assays have been in use for decades and a substantial amount of data demonstrating their performance is available in the literature. However, for the standardised use of these assays for regulatory purposes, a formal evaluation of the assays, in particular focusing on development of standardised transferable protocols and further information on assay reproducibility, was considered important to serve as a basis for the drafting of generally accepted OECD test guidelines. To address this issue, a prevalidation study of the CTAs using the BALB/c 3T3 cell line, SHE cells at pH 6.7, and SHE cells at pH 7.0 was coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and focused on issues of standardisation of protocols, test method transferability and within- and between-laboratory reproducibility. The study resulted in the availability of standardised protocols that had undergone prevalidation . The results of the ECVAM study demonstrated that for the BALB/c 3T3 method, some modifications to the protocol were needed to obtain reproducible results between laboratories, while the SHE pH 6.7 and the SHE pH 7.0 protocols are transferable between laboratories, and results are reproducible within- and between-laboratories. It is recommended that the BALB/c 3T3 and SHE protocols as instituted in this prevalidation study should be used in future applications of these respective transformation assays. To support their harmonised use and regulatory application, the development of an OECD test guideline for the SHE CTAs, based on the protocol published in this issue, is recommended. The development of an OECD test guideline for the BALB/c 3T3 CTA should likewise be further pursued upon the availability of additional supportive data and improvement of the statistical analysis. [Copyright &y& Elsevier]
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- 2012
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19. New and emerging technologies for genetic toxicity testing.
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Lynch, Anthony M., Sasaki, Jennifer C., Elespuru, Rosalie, Jacobson-Kram, David, Thybaud, Véronique, De Boeck, Marlies, Aardema, Marilyn J., Aubrecht, Jiri, Benz, R. Daniel, Dertinger, Stephen D., Douglas, George R., White, Paul A., Escobar, Patricia A., Fornace, Albert, Honma, Masamitsu, Naven, Russell T., Rusling, James F., Schiestl, Robert H., Walmsley, Richard M., and Yamamura, Eiji
- Subjects
GENETIC toxicology ,MEDICAL technology ,IN vivo toxicity testing ,FOLLOW-up studies (Medicine) ,HEALTH risk assessment - Abstract
The International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing established an Emerging Technologies and New Strategies Workgroup to review the current State of the Art in genetic toxicology testing. The aim of the workgroup was to identify promising technologies that will improve genotoxicity testing and assessment of in vivo hazard and risk, and that have the potential to help meet the objectives of the IVGT. As part of this initiative, HESI convened a workshop in Washington, DC in May 2008 to discuss mature, maturing, and emerging technologies in genetic toxicology. This article collates the abstracts of the New and Emerging Technologies Workshop together with some additional technologies subsequently considered by the workgroup. Each abstract (available in the online version of the article) includes a section addressed specifically to the strengths, weaknesses, opportunities, and threats associated with the respective technology. Importantly, an overview of the technologies and an indication of how their use might be aligned with the objectives of IVGT are presented. In particular, consideration was given with regard to follow-up testing of positive results in the standard IVGT tests (i.e., Salmonella Ames test, chromosome aberration assay, and mouse lymphoma assay) to add weight of evidence and/or provide mechanism of action for improved genetic toxicity risk assessments in humans. Environ. Mol. Mutagen., 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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20. In vitro genotoxicity testing using the micronucleus assay in cell lines, human lymphocytes and 3D human skin models.
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Kirsch-Volders, Micheline, Decordier, Ilse, Elhajouji, Azeddine, Plas, Gina, Aardema, Marilyn J., and Fenech, Michael
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GENETIC toxicology ,NUCLEOLUS ,MICROBIOLOGICAL assay ,CELL lines ,LYMPHOCYTES ,EXTRAPOLATION ,BLOOD testing ,NANOSTRUCTURED materials - Abstract
The toxicological relevance of the micronucleus (MN) test is well defined: it is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events but also some epigenetic effects, which is simple to score, accurate, applicable in different cell types. In addition, it is predictive for cancer, amenable for automation and allows good extrapolation for potential limits of exposure or thresholds and it is easily measured in experimental both in vitro and in vivo systems. Implementation of in vitro micronucleus (IVMN) assays in the battery of tests for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified. Moreover, the final draft of an OECD guideline became recently available for this test. In this review, we discuss the prerequisites for an acceptable MN assay, including the cell as unit of observation, importance of cell membranes, the requirement of a mitotic or meiotic division and the assessment of cell division in the presence of the test substance. Furthermore, the importance of adequate design of protocols is highlighted and new developments, in particular the in vitro 3D human skin models, are discussed. Finally, we address future research perspectives including the possibility of a combined primary 3D human skin and primary human whole blood culture system, and the need for adaptation of the IVMN assays to assess the genotoxic potential of new materials, in particular nanomaterials. [ABSTRACT FROM AUTHOR]
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- 2011
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21. International prevalidation studies of the EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay: Transferability and reproducibility
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Aardema, Marilyn J., Barnett, Brenda C., Khambatta, Zubin, Reisinger, Kerstin, Ouedraogo-Arras, Gladys, Faquet, Brigitte, Ginestet, Anne-Claire, Mun, Greg C., Dahl, Erica L., Hewitt, Nicola J., Corvi, Raffallea, and Curren, Rodger D.
