Toxicology Consulting Services

Bonita Springs, FL, United States

Toxicology Consulting Services

Bonita Springs, FL, United States
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Dertinger S.D.,University of Rochester | Phonethepswath S.,University of Rochester | Franklin D.,University of Rochester | Weller P.,University of Rochester | And 7 more authors.
Toxicological Sciences | Year: 2010

Two endpoints of genetic toxicity, mutation at the X-linked Pig-a gene and chromosomal damage in the form of micronucleated reticulocytes (MN-RETs), were evaluated in blood samples obtained from 28-day repeat-dosing studies typical of those employed in toxicity evaluations. Male Wistar Han rats were treated at 24-h intervals on days 1 through 28 with one of five prototypical genotoxicants: N-ethyl-N-nitrosourea, 7,12-dimethyl-12-benz[a]anthracene, 4-nitroquinoline-1-oxide (4NQO), benzo(a)-pyrene, and N-methyl-N-nitrosourea. Flow cytometric scoring of CD59-negative erythrocytes (indicative of glycosylphosphatidylinositol anchor deficiency and hence Pig-a mutation) was performed using blood specimens obtained on days 21, 15, 29, and 56. Blood specimens collected on days 4 and 29 were evaluated for MN-RET frequency using flow cytometry-based MicroFlow Kits. With the exception of 4NQO, each chemical induced significant increases in the frequency of MN-RETs on days 4 and 29. All five agents increased the frequency of mutant phenotype (CD59 negative) reticulocytes (RETs) and erythrocytes. Mutation responses in RETs occurred earlier than in erythrocytes and tended to peak, or nearly peak, at day 29. In contrast, the mutant phenotype erythrocyte responses were modest on day 29 and required additional time to reach their maximal value. The observed kinetics were expected based on the known turnover of RETs and erythrocytes. The data show that RETs can serve as an appropriate indicator cell population for 28-day studies. Collectively, these data suggest that blood-based genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.


Dertinger S.D.,Litron Laboratories, Ltd. | Phonethepswath S.,Litron Laboratories, Ltd. | Avlasevich S.L.,Litron Laboratories, Ltd. | Torous D.K.,Litron Laboratories, Ltd. | And 6 more authors.
Toxicological Sciences | Year: 2012

The ability to effectively monitor gene mutation and micronucleated reticulocyte (MN-RET) frequency in short-term and repeated dosing schedules was investigated using the recently developed flow cytometric Pig-a mutation assay and flow cytometric micronucleus analysis. Eight reference genotoxicants and three presumed nongenotoxic compounds were studied: chlorambucil, melphalan, thiotepa, cyclophosphamide, azathioprine, 2-acetylaminofluorene, hydroxyurea, methyl methanesulfonate, o-anthranilic acid, sulfisoxazole, and sodium chloride. These experiments extend previously published results with seven other chemicals. Male Sprague Dawley rats were treated via gavage for 3 or 28 consecutive days with several dose levels of each chemical up to the maximum tolerated dose. Blood samples were collected at several time points up to day 45 and were analyzed for Pig-a mutation with a dual-labeling method that facilitates mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. An immunomagnetic separation technique was used to increase the efficiency of scoring mutant cells. Blood samples collected on day 4, and day 29 for the 28-day study, were evaluated for MN-RET frequency. The three nongenotoxicants did not induce Pig-a or MN-RET responses. All genotoxicants except hydroxyurea increased the frequency of Pig-a mutant reticulocytes and erythrocytes. Significant increases in MN-RET frequency were observed for each of the genotoxicants at both time points. Whereas the highest Pig-a responses tended to occur in the 28-day studies, when total dose was greatest, the highest induction of MN-RET was observed in the 3-day studies, when dose per day was greatest. There was no clear relationship between the maximal Pig-a response of a given chemical and its corresponding maximal MN-RET response, despite the fact that both endpoints were determined in the same cell lineage. Taken with other previously published results, these data demonstrate the value of integrating Pig-a and micronucleus endpoints into in vivo toxicology studies, thereby providing information about mutagenesis and chromosomal damage in the same animals from which toxicity, toxicokinetics, and metabolism data are obtained. © The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.


