title: Report on stage III Pig-a mutation assays using N-ethyl-N-nitrosourea-comparison with other in vivo genotoxicity endpoints.
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.
authors.affiliation: Pfizer Global Research and Development, Genetic Toxicology Center of Emphasis, Groton, Connecticut 06350, USA.
authors.forename: Zoryana
authors.initials: Z
authors.lastname: Cammerer
authors.forename: Javed A
authors.initials: JA
authors.lastname: Bhalli
authors.forename: Xuefei
authors.initials: X
authors.lastname: Cao
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Coffing
authors.forename: Donna
authors.initials: D
authors.lastname: Dickinson
authors.forename: Krista L
authors.initials: KL
authors.lastname: Dobo
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Dobrovolsky
authors.forename: Maria
authors.initials: M
authors.lastname: Engel
authors.forename: Ronald D
authors.initials: RD
authors.lastname: Fiedler
authors.forename: William C
authors.initials: WC
authors.lastname: Gunther
authors.forename: Robert H
authors.initials: RH
authors.lastname: Heflich
authors.forename: Mason G
authors.initials: MG
authors.lastname: Pearce
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Shaddock
authors.forename: Thomas
authors.initials: T
authors.lastname: Shutsky
authors.forename: Catherine J
authors.initials: CJ
authors.lastname: Thiffeault
authors.forename: Maik
authors.initials: M
authors.lastname: Schuler
mesh_terms: D000818:Animals; D018957:CD59 Antigens / Q000235:genetics; D002138:Calibration; D003106:Colon / Q000187:drug effects / Q000648:ultrastructure; D020552:Comet Assay / Q000379:methods / Q000592:standards; D003627:Data Interpretation, Statistical; D004052:Diethylnitrosamine / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D023381:Endpoint Determination; D004912:Erythrocytes / Q000187:drug effects / Q000648:ultrastructure; D005434:Flow Cytometry; D008099:Liver / Q000187:drug effects / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D015162:Micronucleus Tests / Q000379:methods / Q000592:standards; D009152:Mutagenicity Tests / Q000379:methods / Q000592:standards; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D009928:Organ Specificity; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D015203:Reproducibility of Results; D012156:Reticulocytes / Q000187:drug effects / Q000648:ultrastructure; D013045:Species Specificity; D013154:Spleen / Q000187:drug effects / Q000648:ultrastructure; D013997:Time Factors
chemical_list: D018957:CD59 Antigens; D008565:Membrane Proteins; D009153:Mutagens; C080086:phosphatidylinositol glycan-class A protein; D004052:Diethylnitrosamine