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CHROMOSOME abnormalities , *EPIDERMIS , *GENETIC toxicology , *SKIN grafting , *PHYSIOLOGICAL effects of chemicals , *COMPARATIVE studies , *DIAGNOSIS - Abstract
Abstract: Recently, a novel in vitro reconstructed skin micronucleus (RSMN) assay incorporating the EpiDerm™ 3D human skin model (Curren et al., Mutat. Res. 607 (2006) 192–204; Mun et al., Mutat. Res. 673 (2009) 92–99) has been shown to produce comparable data when utilized in three different laboratories in the United States (Hu et al., Mutat. Res. 673 (2009) 100–108). As part of a project sponsored by the European cosmetics companies trade association (COLIPA), with a contribution from the European Center for the Validation of Alternative Methods (ECVAM), international prevalidation studies of the RSMN assay have been initiated. The assay was transferred and optimized in two laboratories in Europe, where dose-dependent, reproducibly positive results for mitomycin C and vinblastine sulfate were obtained. Further intra- and inter-laboratory reproducibility of the RSMN assay was established by testing three coded chemicals, N-ethyl-N-nitrosourea, cyclohexanone, and mitomycin C. All chemicals were correctly identified by all laboratories as either positive or negative. These results support the international inter-laboratory and inter-experimental reproducibility of the assay and reinforce the conclusion that the RSMN assay in the EpiDerm™ 3D human skin model is a valuable in vitro method for assessment of genotoxicity of dermally applied chemicals. [Copyright &y& Elsevier]
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- 2010
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22. A review of biomonitoring studies measuring genotoxicity in humans exposed to hair dyes.
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Preston, R. Julian, Skare, Julie A., and Aardema, Marilyn J.
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BIOLOGICAL monitoring ,POISON analysis ,GENETIC toxicology ,HAIR dyeing & bleaching ,HAIR care products - Abstract
Hair dye ingredients frequently produce positive results in short-term in vitro genotoxicity tests, although results from in vivo assays are typically negative, especially for ingredients in use today. The use of hair dyes is quite widespread resulting in the exposure both for persons working in hairdressing salons and for individuals who have their hair dyed. This provides the opportunity to add to the data from standard in vitro and in vivo genotoxicity tests by investigating whether or not genotoxic responses are detected in such exposed individuals. A number of biomonitoring studies of humans exposed to hair dyes have been conducted using either cytogenetic alterations or DNA damage as measures of genotoxicity, or urine mutagenicity as a measure of exposure to genotoxic compounds. In this paper, each study is critically reviewed and interpreted. Overall, there is no consistent evidence of genotoxicity in humans exposed to hair dyes occupationally or through individual use. [ABSTRACT FROM PUBLISHER]
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- 2010
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23. Further development of the EpiDerm™ 3D reconstructed human skin micronucleus (RSMN) assay
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Mun, Greg C., Aardema, Marilyn J., Hu, Ting, Barnett, Brenda, Kaluzhny, Yulia, Klausner, Mitchell, Karetsky, Viktor, Dahl, Erica L., and Curren, Rodger D.
- Subjects
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EPIDERMIS , *BIOLOGICAL models , *NUCLEOLUS , *COSMETICS , *GENETIC toxicology , *ANIMAL models of toxicology , *IN vitro toxicity testing , *PREDICTION models , *BIOLOGICAL assay - Abstract
Abstract: The upcoming ban on testing of cosmetics in animals by the European Union''s 7th Amendment to the Cosmetics Directive will require genotoxicity safety assessments of cosmetics ingredients and final formulations to be based primarily on in vitro genotoxicity tests. The current in vitro test battery produces an unacceptably high rate of false positives, and used by itself would effectively prevent the use and development of many ingredients that are actually safe for human use. To address the need for an in vitro test that is more predictive of genotoxicity in vivo, we have developed an in vitro micronucleus assay using a three-dimensional human reconstructed skin model (EpiDerm™) that more closely mimics the normal dermal exposure route of chemicals. We have refined this model and assessed its ability to predict genotoxicity of a battery of chemicals that have been previously classified as genotoxins or non-genotoxins based on in vivo rodent skin tests. Our reconstructed skin micronucleus assay correctly identified 7 genotoxins and 5 non-genotoxins, demonstrating its potential to have a higher predictive value than currently available in vitro genotoxicity tests, and its utility as part of a comprehensive in vitro genotoxicity testing strategy. [Copyright &y& Elsevier]
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- 2009
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24. An evaluation of the genotoxicity of the antitussive drug Dextromethorphan
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Aardema, Marilyn J., Robison, Steven H., Gatehouse, David, and Johnston, Gail
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GENETIC toxicology , *CARCINOGENICITY testing , *DEXTROMETHORPHAN , *ANTITUSSIVE agents - Abstract