Starr T.B.,NC Associates | MacGregor J.A.,Toxicology Consulting Services | Ehman K.D.,Toxicology Regulatory Services | Nikiforov A.I.,Toxicology Regulatory Services
Regulatory Toxicology and Pharmacology | Year: 2012

The National Toxicology Program (NTP) chronic inhalation bioassay of vanadium pentoxide (V2O5) produced " clear" evidence of lung tumors in B6C3F1 mice, but only " some" and " equivocal" evidence in male and female F344/N rats, respectively. No significant pairwise differences or trends with V2O5 concentration in male or female rat poly-3-adjusted tumor incidence were reported. The " some" and " equivocal" evidence descriptors arose from comparisons of V2O5-exposed group incidence rates with NTP-2000- and NIH-07-fed historical control (HC) group incidence ranges. NTP acknowledged that use of data from NIH-07-fed HC groups could be inappropriate because the V2O5 study used the NTP-2000 diet, but few studies using this newer diet were available then. We supplemented the early NTP-2000 diet HC data with data from 25 additional NTP-2000 diet studies conducted subsequent to the V2O5 bioassay. This widened the HC tumor incidence ranges, thereby weakening the limited evidence for the carcinogenicity of inhaled V2O5 in rats relative to HCs. The male rat control group in the V2O5 study also appeared to be a near-" outlier" relative to the expanded HC database, potentially invalidating any comparisons of exposed group incidence rates with those for HCs. We conclude that there is " no" evidence of V2O5 carcinogenicity in male or female F344/N rats. © 2012 Elsevier Inc.


Dertinger S.D.,Litron Laboratories, Ltd. | Phonethepswath S.,Litron Laboratories, Ltd. | Avlasevich S.L.,Litron Laboratories, Ltd. | Torous D.K.,Litron Laboratories, Ltd. | And 4 more authors.
Environmental and Molecular Mutagenesis | Year: 2014

To evaluate whether blood-based genotoxicity endpoints can provide temporal and dose-response data within the low-dose carcinogenic range that could contribute to carcinogenic mode of action (MoA) assessments, we evaluated the sensitivity of flow cytometry-based micronucleus and Pig-a gene mutation assays at and below tumorigenic dose rate 50 (TD50) levels. The incidence of micronucleated reticulocytes (MN-RET) was used to evaluate chromosomal damage, and the frequency of CD59-negative reticulocytes (RETCD59-) and erythrocytes (RBCCD59-) served as phenotypic reporters of mutation at the X-linked Pig-a gene. Several leukemogenic agents with a presumed genotoxic MoA were studied. Specifically, male Sprague Dawley rats were treated via oral gavage for 28 days with chlorambucil, thiotepa, melphalan, and 1,3-propane sultone at doses corresponding to 0.33x, 1x, and 3x TD50, as well as at the maximum tolerated dose. Frequencies of MN-RET were determined at Days 4 and 29, and RETCD59- and RBCCD59- data were collected pretreatment as well as Days 15/16, 29, and 56/57. Dose-related increases were observed for each endpoint, and time to maximal effect was consistently: MN-RET


Heddle J.A.,York University | Fenech M.,CSIRO | Hayashi M.,Biosafety Research Center | MacGregor J.T.,Toxicology Consulting Services
Mutagenesis | Year: 2011

These are personal reflections on the development of methods to use micronuclei as a measure of genetic damage and their use in research and in toxicology by four people who have been intimately involved with this work, a personal rather than a comprehensive history. About 6000 papers have been published using such methods in many tissues in vivo or in cultured cells of many organisms from plants to humans, but the majority of the work has been on mammalian erythrocytes and human lymphocytes, the areas in which we have worked primarily. Although this is by no means a complete history, those working in the field may be interested in some of the personal events that lie behind the development and acceptance of methods that are now standard. © The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved.


