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1.
Environ Mol Mutagen ; 56(3): 322-32, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25229874

ABSTRACT

Understanding the mutagenic dose response could prove beneficial in the management of pharmaceutically relevant impurities. For most alkyl ester impurities, such as isopropyl methanesulfonate (IPMS), little in vivo mutagenicity data exist for dose analysis. The likelihood of a sublinear dose response for IPMS was assessed by comparing the Swain Scott constant, the SN 1/SN 2 reaction mechanism and the O(6) :N(7) guanine adduct ratio to that of more well-known alkyl esters. Based on available information, IPMS was predicted to have a mutagenic profile most like ethyl nitrosourea. To test this hypothesis, mature male Wistar Han rats were administered IPMS using acute (single administration at 3.5 to 56 mg/kg) or subchronic (28 days at 0.125 to 2 mg/kg/day) exposures. The in vivo Pig-a mutation assay was used to identify mutant phenotype reticulocyte (Ret) and red blood cell (RBC) populations. The maximum mutant response occurred approximately 15 and 28 days after the last dose administration in the mutant Ret and RBC populations respectively in the acute study and on Day 29 and 56 in the mutant Ret and RBC populations, respectively, in the subchronic study. A comparison of RBC mutant frequencies from acute and subchronic protocols suggests a sublinear response; however, this was not substantiated by statistical analysis. A No Observed Effect Level (NOEL) of 0.25 mg/kg/day resulted in a Permitted Daily Exposure equivalent to the Threshold of Toxicological Concern. An estimate of the NOEL based on the previously mentioned factors, in practice, would have pre-empted further investigation of the potent mutagen IPMS.


Subject(s)
Erythrocytes/drug effects , Membrane Proteins/genetics , Mesylates/toxicity , Mutagenicity Tests , Mutagens/toxicity , Reticulocytes/drug effects , Animals , CD59 Antigens/analysis , Erythrocytes/metabolism , Male , Mesylates/administration & dosage , Micronucleus Tests , Mutagenicity Tests/methods , Mutagens/administration & dosage , No-Observed-Adverse-Effect Level , Rats , Rats, Wistar , Reticulocytes/metabolism
2.
Environ Mol Mutagen ; 55(6): 492-9, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24599777

ABSTRACT

Ethyl methanesulfonate (EMS) was evaluated as part of the validation effort for the rat Pig-a mutation assay and compared with other well-established in vivo genotoxicity endpoints. Male Sprague-Dawley (SD) rats were given a daily dose of 0, 6.25, 12.5, 25, 50, or 100 mg/kg/day EMS for 28 days, and evaluated for a variety of genotoxicity endpoints in peripheral blood, liver, and colon. Blood was sampled pre-dose (Day 1) and at various time points up to Day 105. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies. The first statistically significant increases in mutant frequencies were seen in RETs on Day 15 and in RBCs on Day 29 with the maximum RET(CD59-) on Day 29 and of RBC(CD59-) on Day 55. The lowest dose producing a statistically significant increase of RET(CD59-) was 12.5 mg/kg on Day 55 and 25 mg/kg for RBC(CD59-) on Day 55. EMS also induced significant increases in % micronucleated RETs (MN-RETs) in peripheral blood on Days 3, 15, and 28. No statistically significant increases in micronuclei were seen in liver or colon. Results from the in vivo Comet assay on Day 29 showed generally weak increases in DNA damage in all tissues evaluated with little evidence for accumulation of damage seen over time. The results with EMS indicate that the assessment of RBC(CD59-) and/or RET(CD59-) in the Pig-a assay could be a useful and sensitive endpoint for a repeat dose protocol and complements other genotoxicity endpoints.


Subject(s)
Comet Assay/methods , Ethyl Methanesulfonate/toxicity , Membrane Proteins/genetics , Micronucleus Tests/methods , Animals , Colon/cytology , Colon/drug effects , DNA Damage/drug effects , Dose-Response Relationship, Drug , Endpoint Determination , Erythrocytes/drug effects , Liver/cytology , Liver/drug effects , Male , Membrane Proteins/drug effects , Mutation Rate , Rats , Rats, Sprague-Dawley , Reticulocytes/drug effects
3.
Environ Mol Mutagen ; 52(9): 721-30, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22167886

ABSTRACT

N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon. Blood was sampled predose (Day-1) and at various time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD592-) and RET(CD592-) frequencies. Consistent with the results from a reference laboratory, RBC(CD592-) and RET(CD592-) frequencies increased in a dose and time-dependent manner, producing significant increases at all doses by Day 15, with similar frequencies seen in both rat strains. ENU also induced small but significant increases in % micronucleated RETs on Days 4 and 29. No significant increases in micronuclei were seen in the liver or colon of the ENU-treated SD rats. Hprt and Pig-a lymphocyte mutation assays conducted on splenocytes from Day 56 F344 rats detected two- to fourfold stronger responses for Hprt than Pig-a mutations. Results from the in vivo Comet assay in SD rats at Day 29 showed generally weak increases in DNA damage in all tissues evaluated. The results with ENU indicate that the Pig-a RET and RBC assays are reproducible, transferable, and complement other genotoxicity endpoints that could potentially be integrated into 28-day repeat dose rat studies.