Abstract: Dextromethorphan (DMP) is an effective and widely used antitussive drug. While DMP has over a 50 year safe-marketing history, the only available genotoxicity data was an unpublished, negative Ames assay (Roche). Lack of a complete genotoxicity profile on DMP, specifically covering the chromosomal damage endpoint, prompted a regulatory request for an in vitro chromosome aberration assay. In accordance with EC and CPMP Guidance, we evaluated data for a number of chemicals with a structural relationship to DMP. DMP contains no structural alerts for genotoxicity or carcinogenicity using the Deductive Estimation of Risk from Existing Knowledge (DEREK) software tool, confirming the negative results obtained in the existing Ames assay. This is also consistent with the mostly negative genotoxicity and carcinogenicity data available on structurally related chemicals including morphine, codeine, nalbuphine, buprenorphine, naloxone, hydromorphone, levorphanol, and oxycodone. A state-of-the-science, in vitro chromosome aberration assay was also conducted, which demonstrated a lack of genotoxicity for DMP. The overall weight of evidence for DMP and its structural analogues, supports the conclusion that this class of phenanthrene-based chemicals, and DMP, in particular, are not genotoxic in vitro or in vivo, and do not represent a carcinogenic risk to patients. [Copyright &y& Elsevier]
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- 2008
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25. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: II. Further analysis of mammalian cell results, relative predictivity and tumour profiles
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Kirkland, David, Aardema, Marilyn, Müller, Lutz, and Hayashi, Makoto
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GENETIC toxicology , *RODENTS , *CARCINOGENS , *TUMORS - Abstract
Abstract: One of the consequences of the low specificity of the in vitro mammalian cell genotoxicity assays reported in our previous paper [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1–256] is industry and regulatory agencies dealing with a large number of false-positive results during the safety assessment of new chemicals and drugs. Addressing positive results from in vitro genotoxicity assays to determine which are “false” requires extensive resources, including the conduct of additional animal studies. In order to reduce animal usage, and to conserve industry and regulatory agency resources, we thought it was important to raise the question as to whether the protocol requirements for a valid in vitro assay or the criteria for a positive result could be changed in order to increase specificity without a significant loss in sensitivity of these tests. We therefore analysed some results of the mouse lymphoma assay (MLA) and the chromosomal aberration (CA) test obtained for rodent carcinogens and non-carcinogens in more detail. For a number of chemicals that are positive only in either of these mammalian cell tests (i.e. negative in the Ames test) there was no correlation between rodent carcinogenicity and level of toxicity (we could not analyse this for the CA test as insufficient data were available in publications), magnitude of response or lowest effective positive concentration. On the basis of very limited in vitro and in vivo data, we could also find no correlation between the above parameters and formation of DNA adducts. Therefore, a change to the current criteria for required level of toxicity in the MLA, to limit positive calls to certain magnitudes of response, or to certain concentration ranges would not improve the specificity of the tests without significantly reducing the sensitivity. We also investigated a possible correlation between tumour profile (trans-species, trans-sex and multi-site versus single-species, single-sex and single-site) and pattern of genotoxicity results. Carcinogens showing the combination of trans-species, trans-sex and multi-site tumour profile were much more prevalent (70% more) in the group of chemicals giving positive results in all three in vitro assays than amongst those giving all negative results. However, single-species, single-sex, single-site carcinogens were not very prevalent even amongst those chemicals giving three negative results in vitro. Surprisingly, when mixed positive and negative results were compared, multi-site carcinogens were highly prevalent amongst chemicals giving only a single positive result in the battery of three in vitro tests. Finally we extended our relative predictivity (RP) calculations to combinations of positive and negative results in the genotoxicity battery. For two out of three tests positive, the RP for carcinogenicity was no higher than 1.0 and for 2/3 tests negative the RP for non-carcinogenicity was either zero (for Ames+MLA+MN) or 1.7 (for Ames+MLA+CA). Thus, all values were less than a meaningful RP of two, and indicate that it is not possible to predict outcome of the rodent carcinogenicity study when only 2/3 genotoxicity results are in agreement. [Copyright &y& Elsevier]
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- 2006
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26. Toxicology and genetic toxicology in the new era of “toxicogenomics”: impact of “-omics” technologies
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Aardema, Marilyn J. and MacGregor, James T.