Morita T.,Japan National Institute of Health Sciences | MacGregor J.T.,Toxicology Consulting Services | Hayashi M.,Biosafety Research Center
Mutagenesis | Year: 2011

This report updates previous reviews that were conducted as part of the third and fourth International Workshops on Genetic Toxicology Testing of micronucleus (MN) assays in rodent tissues other than bone marrow. Tissues discussed here are liver, lung, skin, colon, spleen, testes and foetal/neonatal tissues with transplacental exposure. Previous reviews have been updated to include literature published after 2000. In addition to the previously described tissues, MN assays in bladder, buccal mucosal cells, stomach and vagina are also included. MN assays using tissues other than bone marrow are critical for risk assessments, for in situ evaluation and for studies of systemic genotoxic effects and modes of action. Protocols for the majority of assays in tissues other than bone marrow have not yet been well standardised and validated for regulatory application, and further development is needed to support regulatory studies. © The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved.


Dertinger S.D.,Litron Laboratories, Ltd. | Torous D.K.,Litron Laboratories, Ltd. | Hayashi M.,Biosafety Research Center | MacGregor J.T.,Toxicology Consulting Services
Mutagenesis | Year: 2011

The relative simplicity of the micronucleated erythrocyte endpoint has made it amenable to automated scoring approaches. Flow cytometry is one such scoring platform that has been employed successfully. This review describes the evolution and properties of flow cytometry-based scoring of micronucleated erythrocytes. The methodology has become widely applied to rodent blood specimens and the high throughput nature of the technology provides a number of advantages over manual microscopic scoring. For instance, the ability to efficiently survey many dose levels and many more cells per specimen relative to microscopy benefits studies that are designed to identify no observable effect levels or lowest observable effect levels. Furthermore, flow cytometry makes it practical to study species with low spontaneous reticulocyte (RET) counts and micronucleus (MN) frequencies, thereby facilitating integration of blood-based micronucleated reticulocyte (MN-RET) frequency measurements into experiments conducted across species of toxicological interest. This capability enhances genotoxicity assessments that have historically been made in dedicated MN tests performed in one species. Importantly, the feasibility of using MN-RET frequencies in blood from humans as an index of genetic damage in bone marrow opens a critical area of application that had not been practical previously. We conclude with recommendations for additional work that is needed to more fully realise the potential of flow cytometric in vivo MN scoring. © The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved.


Starr T.B.,NC Associates | MacGregor J.A.,Toxicology Consulting Services
Regulatory Toxicology and Pharmacology | Year: 2014

The US Environmental Protection Agency (USEPA) is currently conducting a toxicological review of vanadium pentoxide (V2O5). As part of that effort, the Agency will need to address the fact that while a National Toxicology Program (NTP) chronic inhalation bioassay of V2O5 produced clear evidence of treatment-related lung tumors in both male and female B6C3F1 mice, neither of these responses were dose-related across the groups exposed to 1, 2, and 4mg/m3. While lung tumor incidence was significantly elevated in all three exposed groups relative to that in the control groups, it was essentially flat across them. Herein we report results from computing poly-3-adjusted Cochran-Armitage trend test statistics with and without inclusion of the lung tumor incidence data from control group mice. These results confirm the absence of any significant dose-related effect on mouse lung tumor incidence in the study groups exposed to V2O5. We also considered two estimates of area under the vanadium lung burden versus time curve as plausible alternative dose metrics to the V2O5 chamber concentration. However, these alternative dose metrics were so highly correlated with the V2O5 chamber concentration (r=0.998) that nothing is to be gained from their use in place of the V2O5 chamber concentration in attempts to perform dose-response modeling of the tumor incidence or unit cancer risk computations. At the present time, there is no scientific basis to support linear (or nonlinear) extrapolations of estimated cancer risks to V2O5 exposure levels below 1mg/m3. Additional tumor data at multiple V2O5 concentrations lower than 1mg/m3 are required to support such extrapolations. © 2014 Elsevier Inc.


