title: International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories.
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.
authors: Litron Laboratories, Rochester, New York, USA. sdertinger@litronlabs.com
authors: Stephen D
authors: SD
authors: Dertinger
authors: Souk
authors: S
authors: Phonethepswath
authors: Pamela
authors: P
authors: Weller
authors: John
authors: J
authors: Nicolette
authors: Joel
authors: J
authors: Murray
authors: Paul
authors: P
authors: Sonders
authors: Hans-Werner
authors: HW
authors: Vohr
authors: Jing
authors: J
authors: Shi
authors: Ljubica
authors: L
authors: Krsmanovic
authors: Carol
authors: C
authors: Gleason
authors: Laura
authors: L
authors: Custer
authors: Andrew
authors: A
authors: Henwood
authors: Kevin
authors: K
authors: Sweder
authors: Leon F
authors: LF
authors: Stankowski
authors: Daniel J
authors: DJ
authors: Roberts
authors: Amanda
authors: A
authors: Giddings
authors: Julia
authors: J
authors: Kenny
authors: Anthony M
authors: AM
authors: Lynch
authors: Céline
authors: C
authors: Defrain
authors: Fabrice
authors: F
authors: Nesslany
authors: Bas-jan M
authors: BJ
authors: van der Leede
authors: Terry
authors: T
authors: Van Doninck
authors: Ann
authors: A
authors: Schuermans
authors: Kentaro
authors: K
authors: Tanaka
authors: Yoshie
authors: Y
authors: Hiwata
authors: Osamu
authors: O
authors: Tajima
authors: Eleanor
authors: E
authors: Wilde
authors: Azeddine
authors: A
authors: Elhajouji
authors: William C
authors: WC
authors: Gunther
authors: Catherine J
authors: CJ
authors: Thiffeault
authors: Thomas J
authors: TJ
authors: Shutsky
authors: Ronald D
authors: RD
authors: Fiedler
authors: Takafumi
authors: T
authors: Kimoto
authors: Javed A
authors: JA
authors: Bhalli
authors: Robert H
authors: RH
authors: Heflich
authors: James T
authors: JT
authors: MacGregor
mesh_terms: D000818:Animals; D018957:CD59 Antigens / Q000235:genetics; D002138:Calibration; D003627:Data Interpretation, Statistical; D023381:Endpoint Determination; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D005038:Ethylnitrosourea / Q000633:toxicity; D005434:Flow Cytometry / Q000379:methods / Q000592:standards; D007391:International Cooperation; D007753:Laboratories / Q000592:standards; D008565:Membrane Proteins / Q000235:genetics*; D009152:Mutagenicity Tests / Q000379:methods / Q000592:standards; D009153:Mutagens / Q000633:toxicity; D009154:Mutation; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; 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; D005038:Ethylnitrosourea