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GENETIC toxicology , *MOLECULAR biology - Abstract
The unprecedented advances in molecular biology during the last two decades have resulted in a dramatic increase in knowledge about gene structure and function, an immense database of genetic sequence information, and an impressive set of efficient new technologies for monitoring genetic sequences, genetic variation, and global functional gene expression. These advances have led to a new sub-discipline of toxicology: “toxicogenomics”. We define toxicogenomics as “the study of the relationship between the structure and activity of the genome (the cellular complement of genes) and the adverse biological effects of exogenous agents”. This broad definition encompasses most of the variations in the current usage of this term, and in its broadest sense includes studies of the cellular products controlled by the genome (messenger RNAs, proteins, metabolites, etc.). The new “global” methods of measuring families of cellular molecules, such as RNA, proteins, and intermediary metabolites have been termed “-omic” technologies, based on their ability to characterize all, or most, members of a family of molecules in a single analysis. With these new tools, we can now obtain complete assessments of the functional activity of biochemical pathways, and of the structural genetic (sequence) differences among individuals and species, that were previously unattainable. These powerful new methods of high-throughput and multi-endpoint analysis include gene expression arrays that will soon permit the simultaneous measurement of the expression of all human genes on a single “chip”. Likewise, there are powerful new methods for protein analysis (proteomics: the study of the complement of proteins in the cell) and for analysis of cellular small molecules (metabonomics: the study of the cellular metabolites formed and degraded under genetic control). This will likely be extended in the near future to other important classes of biomolecules such as lipids, carbohydrates, etc. These assays provide a general capability for global assessment of many classes of cellular molecules, providing new approaches to assessing functional cellular alterations. These new methods have already facilitated significant advances in our understanding of the molecular responses to cell and tissue damage, and of perturbations in functional cellular systems.As a result of this rapidly changing scientific environment, regulatory and industrial toxicology practice is poised to undergo dramatic change during the next decade. These advances present exciting opportunities for improved methods of identifying and evaluating potential human and environmental toxicants, and of monitoring the effects of exposures to these toxicants. These advances also present distinct challenges. For example, the significance of specific changes and the performance characteristics of new methods must be fully understood to avoid misinterpretation of data that could lead to inappropriate conclusions about the toxicity of a chemical or a mechanism of action. We discuss the likely impact of these advances on the fields of general and genetic toxicology, and risk assessment. We anticipate that these new technologies will (1) lead to new families of biomarkers that permit characterization and efficient monitoring of cellular perturbations, (2) provide an increased understanding of the influence of genetic variation on toxicological outcomes, and (3) allow definition of environmental causes of genetic alterations and their relationship to human disease. The broad application of these new approaches will likely erase the current distinctions among the fields of toxicology, pathology, genetic toxicology, and molecular genetics. Instead, a new integrated approach will likely emerge that involves a comprehensive understanding of genetic control of cellular functions, and of cellular responses to alterations in normal molecular structure and function. [Copyright &y& Elsevier]
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- 2002
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27. A weight of evidence assessment of the genotoxic potential of 4-methylimidazole as a possible mode of action for the formation of lung tumors in exposed mice.
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Brusick, David, Aardema, Marilyn J., Allaben, William T., Kirkland, David J., Williams, Gary, Llewellyn, G. Craig, Parker, Julia M., and Rihner, Marisa O.
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LUNG cancer , *CARCINOGENICITY , *PANEL analysis , *GENETIC toxicology , *EVIDENCE , *MICE - Abstract
4-Methylimidazole (4-MeI) is a byproduct formed during the cooking of foods containing carbohydrates and amino acids, including the production of flavors and coloring substances, e.g., class III and IV caramel colors, used in many food products with extensive human exposure. Two-year rodent bioassays via oral exposure conducted by the National Toxicology Program reported evidence of carcinogenicity only in B6C3F 1 mice (increased alveolar/bronchial neoplasms). In 2011, the International Agency for Research on Cancer classified 4-MeI as Group 2B, "possibly carcinogenic to humans". An expert panel was commissioned to assess the genotoxic potential of 4-MeI and the plausibility of a genotoxic mode of action in the formation of lung tumors in mice when exposed to high doses of 4-MeI. The panel defined and used a weight-of-evidence (WOE) approach that included thorough evaluation of studies assessing the genotoxic potential of 4-MeI. The panelists categorized each study, consisting of study weight, degree of technical performance, study reliability, and contribution to the overall WOE. Based on the reviewed studies' weighted contribution, the panel unanimously concluded that the WOE supports no clear evidence of in vivo genotoxicity of 4-MeI and no association for a genotoxic mode of action in the formation of mouse lung tumors. • An expert panel assessed the genotoxic potential of 4-methylimidazole (4-MeI). • The panel used a weight-of-evidence (WOE) approach for 4-MeI study evaluation. • The expert panel evaluated study reliability and contribution to the overall WOE. • The expert panel found no clear evidence of in vivo genotoxicity of 4-MeI. • Genotoxic mode of action for mouse lung tumor formation is not plausible for 4-MeI. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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28. Corrigendum to "Simulation of mouse and rat spermatogenesis to inform genotoxicity testing using OECD test guideline 488" [Mutat. Res. 832–833 (2018) 19–28].