Benson J.M.,Lovelace Respiratory Research Institute | Gigliotti A.P.,Lovelace Respiratory Research Institute | March T.H.,Lovelace Respiratory Research Institute | Barr E.B.,Lovelace Respiratory Research Institute | And 4 more authors.
Journal of Toxicology and Environmental Health - Part A: Current Issues | Year: 2011

Chronic inhalation studies were conducted to compare the toxicity and potential carcinogenicity of evaporative emissions from unleaded gasoline (GVC) and gasoline containing the oxygenate methyl tertiary-butyl ether (MTBE; GMVC). The test materials were manufactured to mimic vapors people would be exposed to during refueling at gas stations. Fifty F344 rats per gender per exposure level per test article were exposed 6 h/d, 5 d/wk for 104 wk in whole body chambers. Target total vapor concentrations were 0, 2, 10, or 20 g/m3 for the control, low-, mid-, and high-level exposures, respectively. Endpoints included survival, body weights, clinical observations, organs weights, and histopathology. GVC and GMVC exerted no marked effects on survival or clinical observations and few effects on organ weights. Terminal body weights were reduced in all mid-and high-level GVC groups and high-level GMVC groups. The major proliferative lesions attributable to gasoline exposure with or without MTBE were renal tubule adenomas and carcinomas in male rats. GMV exposure led to elevated testicular mesothelioma incidence and an increased trend for thyroid carcinomas in males. GVMC inhalation caused an increased trend for testicular tumors with exposure concentration. Mid-and high-level exposures of GVC and GMVC led to elevated incidences of nasal respiratory epithelial degeneration. Overall, in these chronic studies conducted under identical conditions, the health effects in F344 rats following 2 yr of GVC or GMVC exposure were comparable in the production of renal adenomas and carcinomas in male rats and similar in other endpoints. Copyright © Taylor & Francis Group, LLC.


Macgregor J.T.,Toxicology Consulting Services | Dertinger S.D.,Litron Laboratories, Ltd.
Mutagenesis | Year: 2014

Determination of the mode of action of carcinogenic agents is an important factor in risk assessment and regulatory practice. To assess the ability of the erythrocyte-based Pig-a mutation assay to discriminate between genotoxic and non-genotoxic modes of action, the mutagenic response of Sprague Dawley rats exposed to methyl carbamate (MC) or ethyl carbamate (EC) was investigated. EC, a potent carcinogen, is believed to induce DNA damage through the formation of a DNA-reactive epoxide group, whereas the closely structurally related compound, MC, cannot form this epoxide and its weaker carcinogenic activity is thought to be secondary to inflammation and promotion of cell proliferation. The frequency of Pig-a mutant phenotype cells was monitored before, during, and after 28 consecutive days of oral gavage exposure to either MC (doses ranging from 125 to 500mg/kg/day) or EC (250mg/kg/day). Significant increases in the frequency of mutant reticulocytes were observed from Days 15 through 43, with a peak mean frequency of 19.9×10-6 on Day 29 (i.e. 24.9-fold increase relative to mean vehicle control across all four sampling times). As expected, mutant erythrocyte responses lagged behind mutant reticulocyte responses, with a maximal mean frequency of 8.2×10-6 on Day 43 (i.e. 16.4-fold increase). No mutagenic effects were observed with MC. A second indicator of in vivo genotoxicity, peripheral blood micronucleated reticulocytes, was also studied. This endpoint was responsive to EC (3.3-fold mean increase), but not to MC. These results support the hypothesis that genotoxicity contributes to the carcinogenicity of EC but not of MC, and illustrates the value of the Pig-a assay for discriminating between genotoxic and non-genotoxic modes of action. © 2015 © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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