title: Assessment of genotoxicity induced by 7,12-dimethylbenz(a)anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies.
abstract: As part of the Stage 3 of the Pig-a international trial, we evaluated 7,12-dimethylbenz(a)anthracene (DMBA) for induction of Pig-a gene mutation using a 28-day repeat dose study design in Sprague-Dawley rats. In the same study, chromosomal damage in peripheral blood and primary DNA damage in liver were also investigated by the micronucleus (MN) assay and the Comet assay, respectively. In agreement with previously published data (Dertinger et al., [2010]: Toxicol Sci 115:401-411), DMBA induced dose-dependent increases of CD59-negative erythrocytes/reticulocytes and micronucleated reticulocytes (MN-RETs). However, there was no significant increase in DNA damage in the liver cells when tested up to 10 mg/kg/day, which appears to be below the maximum tolerated dose. When tested up to 200 mg/kg/day in a follow-up 3 dose study, DMBA was positive in the liver Comet assay. Additionally, we evaluated diethylnitrosamine (DEN), a known mutagen/hepatocarcinogen, for induction of Pig-a mutation, MN and DNA damage in a 28-day study. DEN produced negative results in both the Pig-a mutation assay and the MN assay, but induced dose-dependent increases of DNA damage in the liver and blood Comet assay. In summary, our results demonstrated that the Pig-a mutation assay can be effectively integrated into repeat dose studies and the data are highly reproducible between different laboratories. Also, integration of multiple genotoxicity endpoints into the same study not only provides a comprehensive evaluation of the genotoxic potential of test chemicals, but also reduces the number of animals needed for testing, especially when more than one in vivo genotoxicity tests are required.
authors: BioReliance Corporation, Rockville, Maryland 20850, USA. jing.shi@bioreliance.com
authors: Jing
authors: J
authors: Shi
authors: Ljubica
authors: L
authors: Krsmanovic
authors: Shannon
authors: S
authors: Bruce
authors: Tawney
authors: T
authors: Kelly
authors: Madhav
authors: M
authors: Paranjpe
authors: Kathleen
authors: K
authors: Szabo
authors: Mirna
authors: M
authors: Arevalo
authors: Samual
authors: S
authors: Atta-Safoh
authors: Fekado
authors: F
authors: Debelie
authors: Michelle Klug
authors: MK
authors: LaForce
authors: Jamie
authors: J
authors: Sly
authors: Sandra
authors: S
authors: Springer
mesh_terms: D015127:9,10-Dimethyl-1,2-benzanthracene / Q000633:toxicity*; D000818:Animals; D001853:Bone Marrow / Q000187:drug effects / Q000648:ultrastructure; D018957:CD59 Antigens / Q000235:genetics; D002138:Calibration; 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 / Q000378:metabolism / Q000648:ultrastructure; D005434:Flow Cytometry; D007753:Laboratories / Q000592:standards; 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; 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; D004052:Diethylnitrosamine; D015127:9,10-Dimethyl-1,2-benzanthracene