title: Inter-laboratory validation of the in-vivo flow cytometric micronucleus analysis method (MicroFlow®) in China.
abstract: Although inter-laboratory validation efforts of the in-vivo micronucleus (MN) assay based on flow cytometry (FCM) have taken place in the EU and US, none have been organized in China. Therefore, an inter-laboratory study that included eight laboratories in China and one experienced reference laboratory in the US was coordinated to validate the in-vivo FCM MicroFlow(®) method to determine the frequency of micro-nucleated reticulocytes (MN-RETs) in rat blood. Assay reliability and reproducibility were evaluated with four known genotoxicants, and the results obtained with the FCM method were compared with the outcome of the traditional evaluation of bone-marrow micronuclei by use of microscopy. Each of the four chemicals was tested at three sites (two in China and the one US reference laboratory). After three consecutive daily exposures to a genotoxicant, blood and bone-marrow samples were obtained from rats 24h after the third dose. MN-RET frequencies were measured in 20,000 RET in blood by FCM, and micro-nucleated polychromatic erythrocyte (MN-PCE) frequencies were measured in 2,000 PCEs in bone marrow by microscopy. For both methods, each genotoxicant was shown to induce a statistically significant increase in the frequency of MN after treatment with at least one dose. Where more doses than one caused an increase, responses occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM-based MN-RET vs microscopy-based MN-PCE measurements (eight experiments, 200 paired measurements) was 0.723, indicating a high degree of correspondence between methods and compartments. The rs value for replicate FCM MN-RET measurements performed at the eight collaborative laboratories was 0.940 (n=200), and between the eight FCM laboratories with the reference laboratory was 0.933 (n=200), suggesting that the automated method is very well transferable between laboratories. The FCM micronucleus analysis method is currently used in many countries worldwide, and these data support its use for evaluating the in-vivo genotoxic potential of test chemicals in China.
authors.affiliation: National Shanghai Center for New Drug Safety Evaluation & Research, China State Institute of Pharmaceutical Industry, Shanghai, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chang
authors.affiliation: National Shanghai Center for New Drug Safety Evaluation & Research, China State Institute of Pharmaceutical Industry, Shanghai, China.
authors.forename: Changhui
authors.initials: C
authors.lastname: Zhou
authors.affiliation: Center for Drug Evaluation, China Food and Drug Administration, Beijing, China.
authors.forename: Fanghua
authors.initials: F
authors.lastname: Huang
authors.affiliation: Litron Laboratories, Rochester, NY, US.
authors.forename: Dorothea K
authors.initials: DK
authors.lastname: Torous
authors.affiliation: Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Luan
authors.affiliation: Beijing Institute of Pharmacology and Toxicology, National Beijing Center for Drug Safety Evaluation and Research, Beijing, China.
authors.forename: Chang
authors.initials: C
authors.lastname: Shi
authors.affiliation: Joinn Laboratories, Beijing, China.
authors.forename: Hongling
authors.initials: H
authors.lastname: Wang
authors.affiliation: Institute for Food and Drug Safety Evaluation, National Institute for Food and Drug Control, Beijing, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: National Chengdu Center for Safety Evaluation of Drugs, Chengdu, Sichuan, China.
authors.forename: Na
authors.initials: N
authors.lastname: Wei
authors.affiliation: Center for Evaluation of Drug Safety, Second Military Medical University, Shanghai, China.
authors.forename: Zhenna
authors.initials: Z
authors.lastname: Xia
authors.affiliation: WuXi AppTec (Suzhou) Co., Ltd., Suzhou, Jiangsu, China.
authors.forename: Zhaomin
authors.initials: Z
authors.lastname: Zhong
authors.affiliation: National Shanghai Center for New Drug Safety Evaluation & Research, China State Institute of Pharmaceutical Industry, Shanghai, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Joinn Laboratories, Beijing, China.
authors.forename: Fengjun
authors.initials: F
authors.lastname: An
authors.affiliation: Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Yiyi
authors.initials: Y
authors.lastname: Cao
authors.affiliation: Institute for Food and Drug Safety Evaluation, National Institute for Food and Drug Control, Beijing, China.
authors.forename: Xingchao
authors.initials: X
authors.lastname: Geng
authors.affiliation: WuXi AppTec (Suzhou) Co., Ltd., Suzhou, Jiangsu, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: National Chengdu Center for Safety Evaluation of Drugs, Chengdu, Sichuan, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Ju
authors.affiliation: Beijing Institute of Pharmacology and Toxicology, National Beijing Center for Drug Safety Evaluation and Research, Beijing, China.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Center for Evaluation of Drug Safety, Second Military Medical University, Shanghai, China.
authors.forename: Jiangbo
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Litron Laboratories, Rochester, NY, US.
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Dertinger
authors.affiliation: Institute for Food and Drug Safety Evaluation, National Institute for Food and Drug Control, Beijing, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.affiliation: Beijing Institute of Pharmacology and Toxicology, National Beijing Center for Drug Safety Evaluation and Research, Beijing, China.
authors.forename: Mingyang
authors.initials: M
authors.lastname: Liao
authors.affiliation: Center for Evaluation of Drug Safety, Second Military Medical University, Shanghai, China.
authors.forename: Bojun
authors.initials: B
authors.lastname: Yuan
authors.affiliation: Center for Evaluation of Drug Safety, Second Military Medical University, Shanghai, China.
authors.forename: Tianbao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Becton Dickinson Medical Devices (Shanghai) Co Ltd., Shanghai, China.
authors.forename: Jinbao
authors.initials: J
authors.lastname: Yu
authors.affiliation: Becton Dickinson Medical Devices (Shanghai) Co Ltd., Shanghai, China.
authors.forename: Zhongchun
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Center for Drug Evaluation, China Food and Drug Administration, Beijing, China. Electronic address: wangql@cde.org.cn.
authors.forename: Qingli
authors.initials: Q
authors.lastname: Wang
authors.affiliation: National Shanghai Center for New Drug Safety Evaluation & Research, China State Institute of Pharmaceutical Industry, Shanghai, China. Electronic address: jma@ncdser.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Ma
mesh_terms: D000818:Animals; D002681:China; D004249:DNA Damage; D004900:Erythroblasts / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D008297:Male; D048629:Micronuclei, Chromosome-Defective; D009153:Mutagens / Q000009:adverse effects* / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
chemical_list: D009153:Mutagens
keywords: Aneugen; Clastogen; Flow cytometry; Genotoxicity; Micronucleus; Validation