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Marchetti, Francesco, Aardema, Marilyn J., Beevers, Carol, van Benthem, Jan, Douglas, George R., Godschalk, Roger, Yauk, Carole L., Young, Robert, and Williams, Andrew
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SPERMATOGENESIS , *RATS , *GENETIC toxicology , *MICE , *TEST interpretation , *GUIDELINES - Published
- 2019
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29. Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics
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Vanparys, Philippe, Corvi, Raffaella, Aardema, Marilyn J., Gribaldo, Laura, Hayashi, Makoto, Hoffmann, Sebastian, and Schechtman, Leonard
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GENETIC toxicology , *CELL transformation , *PHYSIOLOGICAL effects of cosmetics , *CARCINOGENICITY testing , *LABORATORY mice , *MUTAGENS , *NUCLEOLUS , *HAMSTERS as laboratory animals - Abstract
Abstract: Two year rodent bioassays play a key role in the assessment of carcinogenic potential of chemicals to humans. The seventh amendment to the European Cosmetics Directive will ban in 2013 the marketing of cosmetic and personal care products that contain ingredients that have been tested in animal models. Thus 2-year rodent bioassays will not be available for cosmetics/personal care products. Furthermore, for large testing programs like REACH, in vivo carcinogenicity testing is impractical. Alternative ways to carcinogenicity assessment are urgently required. In terms of standardization and validation, the most advanced in vitro tests for carcinogenicity are the cell transformation assays (CTAs). Although CTAs do not mimic the whole carcinogenesis process in vivo, they represent a valuable support in identifying transforming potential of chemicals. CTAs have been shown to detect genotoxic as well as non-genotoxic carcinogens and are helpful in the determination of thresholds for genotoxic and non-genotoxic carcinogens. The extensive review on CTAs by the OECD (OECD (2007) Environmental Health and Safety Publications, Series on Testing and Assessment, No. 31) and the proven within- and between-laboratories reproducibility of the SHE CTAs justifies broader use of these methods to assess carcinogenic potential of chemicals. [Copyright &y& Elsevier]
- Published
- 2012
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30. Workshop summary: Top concentration for in vitro mammalian cell genotoxicity assays; and report from working group on toxicity measures and top concentration for in vitro cytogenetics assays (chromosome aberrations and micronucleus)
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Galloway, Sheila, Lorge, Elisabeth, Aardema, Marilyn J., Eastmond, David, Fellows, Mick, Heflich, Robert, Kirkland, David, Levy, Dan D., Lynch, Anthony M., Marzin, Daniel, Morita, Takeshi, Schuler, Maik, and Speit, Günter
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GENETIC toxicology , *FIRE assay , *CYTOKINESIS , *CELL populations , *CELL growth , *NUCLEOLUS , *CLINICAL trials , *CYTOGENETICS ,MAMMAL cytology - Abstract
Abstract: The selection of maximum concentrations for in vitro mammalian cell genotoxicity assays was reviewed at the 5th International Workshop on Genotoxicity Testing (IWGT), 2009. Currently, the top concentration recommended when toxicity is not limiting is 10mM or 5mg/ml, whichever is lower. The discussion was whether to reduce the limit, and if so whether the 1mM limit proposed for human pharmaceuticals was appropriate for testing other chemicals. The consensus was that there was reason to consider reducing the 10mM limit, and many, but not all, attendees favored a reduction to 1mM. Several proposals are described here for the concentration limit. The in vitro cytogenetics expert working group also discussed appropriate measures and level of cytotoxicity. Data were reviewed from a multi-laboratory trial of the in vitro micronucleus (MN) assay with multiple cell types and several types of toxicity measurements. The group agreed on a preference for toxicity measures that take cell proliferation after the beginning of treatment into account (relative increase in cell counts, relative population doubling, cytokinesis block proliferation index or replicative index), and that this applies both to in vitro MN assays and to in vitro chromosome aberration assays. Since relative cell counts (RCC) underestimate toxicity, many group members favored making a recommendation against the use of RCC as a toxicity measure for concentration selection. All 14 chemicals assayed for MN induction in the multi-laboratory trial were detected without exceeding 50% toxicity by any measure, but some were positive only at concentrations with toxicity quite close to 50%. The expert working group agreed to accept the cytotoxicity range recommended by OECD guideline 487 (55±5% toxicity at the top concentration scored). This also reinforces the original intent of the guidance for the in vitro chromosome aberration assay, where “>50%” was intended to target the range close to 50% toxicity. [Copyright &y& Elsevier]
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- 2011
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31. A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: Genotoxicity. A COLIPA analysis
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Pfuhler, Stefan, Kirst, Annette, Aardema, Marilyn, Banduhn, Norbert, Goebel, Carsten, Araki, Daisuke, Costabel-Farkas, Margit, Dufour, Eric, Fautz, Rolf, Harvey, James, Hewitt, Nicola J., Hibatallah, Jalila, Carmichael, Paul, Macfarlane, Martin, Reisinger, Kerstin, Rowland, Joanna, Schellauf, Florian, Schepky, Andreas, and Scheel, Julia
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ALTERNATIVE toxicity testing , *ANIMAL experimentation , *COSMETICS , *GENETIC toxicology , *HEALTH risk assessment , *BIOLOGICAL assay , *IN vitro toxicity testing , *HAZARDOUS substances - Abstract
Abstract: For the assessment of genotoxic effects of cosmetic ingredients, a number of well-established and regulatory accepted in vitro assays are in place. A caveat to the use of these assays is their relatively low specificity and high rate of false or misleading positive results. Due to the 7th amendment to the EU Cosmetics Directive ban on in vivo genotoxicity testing for cosmetics that was enacted March 2009, it is no longer possible to conduct follow-up in vivo genotoxicity tests for cosmetic ingredients positive in in vitro genotoxicity tests to further assess the relevance of the in vitro findings. COLIPA, the European Cosmetics Association, has initiated a research programme to improve existing and develop new in vitro methods. A COLIPA workshop was held in Brussels in April 2008 to analyse the best possible use of available methods and approaches to enable a sound assessment of the genotoxic hazard of cosmetic ingredients. Common approaches of cosmetic companies are described, with recommendations for evaluating in vitro genotoxins using non-animal approaches. A weight of evidence approach was employed to set up a decision-tree for the integration of alternative methods into tiered testing strategies. [Copyright &y& Elsevier]
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- 2010
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32. Evaluation of the genotoxicity of the imidazole antifungal climbazole: Comparison to published results for other azole compounds
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Pérez-Rivera, Alex A., Hu, Ting, Aardema, Marilyn J., and Nash, J.F.
