title: Predictive capacity of a non-radioisotopic local lymph node assay using flow cytometry, LLNA:BrdU-FCM: Comparison of a cutoff approach and inferential statistics.
abstract: In order for a novel test method to be applied for regulatory purposes, its reliability and relevance, i.e., reproducibility and predictive capacity, must be demonstrated. Here, we examine the predictive capacity of a novel non-radioisotopic local lymph node assay, LLNA:BrdU-FCM (5-bromo-2'-deoxyuridine-flow cytometry), with a cutoff approach and inferential statistics as a prediction model. 22 reference substances in OECD TG429 were tested with a concurrent positive control, hexylcinnamaldehyde 25%(PC), and the stimulation index (SI) representing the fold increase in lymph node cells over the vehicle control was obtained. The optimal cutoff SI (2.7≤cutoff <3.5), with respect to predictive capacity, was obtained by a receiver operating characteristic curve, which produced 90.9% accuracy for the 22 substances. To address the inter-test variability in responsiveness, SI values standardized with PC were employed to obtain the optimal percentage cutoff (42.6≤cutoff <57.3% of PC), which produced 86.4% accuracy. A test substance may be diagnosed as a sensitizer if a statistically significant increase in SI is elicited. The parametric one-sided t-test and non-parametric Wilcoxon rank-sum test produced 77.3% accuracy. Similarly, a test substance could be defined as a sensitizer if the SI means of the vehicle control, and of the low, middle, and high concentrations were statistically significantly different, which was tested using ANOVA or Kruskal-Wallis, with post hoc analysis, Dunnett, or DSCF (Dwass-Steel-Critchlow-Fligner), respectively, depending on the equal variance test, producing 81.8% accuracy. The absolute SI-based cutoff approach produced the best predictive capacity, however the discordant decisions between prediction models need to be examined further.
authors.affiliation: College of Pharmacy, Ewha Womans University, Republic of Korea.
authors.forename: Da-Eun
authors.initials: DE
authors.lastname: Kim
authors.affiliation: College of Pharmacy, Ewha Womans University, Republic of Korea.
authors.forename: Hyeri
authors.initials: H
authors.lastname: Yang
authors.affiliation: Medical Beauty Research Division, Amorepacific Corp. R&D Center, Republic of Korea.
authors.forename: Won-Hee
authors.initials: WH
authors.lastname: Jang
authors.affiliation: Medical Beauty Research Division, Amorepacific Corp. R&D Center, Republic of Korea.
authors.forename: Kyoung-Mi
authors.initials: KM
authors.lastname: Jung
authors.affiliation: Medical Beauty Research Division, Amorepacific Corp. R&D Center, Republic of Korea.
authors.forename: Miyoung
authors.initials: M
authors.lastname: Park
authors.affiliation: Medical Beauty Research Division, Amorepacific Corp. R&D Center, Republic of Korea.
authors.forename: Jin Kyu
authors.initials: JK
authors.lastname: Choi
authors.affiliation: Pharmacology Efficacy Team, Biotoxtech Co., Ltd., Republic of Korea.
authors.forename: Mi-Sook
authors.initials: MS
authors.lastname: Jung
authors.affiliation: Pharmacology Efficacy Team, Biotoxtech Co., Ltd., Republic of Korea.
authors.forename: Eun-Young
authors.initials: EY
authors.lastname: Jeon
authors.affiliation: Department of Occupational Health, College of Natural Sciences, Catholic University of Daegu, Republic of Korea.
authors.forename: Yong
authors.initials: Y
authors.lastname: Heo
authors.affiliation: Department of Occupational Health, College of Natural Sciences, Catholic University of Daegu, Republic of Korea.
authors.forename: Kyung-Wook
authors.initials: KW
authors.lastname: Yeo
authors.affiliation: Department of Occupational Health, College of Natural Sciences, Catholic University of Daegu, Republic of Korea.
authors.forename: Ji-Hoon
authors.initials: JH
authors.lastname: Jo
authors.affiliation: Department of Occupational Health, College of Natural Sciences, Catholic University of Daegu, Republic of Korea.
authors.forename: Jung Eun
authors.initials: JE
authors.lastname: Park
authors.affiliation: National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Republic of Korea.
authors.forename: Soo Jung
authors.initials: SJ
authors.lastname: Sohn
authors.affiliation: National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Republic of Korea.
authors.forename: Tae Sung
authors.initials: TS
authors.lastname: Kim
authors.affiliation: National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Republic of Korea.
authors.forename: Il Young
authors.initials: IY
authors.lastname: Ahn
authors.affiliation: College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea.
authors.forename: Tae-Cheon
authors.initials: TC
authors.lastname: Jeong
authors.affiliation: College of Pharmacy, Ewha Womans University, Republic of Korea. Electronic address: kmlim@ewha.ac.kr.
authors.forename: Kyung-Min
authors.initials: KM
authors.lastname: Lim
authors.affiliation: College of Pharmacy, Ewha Womans University, Republic of Korea. Electronic address: sjbae@ewha.ac.kr.
authors.forename: SeungJin
authors.initials: S
authors.lastname: Bae
mesh_terms: D000818:Animals; D001973:Bromodeoxyuridine / Q000032:analysis* / Q000737:chemistry*; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005544:Forecasting; D023441:Local Lymph Node Assay; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D001973:Bromodeoxyuridine
keywords: Descriptive and inferential statistics; LLNA:BrdU–FCM; Local lymph node assay; Prediction model; Predictive capacity; Skin sensitization