title: Evaluation of the Pig-a, micronucleus, and comet assay endpoints in a 28-day study with ethyl methanesulfonate.
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.
authors.affiliation: Pfizer Global Research and Development, Genetic Toxicology, Groton, Connecticut.
authors.forename: William C
authors.initials: WC
authors.lastname: Gunther
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Coffing
authors.forename: Donna A
authors.initials: DA
authors.lastname: Dickinson
authors.forename: Maria E
authors.initials: ME
authors.lastname: Engel
authors.forename: Ronald D
authors.initials: RD
authors.lastname: Fiedler
authors.forename: Susan D
authors.initials: SD
authors.lastname: O'Lone
authors.forename: Kelley E
authors.initials: KE
authors.lastname: Sanok
authors.forename: Catherine J
authors.initials: CJ
authors.lastname: Thiffeault
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Shutsky
authors.forename: Maik J
authors.initials: MJ
authors.lastname: Schuler
authors.forename: Krista L
authors.initials: KL
authors.lastname: Dobo
mesh_terms: D000818:Animals; D003106:Colon / Q000166:cytology / Q000187:drug effects; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D023381:Endpoint Determination; D004912:Erythrocytes / Q000187:drug effects; D005020:Ethyl Methanesulfonate / Q000633:toxicity*; D008099:Liver / Q000166:cytology / Q000187:drug effects; D008297:Male; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics*; D015162:Micronucleus Tests / Q000379:methods*; D059645:Mutation Rate; D051381:Rats; D017207:Rats, Sprague-Dawley; D012156:Reticulocytes / Q000187:drug effects
chemical_list: D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein; D005020:Ethyl Methanesulfonate
keywords: in vivo genotoxicity; in vivo mutation assay; integration