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IMIDAZOLES , *ANTIFUNGAL agents , *GENETIC toxicology , *LYMPHOMAS in animals , *NUCLEOLUS , *IN vitro toxicity testing , *SALMONELLA typhimurium , *ESCHERICHIA coli , *ANTIBACTERIAL agents , *DNA damage - Abstract
Abstract: Climbazole is an imidazole antifungal agent that can provide anti-dandruff benefits when incorporated into a shampoo matrix. A series of genotoxicity studies were performed to support the human safety of this azole antifungal drug. Climbazole was not mutagenic in the Salmonella typhimurium or Escherichia coli Ames assay and did not induce micronuclei in human lymphocytes. In the mouse lymphoma assay (MLA), climbazole was negative (non-mutagenic) with and without metabolic (S9) activation after a 4h exposure; an increase in small colony mutants was observed without metabolic activation after a 24h exposure at concentrations of 15 and 17.5μg/mL. An in vivo mouse micronucleus test was negative up to a maximum tolerated dose (MTD) of 150mg/kg climbazole administered orally. In the in vivo/in vitro unscheduled DNA synthesis assay, climbazole showed no evidence of DNA damage in the livers of rats at doses up to the MTD of 200mg/kg orally. A toxicokinetic study was performed in mice with oral administration of [14C]-climbazole (150mg/kg). Radioactivity (20.42μg-equiv./g plasma) was detected 15min after oral administration of [14C]-climbazole, and the peak concentration was 62.96μg-equiv./g plasma at 8h after dosing. The measured amounts of radioactivity in plasma, at all sample times from 15min up to 24h, exceeded the concentrations that induced increases in mutation frequency after 24h exposure of mouse lymphoma cells in vitro (15 and 17.5μg/mL). These observations lend support to the conclusion that climbazole does not present a genotoxic risk in vivo. Furthermore, these data are consistent with the published data for other azole antifungals that work by preventing the synthesis of ergosterol and, as a class, are generally non-genotoxic, except some isolated positive results of questionable significance. Collectively, these data are supportive of the view that climbazole does not present a genotoxic or carcinogenic risk to humans. [Copyright &y& Elsevier]
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- 2009
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33. Report from the in vitro micronucleus assay working group
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Kirsch-Volders, Micheline, Sofuni, Toshio, Aardema, Marilyn, Albertini, Silvio, Eastmond, David, Fenech, Michael, Ishidate Jr., Motoi, Kirchner, Stephan, Lorge, Elisabeth, Morita, Takeshi, Norppa, Hannu, Surrallés, Jordi, Vanhauwaert, Annelies, and Wakata, Akihiro
- Subjects
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GENETIC toxicology , *NUCLEOLUS , *CONFERENCES & conventions - Abstract
At the Washington “2nd International Workshop on Genotoxicity Testing” (25–26 March 1999) current methodologies and data for the in vitro micronucleus test were reviewed. As a result, guidelines for the conduct of specific aspects of the protocol were developed. Agreement was achieved on the following topics: choice of cells, slide preparation, analysis of micronuclei, toxicity, use of cytochalasin-B, number of doses, and treatment/harvest times [Environ. Mol. Mutagen. 35 (2000) 167]. Because there were a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at that time. These studies have now been completed and the data were reviewed at the Plymouth “3rd International Workshop on Genotoxicity Testing” (28–29 June 2002). Data from studies coordinated by the French Society of Genetic Toxicology, Japanese collaborative studies, European pharmaceutical industry validation studies, along with data from Lilly Research Laboratories were used to prepare conclusions on the main aspects of the in vitro micronucleus protocol. In this paper, the consensus agreements on the protocol for performing the in vitro micronucleus assay are presented. The major recommendations concern:
[Copyright &y& Elsevier]- Demonstration of cell proliferation: both cell lines and lymphocytes can be used, but demonstration of cell proliferation in both control and treated cells is compulsory for the acceptance of the test.
- Assessment of toxicity and dose range finding: assessment of toxicity should be performed by determining cell proliferation, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; and by determining other markers for cytotoxicity (confluency, apoptosis, necrosis) which can provide valuable additional information.
- Treatment schedules for cell lines and lymphocytes.
- Choice of positive controls: without S9-mix both a clastogen (e.g. mitomycin C or bleomycin) and an aneugen (e.g. colchicine) should be included as positive controls and a clastogen that requires S9 for activity when S9-mix is used (e.g. dimethylnitrosamine, or cyclophosphamide in those cell types that cannot activate this agent directly).
- Duplicate cultures and number of cells to be scored.
- Repeat experiments: in lymphocytes, for each experiment blood from 2 different healthy young and non-smoking donors should be compared. In cell lines, the experiments need only to be repeated if the first one is negative.
- Statistics: statistical significance should not be the sole factor for determining positive results. Biological meaning should serve as a guideline. Examples of statistical analyses are given.
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- 2003
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34. A comparison of transgenic rodent mutation and in vivo comet assay responses for 91 chemicals.