Subject(s)
Diethylnitrosamine/toxicity , Membrane Proteins/genetics , Mutagenicity Tests , Mutagens/toxicity , Mutation , Animals , CD59 Antigens/genetics , Calibration , Colon/drug effects , Colon/ultrastructure , Comet Assay/methods , Comet Assay/standards , Data Interpretation, Statistical , Dose-Response Relationship, Drug , Endpoint Determination , Erythrocytes/drug effects , Erythrocytes/ultrastructure , Flow Cytometry , Liver/drug effects , Liver/ultrastructure , Male , Micronucleus Tests/methods , Micronucleus Tests/standards , Mutagenicity Tests/methods , Mutagenicity Tests/standards , Organ Specificity , Rats , Rats, Inbred F344 , Rats, Sprague-Dawley , Reference Standards , Reproducibility of Results , Reticulocytes/drug effects , Reticulocytes/ultrastructure , Species Specificity , Spleen/drug effects , Spleen/ultrastructure , Time Factors
4.
Environ Mol Mutagen ; 52(9): 690-8, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21910140

ABSTRACT

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RET(CD59-) , and frequency of RBC(CD59-) . The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87-0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories.


Subject(s)
Flow Cytometry , Laboratories , Membrane Proteins/genetics , Mutagenicity Tests , Mutation , Animals , CD59 Antigens/genetics , Calibration , Data Interpretation, Statistical , Endpoint Determination , Erythrocytes/drug effects , Erythrocytes/metabolism , Erythrocytes/ultrastructure , Ethylnitrosourea/toxicity , Flow Cytometry/methods , Flow Cytometry/standards , International Cooperation , Laboratories/standards , Mutagenicity Tests/methods , Mutagenicity Tests/standards , Mutagens/toxicity , Rats , Rats, Inbred F344 , Rats, Sprague-Dawley , Rats, Wistar , Reference Standards , Reproducibility of Results , Reticulocytes/drug effects , Reticulocytes/metabolism , Reticulocytes/ultrastructure , Risk Assessment , Time Factors
5.
Mutat Res ; 725(1-2): 13-21, 2011 Oct 09.
Article in English | MEDLINE | ID: mdl-21729764

ABSTRACT

In recent years, experimental evidence has accumulated that supports the existence of sublinear dose-response relationships at low doses of DNA reactive mutagens. However, creating the in vivo data necessary to allow for a more detailed dose-response modeling with the currently available tools might not always be practical. The purpose of the current work was to evaluate the utility of the Pig-a gene mutation assay to rapidly identify dose-response relationships for direct acting genotoxicants. The induction of mutations in the peripheral blood of rats was evaluated following 28 days of exposure down to low doses of the direct acting alkylating agents ethyl methane sulfonate (EMS) and ethylnitrosourea (ENU). Using statistical modeling based on the 28-day studies, a threshold for mutation induction for EMS was estimated to be 21.9mg/kg, whereas for the more potent ENU, the threshold was estimated to be 0.88mg/kg. Comparing mutation frequencies from acute and sub-chronic dosing indicated less than additive dose-response relationships, further confirming the possibility of a threshold dose-response relationship for both compounds. In conclusion, the work presented provides evidence that the Pig-a assay might be a practical alternative to other in vivo mutation assays when assessing dose-response relationships for direct acting mutagens and that an experimental approach using fractionated dosing could be used to substantiate a biological mechanism responsible for the observation of a sublinear dose-response relationship.


Subject(s)
Dose-Response Relationship, Drug , Ethyl Methanesulfonate/toxicity , Ethylnitrosourea/toxicity , Membrane Proteins/genetics , Mutagenicity Tests/methods , Mutagens/toxicity , Animals , Rats
6.
Environ Mol Mutagen ; 52(4): 269-79, 2011 May.
Article in English | MEDLINE | ID: mdl-20872852

ABSTRACT

The in vivo bone marrow (BM) micronucleus assay is one of the three tests in the standard test battery to assess the genotoxic potential of a pharmaceutical candidate. In some cases, depending on results of in vitro studies, the route of administration or the degree of systemic exposure, in vivo assessment of genotoxicity in the BM alone may not be sufficient. Based on the potential for high gut exposures to orally administered compounds with low systemic exposures as well as the potential susceptibility of rapidly dividing cells of the intestinal tissues, we have developed a modified technique for evaluating micronuclei formation in both the duodenum and colon of rats based on earlier publications. Adult male Sprague Dawley rats were treated once daily for 2 days with either vehicle control or with the test articles acetyl salicylic acid (ASA), carbendazim (CAR), cyclophosphamide (CP), dimethylhydrazine (DMH), mitomycin C (MMC) or vinblastine sulfate (VIN). The duodenum, colon, and BM were harvested, processed, and analyzed for micronucleus induction. Results from these studies demonstrated differences in the susceptibility for different test compounds in the three tissues tested. When MMC and VIN were dosed by different routes at the same dose levels both compounds produced positive results in all three tissues by intraperitoneal injection but not oral administration. These studies suggest that overall the GI micronucleus assay might be a useful tool for clastogenic and aneugenic compounds that are expected to produce high sustained concentrations in the gastrointestinal tract with little systemic exposure.


Subject(s)
Colon/drug effects , Duodenum/drug effects , Micronucleus Tests/methods , Animals , Aspirin/adverse effects , Aspirin/pharmacology , Benzimidazoles/adverse effects , Benzimidazoles/pharmacology , Carbamates/adverse effects , Carbamates/pharmacology , Cyclophosphamide/adverse effects , Cyclophosphamide/pharmacology , DNA Damage/drug effects , Dimethylhydrazines/adverse effects , Dimethylhydrazines/pharmacology , Male , Mitomycin/adverse effects , Mitomycin/pharmacology , Mutagenicity Tests/methods , Mutagens/adverse effects , Mutagens/pharmacology , Rats , Rats, Sprague-Dawley , Vinblastine/adverse effects , Vinblastine/pharmacology
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