title: International Pig-a gene mutation assay trial (stage III): results with N-methyl-N-nitrosourea.
abstract: N-methyl-N-nitrosourea (MNU) was evaluated in the in vivo Pig-a mutation assay as part of an International Collaborative Trial to investigate laboratory reproducibility, 28-day study integration, and comparative analysis with micronucleus (MN), comet, and clinical pathology endpoints. Male Sprague Dawley rats were treated for 28 days with doses of 0, 2.5, 5, and 10 mg MNU/kg/day in two independent laboratories, GlaxoSmithKline (GSK) and Bristol Myers Squibb (BMS). Additional studies investigated the low-dose region (<2.5 mg/kg/day). Reticulocytes were evaluated for Pig-a phenotypic mutation, CD59-negative reticulocytes/erythrocytes (RETs(CD592-)/ RBCs(CD592-)) on Days 1, 4, 15, 29, 43, and 57, and for micronucleated reticulocytes (MN-RETs) on Days 4 and 29. Comet analysis was conducted for liver and whole blood, and hematology and clinical chemistry was investigated. Dose-dependent increases in the frequency of RETs(CD592-) and RBCs(CD592-) were observed by Day 15 or 29, respectively. Dose-dependent increases were observed in %MN-RET on Days 4 and 29, and in mean %tail intensity in liver and in blood. Hematology/clinical chemistry data demonstrated bone marrow toxicity. Data comparison between GSK and BMS indicated a high degree of concordance with the Pig-a mutation assay results, consistent with previous observations with MNU and N-ethyl-N-nitrosourea. These data confirm that complementary genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that can provide information on gene mutation, chromosome damage, and DNA strand breaks in a single repeat dose rodent study. Collectively, this would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints.
authors.affiliation: GlaxoSmithKline, Ware, Hertfordshire, United Kingdom. anthony.m.lynch@gsk.com
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Lynch
authors.forename: Amanda
authors.initials: A
authors.lastname: Giddings
authors.forename: Laura
authors.initials: L
authors.lastname: Custer
authors.forename: Carol
authors.initials: C
authors.lastname: Gleason
authors.forename: Andrew
authors.initials: A
authors.lastname: Henwood
authors.forename: Mike
authors.initials: M
authors.lastname: Aylott
authors.forename: Julia
authors.initials: J
authors.lastname: Kenny
mesh_terms: D000818:Animals; D018957:CD59 Antigens / Q000235:genetics; D002138:Calibration; D020552:Comet Assay / Q000379:methods / Q000592:standards; D003627:Data Interpretation, Statistical; D023381:Endpoint Determination; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D007391:International Cooperation; D007753:Laboratories / Q000592:standards; D008099:Liver / Q000187:drug effects / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008770:Methylnitrosourea / Q000633:toxicity*; D015162:Micronucleus Tests / Q000379:methods / Q000592:standards; D009152:Mutagenicity Tests / Q000379:methods / Q000592:standards; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D015203:Reproducibility of Results; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D018570:Risk Assessment; D013997:Time Factors
chemical_list: D018957:CD59 Antigens; D008565:Membrane Proteins; D009153:Mutagens; C080086:phosphatidylinositol glycan-class A protein; D008770:Methylnitrosourea