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Kirkland, David, Levy, Dan D., LeBaron, Matthew J., Aardema, Marilyn J., Beevers, Carol, Bhalli, Javed, Douglas, George R., Escobar, Patricia A., Farabaugh, Christopher S., Guerard, Melanie, Johnson, George E., Kulkarni, Rohan, Le Curieux, Frank, Long, Alexandra S., Lott, Jasmin, Lovell, David P., Luijten, Mirjam, Marchetti, Francesco, Nicolette, John J., and Pfuhler, Stefan
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DNA damage , *BONE marrow cells , *GENETIC toxicology , *MUTAGENS , *COLON (Anatomy) - Abstract
Highlights • In vivo comet assay compared with transgenic rodent assay for 91 chemicals. • Comparison focussed on evaluation of in vivo genotoxicity per se. • Fair to good agreement between assays in GI tract (stomach and colon) and liver. • Poor to no agreement between assays in bone marrow. Abstract A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals. [ABSTRACT FROM AUTHOR]
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- 2019
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35. The EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay: Historical control data and proof of principle studies for mechanistic assay adaptations.
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Roy, Shambhu, Kulkarni, Rohan, Hewitt, Nicola J., and Aardema, Marilyn J.
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NUCLEOLUS , *GENETIC toxicology , *CYTOKINES , *CARCINOGENS , *BIOMARKERS - Abstract
The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm™ is a promising novel animal alternative for evaluating genotoxicity of topically applied chemicals. It is particularly useful for assessing cosmetic ingredients that can no longer be tested using in vivo assays. To advance the use of this test especially for regulatory decision-making, we have established the RSMN assay in our laboratory according to Good Laboratory Practice and following the principles of the OECD test guideline 487 in vitro mammalian cell micronucleus test. Proficiency with the assay was established by correctly identifying direct-acting genotoxins and genotoxins requiring metabolism, as well as non-genotoxic/non-carcinogenic chemicals. We also report the analysis of our historical control data that demonstrate vehicle control and positive control values for %micronuclei in binucleated cells are in the ranges reported previously. Technical issues including evaluating various solvents with both 48 h and 72 h treatment regimens were investigated. For the first time, mechanistic studies using CREST analysis revealed that the RSMN assay is suitable for distinguishing aneugens and clastogens. Moreover, the assay is also suitable for measuring cytokines as markers for proliferative and toxic effects of chemicals. [ABSTRACT FROM AUTHOR]
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- 2016
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36. Improvement of in vivo genotoxicity assessment: Combination of acute tests and integration into standard toxicity testing
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Rothfuss, Andreas, Honma, Masamitu, Czich, Andreas, Aardema, Marilyn J., Burlinson, Brian, Galloway, Sheila, Hamada, Shuichi, Kirkland, David, Heflich, Robert H., Howe, Jonathan, Nakajima, Madoka, O’Donovan, Mike, Plappert-Helbig, Ulla, Priestley, Catherine, Recio, Leslie, Schuler, Maik, Uno, Yoshifumi, and Martus, Hans-Jörg
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GENETIC toxicology , *TOXICITY testing , *NUCLEOLUS , *TECHNICAL specifications , *BIOLOGICAL assay , *MEDICAL protocols , *HEMORRHAGE , *DOSE-response relationship in poisons - Abstract
Abstract: A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin. [Copyright &y& Elsevier]
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- 2011
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37. The reconstructed skin micronucleus assay (RSMN) in EpiDerm™: Detailed protocol and harmonized scoring atlas
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Dahl, Erica L., Curren, Rodger, Barnett, Brenda C., Khambatta, Zubin, Reisinger, Kerstin, Ouedraogo, Gladys, Faquet, Brigitte, Ginestet, Anne-Claire, Mun, Greg, Hewitt, Nicola J., Carr, Greg, Pfuhler, Stefan, and Aardema, Marilyn J.
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NUCLEOLUS , *BIOTECHNOLOGY laboratories , *EPIDERMAL growth factor , *FIRE assay , *GENETIC toxicology , *CELL culture , *CELL lines - Abstract
Abstract: The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm™ for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm™ and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test. [Copyright &y& Elsevier]
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- 2011
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38. Dermal penetration and metabolism of p-aminophenol and p-phenylenediamine: Application of the EpiDerm™ human reconstructed epidermis model
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Hu, Ting, Bailey, Ruth E., Morrall, Stephen W., Aardema, Marilyn J., Stanley, Lesley A., and Skare, Julie A.
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BIOLOGICAL assay , *DERMATOTOXICOLOGY , *COSMETICS , *GENETIC toxicology , *BIOTRANSFORMATION (Metabolism) , *AROMATIC amines , *EPIDERMIS , *HAIR dyeing & bleaching - Abstract
Abstract: To address the provision of the 7th Amendment to the EU Cosmetics Directive banning the use of in vivo genotoxicity assays for testing cosmetic ingredients in 2009, the 3D EpiDerm™ reconstructed human skin micronucleus assay has been developed. To further characterise the EpiDerm™ tissue for potential use in genotoxicity testing, we have evaluated the dermal penetration and metabolism of two hair dye ingredients, p-aminophenol (PAP) and p-phenylenediamine (PPD) in this reconstructed epidermis model. When EpiDerm™ tissue was topically exposed to PAP or PPD for 30min (typical for a hair dye exposure), the majority (80–>90%) of PAP or PPD was excluded from skin tissue and removed by rinsing. After a 23.5h recovery period, the PAP fraction that did penetrate was completely N-acetylated to acetaminophen (APAP). Similarly, 30min topical application of PPD resulted in the formation of the N-mono- and N,N′-diacetylated metabolites of PPD. These results are consistent with published data on the dermal metabolism of these compounds from other in vitro systems as well as from in vivo studies. When tissue was exposed topically (PAP) or via the culture media (PPD) for 24h, there was good batch-to-batch and donor-to-donor reproducibility in the penetration and metabolism of PAP and PPD. Overall, the results demonstrate that these two aromatic amines are biotransformed in 3D EpiDerm™ tissue via N-acetylation. Characterising the metabolic capability of EpiDerm™ tissue is important for the evaluation of this model for use in genotoxicity testing. [Copyright &y& Elsevier]
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- 2009
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39. Intralaboratory and interlaboratory evaluation of the EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay
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Hu, Ting, Kaluzhny, Yulia, Mun, Greg C., Barnett, Brenda, Karetsky, Viktor, Wilt, Nathan, Klausner, Mitchell, Curren, Rodger D., and Aardema, Marilyn J.
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EPIDERMIS , *BIOLOGICAL models , *NUCLEOLUS , *BIOLOGICAL assay , *GENETIC toxicology , *IN vitro toxicity testing , *COSMETICS , *MITOMYCIN C , *VINBLASTINE , *METHANESULFONATES - Abstract
Abstract: A novel in vitro human reconstructed skin micronucleus (RSMN) assay has been developed using the EpiDerm™ 3D human skin model [R. D. Curren, G. C. Mun, D. P. Gibson, and M. J. Aardema, Development of a method for assessing micronucleus induction in a 3D human skin model EpiDerm™, Mutat. Res. 607 (2006) 192–204]. The RSMN assay has potential use in genotoxicity assessments as a replacement for in vivo genotoxicity assays that will be banned starting in 2009 according to the EU 7th Amendment to the Cosmetics Directive. Utilizing EpiDerm™ tissues reconstructed with cells from four different donors, intralaboratory and interlaboratory reproducibility of the RSMN assay were examined. Seven chemicals were evaluated in three laboratories using a standard protocol. Each chemical was evaluated in at least two laboratories and in EpiDerm™ tissues from at least two different donors. Three model genotoxins, mitomycin C (MMC), vinblastine sulfate (VB) and methyl methanesulfonate (MMS) induced significant, dose-related increases in cytotoxicity and MN induction in EpiDerm™ tissues. Conversely, four dermal non-carcinogens, 4-nitrophenol (4-NP), trichloroethylene (TCE), 2-ethyl-1,3-hexanediol (EHD), and 1,2-epoxydodecane (EDD) were negative in the RSMN assay. Results between tissues reconstructed from different donors were comparable. These results indicate the RSMN assay using the EpiDerm™ 3D human skin model is a promising new in vitro genotoxicity assay that allows evaluation of chromosome damage following “in vivo-like” dermal exposures. [Copyright &y& Elsevier]
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- 2009
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40. Identification of a gene expression profile that discriminates indirect-acting genotoxins from direct-acting genotoxins
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Hu, Ting, Gibson, David P., Carr, Gregory J., Torontali, Suzanne M., Tiesman, Jay P., Chaney, Joel G., and Aardema, Marilyn J.
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GENE expression , *GENETIC toxicology , *DNA , *LYMPHOMAS - Abstract
During the safety evaluation process of new drugs and chemicals, a battery of genotoxicity tests is conducted starting with in vitro genotoxicity assays. Obtaining positive results in in vitro genotoxicity tests is not uncommon. Follow-up studies to determine the biological relevance of positive genotoxicity results are costly, time consuming, and utilize animals. More efficient methods, especially for identifying a putative mode of action like an indirect mechanism of genotoxicity (where DNA molecules are not the initial primary targets), would greatly improve the risk assessment for genotoxins. To this end, we are participating in an International Life Sciences Institute (ILSI) project involving studies of gene expression changes caused by model genotoxins. The purpose of the work is to evaluate gene expression tools in general, and specifically for discriminating genotoxins that are direct-acting from indirect-acting. Our lab has evaluated gene expression changes as well as micronuclei (MN) in L5178Y TK+/− mouse lymphoma cells treated with six compounds. Direct-acting genotoxins (where DNA is the initial primary target) that were evaluated included the DNA crosslinking agents, mitomycin C (MMC) and cisplatin (CIS), and an alkylating agent, methyl methanesulfonate (MMS). Indirect-acting genotoxins included hydroxyurea (HU), a ribonucleotide reductase inhibitor, taxol (TXL), a microtubule inhibitor, and etoposide (ETOP), a DNA topoisomerase II inhibitor. Microarray gene expression analysis was conducted using Affymetrix mouse oligonucleotide arrays on RNA samples derived from cells which were harvested immediately after the 4 h chemical treatment, and 20 h after the 4 h chemical treatment. The evaluation of these experimental results yields evidence of differentially regulated genes at both 4 and 24 h time points that appear to have discriminating power for direct versus indirect genotoxins, and therefore may serve as a fingerprint for classifying chemicals when their mechanism of action is unknown. [Copyright &y& Elsevier]
- Published
- 2004
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